AN ABSTRACT OF THE DISSERTATION OF
Brian Nowak-Thompson for the degree of Doctor of Philosophy in
Biochemistry and Biophysics presented on March 14,1997. Title: An Integrative
Approach to Understanding Pyoluteorin Biosynthesis in the Biological
Control Bacterium Pseudomonas fluorescens Pf-5.
Redacted for Privacy
Abstract Approved:
Pyoluteorin is a chlorinated, mixed polyketide metabolite produced by
Pseudomonas fluorescens Pf-5. Pyoluteorin exhibits antifungal activity against
Pythium ultimum, the causal agent of preemergence damping-off disease.
Biosynthesis of this compound was studied using an approach that integrated
nucleotide sequence analysis of a region required for pyoluteorin production
and isotopic labelling studies of proposed pathway intermediates. ['COOKL-Proline was demonstrated to be the primary precursor of the chlorinated
pyrrole moiety within pyoluteorin. Hypothetical pathway intermediates were
synthesized and tested in vivo for their incorporation into pyoluteorin. The
results of these studies, however, were equivocal and definitive conclusions
regarding the biochemical pathway leading to pyoluteorin could not be made.
Nucleotide sequencing of the region required for pyoluteorin production
identified several clustered biosynthetic genes. pltB and pltC encode a Type I
polyketide synthase required for pyoluteorin biosynthesis. A pathway for
resorcinol ring formation is proposed based on the catalytic domain
organization of the polyketide synthase. The deduced peptide sequences of
pltA, pltD, and a partial open reading frame, orf-S, exhibit similarity to the
halogenases that are involved in tetracycline and pyrrolnitrin biosynthesis.
Although halogenases are a poorly characterized class of enzymes, some
aspects of their catalytic mechanism regarding pyoluteorin biosynthesis are
discussed based on peptide sequence analysis. The regulatory gene pltR was
also identified and appears to encode a transcriptional activator protein with
similarity to the LysR family of transcriptional regulators.
An Integrative Approach to Understanding Pyoluteorin Biosynthesis in the
Biological Control Bacterium Pseudoinonas fluorescens Pf-5.
by
Brian Nowak-Thompson
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Complete March 14, 1997
Commencement June 1997
Doctor of Philosophy dissertation of Brian Nowak-Thompson presented
on March 14, 1997
APPROVED:
Redacted for Privacy
Major Professor, representing Biochemistry and Biophysics
Redacted for Privacy
Head of Department of Biochemistry and Biophysics
Redacted for Privacy
Dean of GradweXe School
I understand that my thesis will become part of the permanent collection
of Oregon State University libraries. My signature below authorizes
release of my thesis to any reader upon request.
Redacted for Privacy
Brian Nowak-Thompson
ACKNOWLEDGMENTS
For their non-scientific contributions:
I gratefully acknowledge the undying patience and loving support
of my life-long companion (who is, fortunately, also my wife), Jo. Her
willingness to endure my lengthy stay in graduate school is not to be
forgotten. Good baby Everett was also a major motivating force behind
the completion of this writing and is the newest person I will probably
ever see. I also thank Dr. Glenn Tinseth for his insightful discussions
on brewing and fermentations and damn him for finishing first!
For their scientific contributions:
I would like to express my gratitude to Nathan Corbell, Nancy
Chaney, Marcella Henkles, Cheryl Whistler, Dr. Mary Hagen, and Dr.
Virginia Stockwell for their research contributions that have provided
the biological context that is such an integral part of this project. I especially
thank Dr. Jennifer Kraus for her initial work that has made this project
so successful. Dr. Chris Melville is also recognized for initiating many
stimulating discussions concerning chemical synthetic methods.
Finally, I am forever indebted to Drs. Joyce Loper and Steven Gould
who have taught me as much about being a scientist as they have about
science. Their confidence and guidance have been very much appreciated.
Dr. Loper is gratefully recognized for fostering the collaborations I have
taken part in and the many professional contacts I have had the
opportunity to make during my tenure.
TABLE OF CONTENTS
rag
Chapter 1. Biological Control of Plant Pathogens: A Paradigm
for Understanding Bacterial Physiology and the Ecological
Role of Secondary Metabolites
1
1.1 Introduction
1
1.2 Antibiosis as a mechanism in biological control
2
1.3 Statement of research objectives
9
Chapter 2. Biochemical Analysis of the Pyoluteorin Pathway
2.1 Introduction
2.1.1 Rudimentary polyketide pathways: the tetraketides
2.1.2 Unusual polyketide starter units
2.1.3 Post-assembly modifications of polyketide metabolites
11
11
11
15
20
2.2 Biosynthetic Analysis of Pyoluteorin
22
2.3 Results and Discussion
25
2.3.1 Preliminary studies: pyoluteorin production
and isolation
2.3.2 Incorporation of proline into pyoluteorin
2.3.3 Involvement of deschloropyoluteorin
2.3.4 Involvement of pyrrole-2-carboxylic acid
2.3.5 Involvement of tetrahydrodeschloropyoluteorin
25
26
28
32
34
2.4 Conclusion
37
2.5 Experimental methods
37
Chapter 3. Nucleotide Sequence Analysis of Loci Required for
Pyoluteorin Biosynthesis
3.1 Introduction
52
52
TABLE OF CONTENTS (Continued)
Page
3.1.1 Genetic organization of polyketide synthases
3.1.2 Halogenating enzymes in microorganisms
3.2 Results and Discussion
3.2.1 Nucleotide sequencing of the pyoluteorin region
3.2.2 Identification and sequence analysis of the
pyoluteorin PKS
3.2.3 Identification and sequence analysis of the
pyoluteorin halogenases
52
55
56
56
58
67
3.3 Conclusions
70
3.4 Experimental methods
71
Chapter 4. Nucleotide Sequence Analysis of a Putative
Transcriptional Activator Regulating
Pyoluteorin Biosynthesis
73
4.1 Introduction
73
4.2 Results and Discussion
75
4.2.1 Nucleotide sequence analysis of pltR
4.2.2 Similarity of PltR to LysR-type transcriptional activators
4.2.3 A putative P1tR binding site within the
pyoluteorin gene cluster
4.2.4 Models for PltR-dependent regulation of
pyoluteorin biosynthesis
75
76
79
80
4.3 Conclusions
81
4.4 Experimental Methods
81
Chapter 5. Concluding Summary
83
5.1 Pyoluteorin biosynthesis
83
5.2 Regulation of pyoluteorin biosynthesis
85
TABLE OF CONTENTS (Continued)
Page
Bibliography
Appendices
Appendix 1. Production of 2,4-Diacetylphloroglucinol by the
Biocontrol Agent Pseudomonas fluorescens Pf-5
Appendix 2. Nucleotide Sequence and Translated Product of p1tB
Appendix 3. Nucleotide Sequence and Translated Product of pltC
Appendix 4. Nucleotide Sequence and Translated Product of pltA
Appendix 5. Nucleotide Sequence and Translated Product of pltD
Appendix 6. Nucleotide Sequence and Translated Product of orfS
Appendix 7. Nucleotide Sequence and Translated Product of pltR
86
110
111
116
126
133
135
138
139
LIST OF FIGURES
Figure
1-1.
Antibiotics with inhibitory activity against plant pathogens
3-1.
Genetic organization and part of the reaction pathway
catalyzed by a Type I PKS
3-2.
3-3.
3-4.
raga
4
53
Genetic organization and part of the reaction pathway
catalyzed by a Type II PKS
54
Genomic region required for pyoluteorin production
and the biosynthetic genes identified by nucleotide analysis
57
Modular organization of the functional domains
identified in P1tB and P1tC
59
Deduced peptide sequence alignment for the three PKS
modules encoded by pltB and pltC.
61
Proposed route for formation of the pyoluteorin
resorcinol moiety.
66
The deduced peptide sequence alignments for the putative
halogenases PltA, PltD, and Orf-S with PrnC and Cts4.
68
Genomic region required for pyoluteorin production
and the regulatory gene identified by nucleotide analysis.
75
4-2.
Gap alignment of P1tR and a LysR profile sequence
77
4-3.
The putative promoter region of pltR
79
3-5.
3-6.
3-7.
4-1.
An Integrative Approach to Understanding Pyoluteorin Biosynthesis in the
Biological Control Bacterium Pseudomonas fluorescens Pf-5.
Chapter 1. Biological Control of Plant Pathogens:
A Paradigm for Understanding Bacterial Physiology
and the Ecological Role of Secondary Metabolites
1.1 INTRODUCTION
Traditionally, research in plant pathology and plant physiology has
focused on two types of microbial interactions, namely parasitism/predation
and mutualism (symbiosis). Because both types of interactions generally
involve a plant host, and since the respective disciplines often emphasize
agricultural applications, the importance of microbe-microbe interactions
within agricultural systems is often overlooked. Nevertheless, an astounding
number of interactions, both direct and indirect, inevitably occur among the
individual populations within any microbial community and can affect
microbe-plant interactions. It is possible within certain agricultural systems
to exploit the microbial interactions in order to biologically control plant
pathogenic organisms that are not managed adequately by current control
practices.
Biological control is defined as the reduction in the amount of inoculum
or the virulence of a pathogen accomplished by one or more organisms,
excluding humans (Cook and Baker, 1983). Biological control of plant pathogens
can be accomplished through modifying cultural practices to increase or
maintain native antagonistic populations or by artificially introducing
antagonists or hypovirulent strains of the pathogen either into the
environment or onto the plant host. Therefore, successfully implementing a
biological control program requires an understanding of not only the agonistantagonist relationship, but also the environmental influences affecting the
establishment of antagonist populations. Many ecologists advocate that
homeostasis of very diverse communities is generally maintained via negative
feedback, wherein an environmentally induced change in one population
2
elicits a response within other communal populations so as to reduce the
severity of the initial environmental stimulus (Bull and Slater, 1982). Because
environmentally induced changes within microbial populations are
manifested as observable phenotypes, biological control systems serve as useful
paradigms for physiological processes that occur in response to environmental
conditions. As a result, biological control research provides a forum for
understanding biochemical processes as well as the ecological mechanisms
regulating these processes.
Biological control organisms utilize a variety of mechanisms to inhibit
their target pathogens. Competitive exclusion of a pathogen by a biocontrol
antagonist results when the ecological niches occupied by both organisms
overlap. The extent of ecological overlap may be nearly complete as in the
competition between a virulent and a hypovirulent strain of a pathogen
(Paulitz et al, 1987; MacDonald and Fulbright, 1991; Chen et al, 1994).
Alternatively, two unrelated organisms may compete for limiting resources
such as available iron (Loper and Buyer, 1991) or carbon sources (Nelson and
Maloney, 1992; Pau litz, 1990). Other mechanisms that have also been implicated
in biological control include parasitism (Adams, 1990), cross protection (Fulton,
1986), and antibiosis (Fravel, 1988). Details of biocontrol systems utilizing
these mechanisms are described within the many comprehensive reviews
that have been published (Weller, 1988; DeFago and Haas, 1990; Gould, 1990;
Gutterson, 1990; Nelson and Maloney, 1992; O'Sullivan and O'Gara, 1992;
Loper et al, 1994; Thomashow and Weller, 1995). It should be emphasized
that in many systems, several biocontrol mechanisms concertedly contribute
to the total biological control activity observed for a single antagonist.
1.2 ANTIBIOSIS AS A MECHANISM IN BIOLOGICAL CONTROL
Antibiotics are relatively small organic compounds produced by
microorganisms via secondary metabolic pathways. Their production generally
is not considered essential for survival of the producing organism, and the
ecological importance of secondary metabolites has been debated for many
years (Gottlieb, 1976; de Lorenzo and Aguilar, 1984; Williams and Vickers,
1986; Maplestone et al, 1992; Stone and Williams, 1992; Vining, 1992). Research
3
with biological control systems has demonstrated that, in some instances,
antibiotics contribute to the ecological competence of the producing organism
(Mazzola et al, 1992). Nevertheless, their production is not an absolute
requirement for survival of bacteria in natural habitats.
Historically, it has been difficult to establish a role for antibiotic production
in the biological control of plant diseases. Although antibiotics produced by
biocontrol agents in vitro clearly supressed growth of target pathogens, there
was little evidence for in situ antibiotic production by biocontrol organisms
inhabiting soil or plant surfaces. Recently, molecular genetic techniques have
been used to construct mutants that are deficient in antibiotic production.
Comparison of the biocontrol activities of the isogenic mutant and the parental
strains has provided further correlations between antibiotic production and
the biocontrol activity of a microorganism in natural habitats. Unfortunately,
selection of the mutants generally has relied upon in vitro screening for
pathogen inhibition without biochemical characterization. Mutations affecting
antibiotic production are not limited to lesions within biosynthetic loci;
antibiotic production is mediated by global regulatory loci as well as genes
closely linked to the biosynthetic gene cluster. Furthermore, lesions within
genes affecting antibiotic precursor biosynthesis can also result in a nonproducing phenotype. Mutants selected for loss of antibiotic production
commonly have pleiotropic phenotypes (Kraus and Loper, 1992; Laville et al,
1992; Haas et al, 1993; Lam et al, 1993; Willis et al, 1994). Therefore,
demonstrating the dependence of biocontrol activity through a molecular
genetic approach requires thoroughly characterized mutants with genetic
lesions in structural genes for antibiotic biosynthesis.
The following examples highlight several secondary metabolites (Figure
1-1) that are produced by organisms exhibiting biological control activity.
Although some of these compounds have not been definitively established
as contributing factors to biological control, they nevertheless serve to illustrate
the importance of molecular genetics in biological control research as well as
the requirement for rigorously characterizing the mutants used in these studies.
4
Agrocin 84. Agrocin 84 is a bacteriostatic antibiotic produced by the
biological control organism Agrobacterium radiobacter K84 that inhibits DNA
synthesis in A. tumefaciens, the causal agent of crown gall disease (Moore
and Warren, 1979; Kerr, 1980; Farrand, 1990). Agrocin 84 production is a
plasmid determined phenotype of A. radiobacter K84. Curing A. radiobacter
of the plasmid required for agrocin 84 production significantly reduces the
biocontrol activity of this bacterium, but non-producing strains partially
suppress crown gall formation when introduced onto the plant host prior to
infection by A. tumefaciens. Although agrocin 84 production is a major
determinant of biocontrol activity, other factors also contribute to disease
suppression. Increasing the agrocin 84 biosynthetic gene dosage, however,
does not further improve biocontrol activity (Shim et al, 1987).
Agrocin 84
000H
N
H
Pyrrolnitrin
Phenazine-1-carboxylic
acid
o
CH 0
OH
HD
CH
2,4-Diacetylphloro­
glucinol
N
OH 0
a
H
Pyoluteorin
Figure 1-1. Antibiotics with inhibitory activity against plant pathogens.
The antifungal metabolite oomycin A has been omitted because its molecular
structure is unknown.
5
Gliovirin. Gliocladium virens is a soil fungus with a demonstrated ability
to parasitize Rhizoctonia solani (Tu and Vaartaja, 1980; Tu, 1980; Howell,
1982). Although G. virens also is able to inhibit the plant pathogenic fungus
Pythium ultimum, initial evidence suggested that a non-parasitic mechanism
was responsible for this activity (Howell, 1982). Subsequently, the antifungal
metabolite gliovirin was isolated and structurally characterized from G. virens
culture extracts (Howell and Stipanovic, 1982). Gliovirin inhibits only
oomycete fungi, such as P. ultimum and Phytopthora species (Stipanovic and
Howell, 1983). Mutagenesis of G. virens has allowed the isolation of gliovirin
non-producing and over-producing strains. Gliovirin non-producing mutants
were significantly less effective at controlling preemergence damping-off in
Pythium infested soils, but the over-producing mutants did not increase disease
suppression as compared to the parental strain. Although it is tempting to
conclude that gliovirin is a contributing factor to the biocontrol activity of G.
virens against P. ultimum, these studies have not demonstrated in situ
gliovirin production. Furthermore, the non-producing and over-producing
mutants have not been thoroughly characterized to ascertain if the genetic
lesions affect only gliovirin production. Therefore, further studies are necessary
before the role of gliovirin in the biological control of P. ultimum can be
unequivocally demonstrated.
Oomycin A. Pseudcmonas fluorescens Hv37a exhibits in vitro antifungal
activity against P. ultimum (Gutterson et al, 1986) and produces several
antifungal metabolites (James and Gutterson, 1986). Although none of the
compounds produced by this strain have been structurally characterized, one
metabolite, oomycin A, is the major contributing factor for biocontrol activity
(Howie and Sus low, 1991). Genetic analysis within Hv37a has identified at
least four unlinked loci required for oomycin A production (Gutterson et al,
1988; Gutterson, 1990). Several of these loci are responsible for induction of
the biosynthetic genes by the oxidation of glucose via glucose dehydrogenase.
The in situ expression of an oomycin A biosynthetic gene was demonstrated
using a lacZ transcriptional fusion in both an oomycin A producing and
non-producing strain (Howie and Sus low, 1991). Additional evidence for the
ecological role of oomycin A production within Hv37a was obtained by placing
6
a constitutive tac promoter upstream of the oomycin A biosynthetic gene
cluster (Howie et al, 1989; Gutterson, 1990). By introducing the unregulated
loci into the Hv37a chromosome, an increase in oomycin A production
correlated with a significant increase in biological control of P. ultimum as
compared to the wild-type strain. Furthermore, transcriptional fusions of the
tac-derived promoter to a lux reporter gene revealed an increase of biosynthetic
gene expression in situ (Gutterson, 1990). Demonstrating biosynthetic gene
expression in situ, however, does not necessarily establish antibiotic production
because other regulatory mechanisms may ultimately control antibiotic
biosynthesis.
Phenazine-1-carboxylic acid. Suppression of the phytopathogenic fungi
Gaeumannomyces graminis var. tritici has been correlated to the production
of phenazine-1-carboxylic acid by the biocontrol organisms Ps. fluorescens
2-79 (Thomashow and Weller, 1988) and Ps. aureofaciens 30-84 (Pierson and
Thomashow, 1993). The production of phenazine-1-carboxylic acid is one of
the few cases in which a direct correlation has been demonstrated between in
situ antibiotic production and biological control activity (Thomashow et al,
1990). Phenazine-1-carboxylic acid biosynthesis also has a long term, positive
influence on the ecological fitness of producing strains (Mazzola et al, 1992).
Furthermore, the efficacy of Ps. fluorescens 2-79 as a biocontrol agent is a
function of the target pathogen sensitivity to phenazine-l-carboxylic acid
(Mazzola et al, 1995). Antibiotic production, therefore, is not the sole
determinant of biocontrol activity since non-producing mutants partially
suppress disease (Thomashow and Weller, 1988).
The in situ production of phenazine-1-carboxylic acid is influenced by
several edaphic factors such as the proportions of sand, organic matter, silt,
and clay, soil pH, metal ions, and total carbon (Thomashow and Weller,
1995). Although these factors affect antibiotic production, it is likely that their
influence is indirect, exerted through complex interactions between the host,
pathogen, biocontrol agent, and soil. Phenazine-l-carboxylic acid production
also is regulated directly in response to cell density (reviewed in Pierson and
Pierson, 1996). Two loci, phzR and phzl, have been identified via nucleotide
sequence analysis and are linked to genes encoding the biosynthetic enzymes
7
of the phenazine pathway (Pierson et al, 1995). PhzR and PhzI belong to the
LuxR/LuxI family of cell-density dependent regulatory proteins and are
required for antibiotic production in Ps. aureofaciens 30-84. PhzI shares
sequence similarity to several N-acyl-L-homoserine lactone synthases. PhzR
is presumably a transcriptional activator that induces phenazine-1-carboxylic
acid production in response to the N-acyl-L-homoserine lactone derivative
produced by PhzI. It is hypothesized that fungal infection is actually required
for the Ps. aureofaciens population to reach the threshold density required
for phenazine production as a result of an increase in root exudates released
from root lesions.
Pyrrolnitrin. Pyrrolnitrin is produced by many Pseudomonas spp.
(Leisinger and Margraff, 1979) and was identified initially as a contributing
factor to biological control in Ps. fluorescens Pf-5 (Howell and Stipanovic,
1979). Pyrrolnitrin inhibits several phytopathogenic fungi; however, it is most
effective in situ against Rhizoctonia solani. Although the severity of R. solani
infection as measured by seedling emergence was reduced by treating seeds
with purified pyrrolnitrin, greater protection was afforded when Pf-5 itself
was used as a seed treatment (Howell and Stipanovic, 1979). These results
suggest that other biocontrol mechanisms may contribute to the inhibition of
R. solani by Pf-5.
Further evidence for the role of pyrrolnitrin production in biological
control has been obtained in studies of Ps. fluorescens BL915 (Ligon et al,
1996). A 6.2-kb genomic region within BL915 is required both for pyrrolnitrin
production and for biological control activity (Hill et al, 1995). Nucleotide
sequence analysis and pyrrolnitrin production in heterologous strains
containing the cloned 6.2-kb region is compelling evidence for the existence
of pyrrolnitrin biosynthetic genes within this region (Ligon et al, 1996; GenBank
accession no. U74493). These results suggest that pyrrolnitrin is a major
contributing factor to the biological control activity of BL915. Pyrrolnitrin
production does not appear to increase the ecological competence of BL915
(Hill et al, 1994); however, the duration of this competition study was relatively
short (12 days) and may not have been sufficient to detect slight differences
between the ecological competence of non-producing and wild-type strains.
8
2,4-Diacetylphloroglucinol. 2,4-Diacetylphloroglucinol is produced by
many isolates of fluorescent pseudomonads and exhibits activity against a
variety of plant pathogenic fungi (Thomashow and Weller, 1995). Production
of this antifungal metabolite is globally regulated by the GacA/ApdA two
component regulatory system within Ps. fluorescens (Laville, et al, 1992; Corbell
and Loper, 1995) as well as by the sigma factors RpoS and RpoD (Schnider et
al, 1995; Sarniguet et al, 1995). In independent studies using two Ps. fluorescens
strains, antibiotic non-producing mutants were obtained by transposon
mutagenesis (Vincent et al, 1991; Fenton et al, 1992). In both cases, the isolated
mutants were less suppressive against P. ultimum or G. grorninis var. tritici
than were the wild-type strains. Subsequently, a genomic DNA region
conferring 2,4-diacetylphloroglucinol production was cloned from both strains
and when transferred into heterologous hosts, significantly increased their
biocontrol activity against the target pathogens (Fenton et al, 1992; Bangera
and Thomashow, 1996). Four open reading frames that encode biosynthetic
enzymes required for 2,4-diacetylphloroglucinol production have been
identified within a 6.5-kb fragment from Ps. fluorescens Q2-87 (Thomashow
et al, 1996; GenBank accession no. U41818). Since the genes expressed
heterologously in the previous studies were specifically involved in 2,4­
diacetylphloroglucinol biosynthesis, it is very likely that this antibiotic is a
major contributing factor of biological control.
Pyoluteorin. Pyoluteorin is produced by the biological control agent Ps.
fluorescens Pf-5 and is active against the plant pathogenic fungus P. ultimum
(Howell and Stipanovic, 1980). Treatment of cotton seeds with either
pyoluteorin or Pf-5 cultures prior to planting in Pythium infested soils results
in similar levels of disease suppression and was initially the basis for
demonstrating the contribution of pyoluteorin towards the biocontrol activity
of Pf-5 (Howell and Stipanovic, 1980). In a later study, Tn5 mutagenesis of
Pf-5 was used in an attempt to further correlate pyoluteorin production in
the biocontrol of P. ultimum on cucumber (Kraus and Loper, 1992). Seven
pyoluteorin non-producing mutants were isolated; however, they were just
as effective in disease suppression as the wild-type strain. More recently a
study using Ps. fluorescens CHAO has demonstrated that pyoluteorin
9
contributes to biological control on cress but not on cucumbi, leading to the
conclusion that the plant host appears to affects pyoluteorin's contribution to
biological control (Maurhofer et al, 1994). Subsequent characterization of the
Pf-5-derived Tn5 mutants localized all seven of the transposon insertions to
within a 21-kb genomic region (Kraus and Loper, 1995). Since it is generally
accepted that biosynthetic genes are closely linked, it was assumed that this
region encoded pyoluteorin biosynthetic enzymes. The expression of this
region in situ was assessed on cotton and cucumber using an ice nucleation
transcriptional reporter gene fusion. Although gene expression occurred on
both plant hosts, expression on cucumber occurred after the infection period
required by P. ultimum (Nelson et al, 1986). In light of the carbon source
effects on pyoluteorin production within Pf-5 (Kraus and Loper, 1992; Nowak-
Thompson et al, 1994), differences in seed exudates could account for the
plant host-dependent pattern of gene expression.
1.3 STATEMENT OF RESEARCH OBJECTIVES
The research described within this treatise represents an integrated
approach to understanding the biosynthesis of pyoluteorin within Ps.
fluorescens Pf-5. Pf-5, originally isolated from a cotton rhizosphere, is effective
at controlling Pythium damping-off of cotton (Howell and Stipanovic, 1980)
and cucumber (Kraus and Loper, 1992), Rhizoctonia damping-off of cotton
(Howell and Stipanovic, 1979), and ascocarp formation by Pyrenophora tritici­
repentis on wheat straw residue (Pfender et al, 1993). In addition to producing
pyoluteorin, Pf-5 also produces pyrrolnitrin (Howell and Stipanovic, 1979),
2,4-diacetylphloroglucinol (Nowak-Thompson et al, 1994; See Appendix 1),
hydrogen cyanide (Kraus and Loper, 1992), and a pyoverdine siderophore
(Kraus and Loper, 1992).
Because of the importance of pyoluteorin in biological control (Section
1.2), characterization of the genomic region required for pyoluteorin
production has been undertaken. Furthermore, pyoluteorin is a structurally
interesting metabolite due to the halogenated pyrrole moiety, a relatively
rare functionality found in terrestrial natural products. Therefore, two
principle objectives guided the research described within this treatise: the
10
elucidation of the pyoluteorin biosynthetic pathway using an isotope feeding
methodology, and the nucleotide sequence analysis of the genomic region
required for pyoluteorin production. Although the results herein do not
provide a complete understanding of pyoluteorin biosynthesis, they
nevertheless have revealed several interesting aspects of this pathway and
have established a solid framework for future studies concerning both the
biochemical pathway and the regulation of pyoluteorin biosynthesis.
11
Chapter 2. Biochemical Analysis of the Pyoluteorin Pathway
2.1 INTRODUCTION
The polyketide origin of natural products was originally proposed by
Collie (1907) based on the observation that successive condensations and
dehydrations of a few simple aldehydes or ketones can give rise to more
complex structures. However, the biological significance of this proposal was
not demonstrated until the introduction of radioisotopes in the 1950's. 14C_
labelling studies provided the necessary evidence for Collie's original
proposal, and also lead to an expansion of the biosynthetic hypothesis as an
aid in the structural elucidation of natural products (Birch, 1957). Many
polyketide biosynthetic pathways have since been probed using a variety of
techniques, and many reviews have highlighted the recent developments
within the realm of polyketide natural products (Robinson, 1991; Fenical,
1993; Plater and Strohl, 1994; Rohr, 1995; Simpson, 1995; O'Hagan, 1995; Khos la
and Zawada, 1996). The structural diversity of the polyketides makes it
necessary to limit this discussion to a few thoroughly characterized metabolic
pathways, some of which represent "mixed" polyketide pathways (i.e. not
solely acetate-derived). Although the polyketides described herein possess
relatively simple molecular structures when compared to higher molecular
weight polyketides, they nevertheless serve to illustrate the biochemical
transformations often encountered in polyketide biosynthesis. The
biochemical reactions required for polyketide chain assembly and their analogy
to fatty acid synthesis have been reviewed (Hopwood and Sherman, 1990),
and will not be presented here.
2.1.1 Rudimentary polyketide pathways: the tetraketides
One of the most thoroughly studied polyketide systems in which both
the genetic and biochemical aspects are understood, is the 6-methylsalicylic
acid (6MSA) pathway found within various Penicillium species. Several
12
independent isotope labelling studies have firmly established the polyketide
origin of 6MSA, 2.1 (Scheme 2-1; Birch 1967; Spencer and Jordan, 1990).
6MSA biosynthesis is processive and requires an NADPH-dependent
reduction before addition of the final malonyl-CoA unit (Dimroth et al,
1970; Scott et al, 1974). Furthermore, elimination of the C-2 malonyl-CoA
methylene hydrogens required for aromatization of the carbocyclic ring occurs
stereospecifically (Spencer and Jordan, 1990; Jordan and Spencer, 1990). 6MSA
synthase has been isolated and characterized (Dimroth et al, 1970; Scott et al,
1974; Spencer and Jordan, 1992a). The gene encoding the 6MSA synthase has
been cloned and sequenced (Beck and Ripka, 1990). The deduced polypeptide
sequence exhibits similarity to several bacterial polyketide synthases and
contains discrete catalytic domains required for polyketide assembly (see
Chapter 3).
Scheme 2-1
0
Ha Dor'SCoA
O
CoAS
0
Ac
Hs soiegLLSCoA
coi
-----3111111.
H3
0c1,rC H3
c 02.
EnZSNrrolTh.rC H3
0
....--....00..
NADPH
0
SEnz
0
O
0
HO
H3
H0
- H2O
?I'
C 01
CoA
C H3
SEnz
0
Ha
C H3
SEnz
00
014
HR.
HO
0
2.1
Orsellinic acid (OA; see Scheme 2-4, pg. 20) is a metabolite produced by
Penicillium spp. that is structurally related to 6MSA, but contains an additional
C4 hydroxyl moiety. OA synthase has been purified, and although it does
not require exogenous NADPH for catalysis, it exhibits properties similar to
6MSA synthase (Gaucher and Shepherd, 1968). The OA synthase gene sequence
13
has not been reported. With the exception of the NADPH-dependent reduction
at C4 of the aromatic ring, OA and 6MSA biosynthesis appear to occur via
identical reaction pathways (Gatenbeck and Mosbach, 1959; Spencer and
Jordan, 1992b).
The bacterial metabolite 2,4-diacetylphloroglucinol (DAPG), 2.2, is
produced by many fluorescent Pseudomonas species (Nowak-Thompson et
al, 1994 and references therein; see Appendix 1). Although the DAPG structure
certainly suggests that it is acetate derived (Birch, 1967; Mann, 1987), this has
never been experimentally proven. Nevertheless, two pieces of experimental
evidence support this hypothesis. The identification of monoacetyl­
phloroglucinol (MAPG), 2.3, and the detection of enzyme activity converting
MAPG to DAPG in a Pseudomonas species argues in favor of DAPG originating
as a simple tetraketide (Shanahan et al, 1993). Furthermore, the biosynthetic
gene required for DAPG production exhibits sequence similarity to several
chalcone synthases (Thomashow et al, 1996), plant enzymes that utilize three
malonyl-CoA equivalents in precursor formation for flavonoid phytoalexins
(Kreuzaler and Halbrock, 1975; Hrazdina et al, 1976). Therefore, DAPG
biosynthesis most likely occurs as depicted in Scheme 2-2.
Scheme 2-2
0
0 C H3
SEnz
CO 2
EnzS.C H3
0
0
OH
HO
C H3 JP"
C H3
IT
0
CH
OH 0
2.3
C H3
OH 0
2.2
DAPG represents a fundamentally different ring structure from 6MSA
due to the folding of the extended polyketide chain prior to cyclization (Scheme
2-3). Cyclization of 6MSA requires C-C bond formation between C2 and C7 of
the polyketide chain, whereas Cl and C6 are involved in bond formation
14
within DAPG. Differences in cyclization regiospecificity are also apparent in
naringenin chalcone, 2.4, and resveratrol, 2.5, plant-specific polyketide
metabolites required for flavonoid and stilbene production, respectively
(Mann, 1987; Kindl, 1985). Oxygenation patterns on the phenol ring highlight
the similarity of naringenin chalcone to DAPG; however, the relationship of
resveratrol to 6MSA is somewhat obscured by an apparent decarboxylation
(Scheme 2-3). Although both chalcone and resveratrol synthase catalyze
polyketide biosynthesis via successive condensations of malonyl-CoA, these
enzymes are distinct from the polyketide synthases (Schroder et al, 1988;
Lanz et al, 1991).
Scheme 2-3
I SEnz
----Imp.
.7.7"­
.=.:
C H3
0
0
0
0
OH 0
OH 0
2.4
OH 0
1
SEnz
OH
-Ow.
7
C H3
(F10)"
Although only two different ring systems are possible for any of the
tetraketides, applying this same principle to larger polyketides greatly increases
the structural variability within this class of natural products. Structural
variation is an important consideration in biocombinatiorial design of novel
compounds (McDaniel et al, 1995). It has also been suggested that folding
patterns may represent biosynthetic and evolutionary relationships within
the multicyclic polyketides (Rohr, 1992).
15
2.1.2 Unusual polyketide starter units
For many years, the contribution of starter units to polyketide structural
variability has been an understated aspect of polyketide biosynthesis. However,
recent proposals regarding biocombinatorial engineering of polyketide natural
products have suggested manipulating the starter unit specificity of the
polyketide synthase as a means of introducing novel structural variations
(Katz and Donadio, 1993; Hutchinson and Fugii, 1995; Rohr, 1995; Khosla
and Zawada, 1995). Alternative starter units can sometimes be introduced
into polyketide structures through precursor-directed biosynthesis (reviewed
by Thiericke and Rohr, 1993).
Of the naturally occurring polyketide starter units, acetate and propionate
are, by far, the most widely used, but some structures exhibit alternative
starter units (Table 2.1; also see Scheme 2-3 for starter units within naringenin
chalcone and resveratrol.). Nevertheless, polyketide natural products
possessing a non-acetate starter unit are encountered relatively infrequently.
Starter units generally can be identified within the polyketide structure except
in cases of extensive post-assembly modifications or structural
rearrangements. Only precursors that have been experimentally demonstrated
as polyketide starter units are listed in Table 2.1. Although this list is extensive,
it should not be considered complete.
The existence of amino acid polyketide starter units is especially
interesting in light of the similar thiotemplate mechanisms used in polyketide
and non-ribosomal peptide biosynthesis (Kleinkauf and von Dohren, 1995
and 1996). The substrates recognized by polyketide synthases and peptide
synthetases are the activated CoA thioester and acyl adenylated amino acids,
respectively. Since the activated amino acids eventually form a thioester
linkage with a 4'-phosphopantetheine prosthetic group in both the synthases
and synthetases, it is tempting to speculate on the possibility of substrate
competition within an organism possessing both biosynthetic pathways. It is
likely that these activation strategies serve to channel substrates to the
individual pathways and avoid direct competition between both pathways.
Nevertheless, the activating enzymes offer a potential point for the
coordinated regulation of polyketide and non-ribosomal peptide biosynthesis.
16
Table 2.1 Non-acetate compounds that incorporate into the starter units of
polyketide natural products. The location of each starter unit is
indicated by darkened bonds within each structure.
Amino Acids
proline
OH 0
H
Pyoluteorin
(B.N-T., J. Loper, and S. Gould,
unpublished results; Cuppels
et al, 1986)
a
if.
(Kawamura et al, 1996)
0
C H3
Pyralomicin
a
02N
OHO H
(Carter et al, 1989)
Dioxapyrrolomycin
o-acetyl-L-serine
3C. _
pN
O%
Rhizoxin
(Kobayashi et al, 1986)
glycine
0
H3C H3C
H3C) rk''')Y1
C F13
C H3
(Carmeli et al, 1993)
Tolytoxin
13phenylalanine
(Omura et al, 1982)
Hitachimycin
17
Table 2.1 (cont'd)
Glycerol
000
(Lee et al, 1987)
0
Aplasmomycin
Linear Carboxylic Acids
malonate
(Jeffs and McWilliams, 1981)
H
Cycloheximide
,
OH
NK4e2
7
al
Nit
OH 0H0 0 0
(Thomas and Williams, 1983)
Oxytetracycline
(Bockholt et al, 1994)
0
N
F1300
H3
Lysolipin
hexanoate
OH 0
OH
110111
0
C H3
(Townsend et al, 1984;
Brobst and Townsend, 1994)
Averfulvin
hexadecanoate
(Walters et al, 1990)
Anacardic Acid
18
Table 2.1 (cont'd)
Branched and Cyclic
Carboxylic Acids
2-methylpropionatel
(Tsou et al, 1989)
2-methylbutanoatel
0
Lathodoratin
1-0
(Al-Douri and Dewick, 1988)
3-hydroxycyclo­
hexanoate2
(Thiericke et al, 1990)
Asukamycin
3,4-dihydroxy­
cyclohexanoate
C H3
(Lowden et al, 1996)
CH
Rapamycin
Aromatic Carboxylic Acids
benzoate3
(Burns et al, 1979)
OH 0
0
3
C H3
Squalistatins
(Jones et al, 1992)
19
Table 2.1 (cont'd)
Aromatic Carboxylic Acids (cont'd)
o-hydroxybenzoate
(Aragozzini et al, 1988)
cD2H
Thermorubin
m-hydroxybenzoate
R= yy
(Bennett and Lee, 1988)
Mangostin
3-amino-5-hydroxy­
benzoate4
(Kibby et al, 1980; Lee et al, 1993;
Ghisalba and Nuesch, 1981)
1 Identical starter units are incorporated into several avermectin antibiotics (Chen et al, 1989;
Schulman et al, 1986).
2 Ansatrienin A is a related antibiotic utilizing this precursor as the polyketide starter unit
(Moore et al, 1993).
3 Benzoate also serves as the starter unit for vulgamycin biosynthesis (Seto et al, 1976).
4 The me ta-C7N unit is also used in other ansamycin antibiotics; see Staley and Rinehart,
1991.
20
2.1.3 Post-assembly modifications of polyketide metabolites
Post-assembly modification of polyketide structures greatly expands the
structural variability of simple polyketides such as 6MSA or OA. The extent
of these modifications range from simple transformations of functional
groups (i.e. methylations or oxidations/reductions) to complex oxidative
cleavages and carbon skeleton rearrangements. Methylation by S-adenosyl
methionine and oxidative decarboxylation of the OA sub-structure are
required for biosynthesis of the fungal benzoquinone, shanorellin, 2.6,
(Scheme 2-4; Wat et al, 1971). Because the carbon skeleton of OA is found
within a variety of fungal metabolites (Simpson, 1991), OA is a likely precursor
to shanorellin. Nevertheless, this possibility has not been tested.
Scheme 2-4
[Ox]
*CH3 -AdoMet
OH
C H3
C 02H
OH
C 02H
C 02H
OA
*C H3-AdoMet
H3C
*C H3
OH
HO
-C 02
-Nap.
C 02H
Two well known examples of an oxidative C-C bond cleavage followed
by a structural rearrangement are found in the pathways leading to patulin,
2.7, and penicillic acid, 2.8 (reviewed by Zamir, 1980 and Mann, 1987). Both
patulin and penicillic acid are carcinogenic Penicillium metabolites derived
from 6MSA and OA, respectively. Patulin biosynthesis requires ring cleavage
at the C2-C3 bond of the aromatic ring. The epoxide intermediate was isolated
from a patulin non-producing mutant and shown to efficiently incorporate
21
into patulin (Scheme 2-5). In contrast, ring cleavage required for penicillic
acid biosynthesis occurs between the C4-05 bond of the aromatic ring (Scheme
2-6). In light of the reaction pathway leading to patulin, it has been suggested
that an epoxyquinone may serve as a penicillic acid precursor (Zamir, 1980).
Scheme 2-5
[Ox]
-C 02
OH
C H2CH
r°F1
HO
C.3
C HO
-110.
0
0
F/32CC HO
2.7
Scheme 2-6
CH
*O H3-AdoMet
C H3
C 02H
[Ox]
*C H30
C H3
C 02H
-CO2
*C H30
a-13
O
Oxidative coupling of phenolic compounds is another post-assembly
modification found in many natural products and is not limited to polyketide
derived structures. Because oxidative coupling has been discussed extensively
elsewhere (Brown, 1967; Geismann and Crout, 1969), it will only be mentioned
22
briefly as it applies to usnic acid biosynthesis. Usnic acid, 2.9, is produced by
several lichen species and labelling studies have established the biosynthetic
pathway (Scheme 2-7; Taguchi et al, 1969). Although 4-methyl-MAPG, 2.10,
is a precursor to 2.9, MAPG itself does not incorporate into usnic acid. It was
therefore concluded that the aromatic methyl group is introduced prior to
cyclization. It is possible that the 4-methyl moiety actually originates via a
propionate extender unit incorporated by the bacterial symbiont of the lichen.
Scheme 2-7
FI3
0
"4111E-110.
H3C
OH
2.10
2.2 BIOSYNTHETIC ANALYSIS OF PYOLUTEORIN
It is apparent from the oxygenation pattern of pyoluteorin, 2.11, that the
resorcinol moiety most likely results from the condensation of three malonylCoA equivalents with proline, or a derivative thereof, serving as the starter
unit for polyketide assembly. The specific incorporation of [1,2-13C2] acetate
into pyoluteorin has demonstrated that pyoluteorin is indeed a polyketide
metabolite (Cuppels et al, 1986) as evidenced by the labelling pattern found
within the resorcinol moiety (Scheme 2-8). Furthermore, the non-uniform
23
labelling of the dichloropyrrole suggested that this moiety originated from a
tricarboxylic acid cycle intermediate, a conclusion that is consistent with a
proline derived starter unit (Scheme 2-9; Cuppels et al, 1986).
Scheme 2-8
[rAscoA1
SEnz
cot
OH
CI
EnzS
CI
N
0
0
H
OH 0
H
2.11
While the assembly of the resorcinol ring is by no means atypical, the
use of an unusual starter unit by the presumed pyoluteorin polyketide
synthase (PKS) justified further study of this system. The rapamycin synthase
is the only PKS utilizing an unusual starter unit that has been characterized
(Aparicio et al, 1996). Considering the current advances in PKS molecular
genetics, a combinatorial approach to drug design is possible, and the ability
to introduce diverse starter units into a polyketide offers an additional variable
for drug design. The basis for starter unit selection by a PKS, however, is
unknown. Therefore, the concurrent genetic characterization of the
pyoluteorin PKS and identification of the required starter unit substrate may
reveal factors influencing starter unit fidelity.
Further justification for this study focuses on formation of the
pyoluteorin dichloropyrrole ring. Over 2000 halogenated natural products
have been reported (Gribble, 1996), but despite the proliferation of these
compounds, little is known regarding the halogenation reactions involved
in their synthesis. Because halogenation is known to increase the
pharmacological effects of many compounds (Neidelman and Geigert, 1987),
it is conceivable that a mechanistic understanding of biohalogenations could
be exploited for the synthesis of new or otherwise costly antibiotics.
24
Scheme 2-9
Initial labelling of TCA cycle inter­
mediates and proline by 13C-acetate:
0
A
COi _v.. '02ey
H3C
Cr
_.
...410,.
in C 02-
0
0
IIP­
C 02
-02,C).C/%C Oi
/
--110.
--ON-
--Do-
a-ketoglutarate
Further incorporation of 13C-acetate into
TCA cycle intermediates and proline:
-O.
N
0
H
proline
.02CN/4440 02.
.02e)fC 02­
CO2­
o
O
[H.
-o2c')(co2HO C 02­
4
cj ,Lco
-02nC0 02HO 0 02-
4
f
%cc02
01
\[0
H3CAO
oey."coi
-o2e)1/4"co2.
4
4
4
4
Ho COI
Ho coi
25
A considerable amount of effort has been directed, therefore, towards
the isolation and characterization of haloperoxidases, enzymes that are capable
of forming carbon-halogen bonds in the presence of halide ions and hydrogen
peroxide (van Pee, 1996). However, it has yet to be demonstrated that any of
the haloperoxidases so far characterized are responsible for the in vivo
halogenation of known natural products. It is quite likely that elucidating
the pyoluteorin biosynthetic pathway will identify a substrate for the
halogenating enzyme(s) involved. Such a discovery would allow the
development of a specific assay required for the isolation and subsequent
characterization of this unknown class of enzymes.
2.3 RESULTS AND DISCUSSION
2.3.1 Preliminary studies: pyoluteorin production and isolation
Pyoluteorin production was evaluated in a variety of liquid culture
media. HPLC and TLC analysis of culture extracts indicated that pyoluteorin
production was greatest in cultures grown on either modified King's medium
B (King et al, 1954) or Bacto Peptone medium (Difco, 1984). Pyoluteorin
production did not differ between these two media, and modified King's
medium B was chosen for further studies.
By varying the peptone source within King's medium B, it was discovered
that phytone resulted in approximately a two-fold increase in pyoluteorin
production as compared to proteose peptone #3. Several carbon sources were
also used in an attempt to further improve production. The addition of
glucose to the production medium appeared to increase DAPG production at
the expense of pyoluteorin. The differential production of these antibiotics
in response to carbon source parallels previous results demonstrating that
transcriptional activity of a promoter within the pyoluteorin gene cluster is
repressed in cultures containing glucose, but induced in cultures
supplemented with glycerol (Loper and Kraus, 1995).
Aeration effects were examined by varying the flask size to medium
volume ratio. In general, flasks containing a smaller volume of medium
26
result in elevated pyoluteorin production, presumably due to an increase in
aeration rate. This result contrasts with an earlier report that pyoluteorin is
produced only in non-aerated flask cultures (Bencini et al, 1983). Lower
temperatures (18 °C vs 27 °C) also supported greater metabolite production.
Although it is not obvious why temperature would serve to increase
production, it is possible that the lower temperature more closely approximates
the native habitat of Pf-5.
A previously developed purification method for pyoluteorin required
the fractionation of a crude extract by both column and thin layer
chromatography followed by derivatization and further purification (Cuppels
et al, 1986). Several alternative solvent systems (see Section 2.5) therefore
were developed that provided pyoluteorin of sufficient purity after a single
silica column chomatography for NMR spectroscopic analysis.
2.3.2 Incorporation of proline into pyoluteorin
In light of the previous biosynthetic study (Cuppels et al, 1986), the
initial objective was to establish if proline is the primary precursor of the
dichloropyrrole moiety. Therefore, an initial study was conducted in which
R
L-proline was administered to growing cultures of Pf-5. After isolation
and recrystallization to a constant specific activity, 19% of the total radioactivity
added to the culture was detected in pyoluteorin. Although it was possible
that proline may have been catabolized and the labelled products subsequently
incorporated into pyoluteorin, the high level of radioactivity in the final
product argued against this possibility. Nevertheless, [1-13C] -L-proline was
administered to Pf-5 cultures in order to demonstrate the specific incorporation
of proline into pyoluteorin. Purification and 13C-NMR analysis of pyoluteorin
revealed a specific enrichment of 5.7% over and above the natural abundance
carbonyl resonance for the 13C spectrum and provided definitive evidence
that the dichloropyrrole is derived from proline (Scheme 2-10).
27
Scheme 2-10
0
CH 0
H
H
2.11
Based on the results of these simple feeding experiments, several
biosynthetic pathways could be envisioned in which proline serves as the
primary precursor to pyoluteorin (Scheme 2-11). The proposed pathways all
involve a pyrrolidine to pyrrole oxidation, the subsequent chlorination of
the pyrrole, and polyketide assembly of the resorcinol moiety. Differences
among the proposed pathways reside in the temporal order of the
transformations. It was expected that biosynthetic feeding studies would
demonstrate involvement of deschloropyoluteorin, 2.12, and either pyrrole­
2-carboxylic acid, 2.13 or tetrahydrodeschloropyoluteorin, 2.14, and provide
evidence for the intermediates in the metabolic pathway leading to
pyoluteorin.
Scheme 2-11
Ho
O
H
proline
0
H
2.13
a
OH 0
2.14
2.12
H
0H n
H
2.11
28
2.3.3 Involvement of deschloropyoluteorin
Deschloropyoluteorin (DCP) was a logical target compound for the
subsequent feeding studies because two of the three proposed pathways
converge at this putative intermediate. Furthermore, DCP was readily
synthesized (Scheme 2-12) by Friedel-Crafts aroylation of pyrrole with 2,6­
dimethoxybenzoyl chloride, 2.15, (Rao and Reddy, 1990) followed by
deprotection of 2.16 with aluminum bromide (Cue et al, 1981). The
intermediacy of DCP in the pyoluteorin pathway was first examined using a
radioisotope trapping method. [1-14C]-Acetate was added to cultures of Pf-5
with the expectation that pyoluteorin pathway intermediates would be
radiolabelled. Shortly after adding the labelled precursor, the cells were
collected, lysed, and unlabelled synthetic DCP was added to the cell lysate.
DCP was subsequently re-isolated from the lysate, but no radioactivity could
be detected in the purified sample.
Scheme 2-12
AlC13; 5 °C
C H30
2.15
0
0
N
H
OC H 3
O.
CH30
0
AIBr3
--Hp.
H
2.16
(40% yield)
2.12 (72% yield;
30% overall)
This unexpected result unlikely was due to insufficient labelling of
pathway intermediates since approximately 3% of the total radioactivity was
detected in pyoluteorin. Other explanations, however, could account for the
lack of evidence from this experiment. The metabolic flux through the
pyoluteorin pathway may have been rapid enough to prevent accumulation
of pathway intermediates. Addition of unlabelled carrier DCP could
sufficiently dilute the radiolabel to render it undetectable. Alternatively, it is
also possible that DCP was not a pyoluteorin pathway intermediate.
Nevertheless, the outcome of this experiment does not allow for any definitive
conclusions regarding the role of DCP in the pyoluteorin pathway.
29
In light of the unsuccessful result from the isotope trapping experiment,
the involvement of DCP in pyoluteorin biosynthesis was further investigated
by isotope labelling studies. Treatment of DCP with 2H-trifluoroacetic acid
afforded the deuterated compound, 2.17 (Scheme 2-13). Greater than 90%
proton-deuterium exchange occurred on the pyrrole ring and approximately
70% proton-deuterium exchange occurred at two of the three positions on
the resorcinol moiety.
Scheme 2-13
2H-TFA
112; 75 °C
2.17
(76% yield)
Higher reaction temperatures resulted in low yields of the desired
product, and this was likely the result of an acid-catalyzed rearrangement of
the acylpyrrole (Carson and Davis, 1981). Attempts to deuterate DCP using
2H20 and BF3-Et20 (McGeady and Croteau, 1993) were unsuccessful. Compound
2.17 was subsequently fed to cultures of Pf-5 and pyoluteorin analyzed for
deuterium enrichment by 2H NMR spectroscopy. Based on the amount of
2.17 used in this feeding experiment (50 mg; 240 gmole) and a minimal
NMR detection limit of 1 gmole 2H-pyoluteorin, the detectable limit for
incorporation of DCP was approximately 0.5%. However, no deuterium was
detected in the pyoluteorin sample isolated. Therefore, no valid conclusions
regarding the intermediacy of DCP in the pyoluteorin pathway could be
made on the basis of this experiment.
Two observations made during the course of the deuterium feeding
experiment offer some insight to the limitations occasionally encountered
in biosynthetic labelling studies. Since DCP is only sparingly soluble in water
and the majority of 2.17 administered to the cell culture was recovered (98%
30
with no loss of the label), it is possible that an insufficient amount of DCP
was able to cross the cell membrane. The outer membrane structure of Gramnegative bacteria is an effective barrier to macromolecules and allows limited
diffusion of hydrophobic compounds (see references within Vaara, 1992).
Several polycationic agents and divalent cation chelators are reported
to decrease antibiotic resistance in Gram-negative bacteria by increasing the
permeability of the outer membrane (Brown and Richards, 1965; Leive, 1967;
Viljanen et al, 1986; Vaara, 1990; Vaara, 1992). Therefore, several cationic
detergents, polylysine, and polymyxin B nonapeptide (PMBN) were tested
for their effects on pyoluteorin production and DCP utilization by Pf-5 cultures.
In addition, the organic solvents toluene and dimethylsulfoxide (DMSO)
were also examined for their peameabilizing properties. It was expected that
by increasing cell permeability to DCP, an increase in pyoluteorin production
would be accompanied by a corresponding decrease in the concentration of
exogenous DCP. Cell pellets from the detergent- and toluene-amended Pf-5
cultures were visibly different from the control culture, and no pyoluteorin
was detected in the corresponding culture extracts. Both PMBN and polylysine
negatively affected pyoluteorin production in MgC12-containing cultures.
However, the suppressive effect of PMBN was not apparent in a culture
medium lacking MgC12 and amended with ethylenediaminetetraacetic acid
(EDTA). Interestingly enough, DMSO did not affect pyoluteorin production
when compared to the control culture. Nevertheless, no changes in
pyoluteorin production were observed when DCP was added to cultures.
In spite of the negative evidence resulting from this study, it was reasoned
that including either PMBN or DMSO in the culture medium may increase
the chances for a obtaining a positive result. Since there was no apparent
change in pyoluteorin production when DCP was added to the cultures
however, the use of radioisotopes to increase sensitivity in future analysis
was considered prudent. A semi-synthetic strategy was initially considered
to obtain radiolabelled DCP by reductive dechlorination of labelled pyoluteorin
obtained from "C-acetate fed cell cultures (Takeda, 1958; Birch et al, 1964).
However, catalytic hydrogenation of pyoluteorin on platinum oxide or
palladium metal was unsuccessful. A total synthesis of 14C -DCP was therefore
undertaken based on the Friedel-Crafts aroylation shown in Scheme 2-12.
Labelled [14C0] -2,6- dimethoxybenzoic acid, 2.18, was readily available (Scheme
31
2-14) by direct metalation of 1,3-dimethoxybenzene, 2.19, followed by treatment
of the organolithium species with 14CO2 generated from Na214CO3 (Gilman
and Ess, 1933; Gilman et al, 1940). Despite reports that addition of
tetramethylenediamine (TMEDA) to the reaction mixture increases the
overall yield and affects the regiospecificity of metalation (Slocum and
Jennings, 1976; Cabiddu et al, 1979; Cabiddu et al, 1981), use of this reagent
resulted in yields of <5%. However, reaction yields were increased ten-fold
when TMEDA was omitted and THF was used as the reaction solvent instead
of diethyl ether; a similar observation has been reported previously (Meyers
and Avila, 1980). Conversion of 2.18 to the acid chloride, 2.15, was achieved
with thionyl chloride (Bosshard et al, 1959), and the labelled product was
used directly for the Friedel-Crafts aroylation without purification.
Scheme 2-14
H3
C H30
2.19
CCH3
1) n-BuLi, THF
2) 4C 02
C H30
0
2.18
SOCl2
C H30
0
2.15
(78% yield)
The labelled DCP thus synthesized was administered to Pf-5 cultures
grown in medium lacking MgC12 and amended with EDTA and PMBN.
Isolation of pyoluteorin from the culture supernatant followed by
recrystallization to constant specific activity revealed that none of the labelled
DCP was incorporated. The lowest detectable limit of incorporation was
calculated as 0.005% based on the amount of pyoluteorin produced (17.1 mg;
62.6 pimole), a weighing accuracy of ± 0.10 mg, and a specific activity of at
least 103 dpm mg-1 for the recovered pyoluteorin.
Despite the considerable amount of effort focused on the possible
involvement of DCP in the pyoluteorin biosynthetic pathway, no evidence
was obtained suggesting that DCP is a pyoluteorin precursor. Although it is
tempting to refute the intermediacy of DCP, such a conclusion based solely
32
on a lack of evidence would be unsound. Therefore, none of the proposed
pathways leading to pyoluteorin could be discounted from these studies and
other putative intermediates were considered.
2.3.4 Involvement of pyrrole-2-carboxylic acid
The putative intermediate pyrrole-2-carboxylic acid (PCA) lies at a branch
point in the hypothetical biosynthetic routes leading to pyoluteorin (Scheme
2-11) and therefore was chosen as the next target for further study. Although
synthesis of PCA is feasible by reacting pyrrole with methyl chloroformate
(Hodge and Rickards, 1963) or with trifluoroacetic anhydride in the presence
of an acid scavenger followed by base hydrolysis (Sonnet, 1971), isotopic
labelling of PCA is not convenient using either of these strategies. [13CO2H]­
PCA, 2.20, was synthesized instead (Scheme 2-15) from pyrrole and 13CO2 via
pyrrylmagnesium bromide (Letellier and Bouthillier, 1957). Subsequently,
2.20 was added to Pf-5 cultures as an aqueous solution, and pyoluteorin was
isolated and analyzed for isotopic enrichment. However, no enrichment of
the carbonyl resonance in the 13C NMR spectrum was observed. The lowest
detectable limit for incorporation of 2.20 into pyoluteorin was calculated as
0.3% based on the amount of pyoluteorin isolated (46.1 mg; 171 limole), a 13C
NMR enrichment of at least 1.1% over natural abundance, and the amount
of 2.20 administered to the culture (75 mg; 681 1.tmole).
Scheme 2-15
13CO2
EtMgBr
MgBr
H
0
2.20
(28% yield)
33
Failure to detect incorporation of the free acid into pyoluteorin provoked
consideration of the initial PKS substrate required for biosynthesis. If the
pyrrole is the actual starter unit for polyketide biosynthesis, it may be
recognized by the PKS only as the ACP-CoA thioester. The use of N­
acetylcysteamine (NAC) thioester derivatives to mimic the ACP-bound
substrate allows recognition of an advanced intermediate by active-sites within
the PKS. This method has been used on occassion to incorporate postulated
polyketide intermediates into several natural products (Yue et al, 1987; Cane
and Ott, 1988; Brobst and Townsend, 1994; Dutton et al, 1994; Hai les et al,
1994a; Hai les et al, 1994b; Cane et al, 1995 and references therein).
Therefore, 2.20 was converted to the NAC thioester 2.21 (Scheme 2-16)
by treatment with dicyclohexaneylcarbodiimide (Brobst and Townsend, 1994).
The labelled product was dissolved in DMSO and administered to a 1 L
culture of Pf-5. Generally, 40-55 mg of pyoluteorin can be recovered from a 1
L culture, however in this instance, only 15 mg was isolated. Nevertheless,
based on the amount of pyoluteorin produced (15 mg; 54.9 umole), a 13C
NMR enrichment of at least 1.1% over natural abundance, and the amount
of 2.21 administered to the culture (93 mg; 440.8 umole), the lowest detectable
limit for incorporation of 2.21 into pyoluteorin was calculated to be 0.1%.
The isolated pyoluteorin, however, was not labelled.
Scheme 2-16
N-acetylcysteamine
H
2.20
0
DC C; DMAP; 0 °C
0
NJrSN..1 NAc
H
0
H
2.21
(41% yield)
The absence of any positive results from these feeding experiments
again obviates any definitive conclusion regarding the role of PCA in the
pyoluteorin pathway. As was the case for results described in Section 2.3.3,
none of the proposed pathways leading to pyoluteorin could be discounted.
34
2.3.5 Involvement of tetrahydrodeschloropyoluteorin
The involvement of the proposed biosynthetic intermediate tetrahydro­
deschloropyoluteorin (THDCP, 2.14 in Scheme 2-11) was examined via isotopic
feeding studies in a final attempt to elucidate the biosynthetic route leading
to pyoluteorin. While the feeding study itself did not present any particular
difficulty, synthesis of labelled THDCP was not as readily accomplished as
was the syntheses of either DCP or PCA. Direct hydrogenation of 2.16 over
palladium or platinum catalysts was unsuccessful, presumably due to the
aromatic resonance stabilization of the pyrrole. However, N protection of
the pyrrole as the tert-butoxycarbonyl derivative 2.22 (Scheme 2-17) provided
sufficient delocalization of the nitrogen electron pair for reduction to occur
(Kaiser and Muchowski, 1984). Although this methodology looked promising,
complete deprotection of 2.23 was unsuccessful despite the variety of
conditions examined (Greene and Wuts, 1991).
Scheme 2.17
OCH3
0 4'LLIDIBu
OCH3
)2
C H30
0
H2
-Do-
K*OtBu
H
C H30
2.16
N
PcVC
0
tBoc
0
C H30
2.22
tBoc
(83% yield)
(75% yield)
OCH3
TFA
BBr3
0°C
C H30
0
N
- 70°C
H2 OOCCF
2.23
(54% yield)
C H30
0
H
(57% yield;
19% overall)
35
An alternative protection strategy was attempted via catalytic
hydrogenation of an N,0,0-tri-tert-butoxycarbonyl derivative of 2.12.
Although complete characterization of the product was not undertaken,
resonances in the 1H NMR spectrum of the isolated product suggested only a
partial reduction of the pyrrole. It is very likely that steric constraints of the
tert-butoxycarbonyl protecting groups were responsible for incomplete
reduction of the pyrrole. Therefore, protection of the resorcinol ring with a
less sterically demanding protecting group was examined within this synthetic
strategy.
Treatment of 2.12 with benzyl bromide resulted in protection of a single
phenolic group as the benzyl aryl ether 2.24 (Scheme 2-18). Conversion of
this ether to 2.25 was afforded by treatment with base and di-tert-butyl
dicarbonate. Catalytic hydrogenation allowed full reduction of the pyrrole to
the pyrrolidine, presumably due to the loss of the benzyl protecting group,
thereby relieving the steric effects limiting pyrrole reduction. Removal of
the remaining tert-butoxycarbonyl groups with trifluoroacetic acid resulted
in THDCP as the desired product. The reaction scheme was then repeated
using a mixture of [13CO]- and [14CO] -DCP.
Scheme 2-18
OtBoc
Cs H5 CH2Br
I
N
Na2CO3
BzO
O
H
BzO
2.24
2.12
0
tBoc
2.25 (74% yield
from DCP)
H2
TFA
Pd/C
0 °C
HD
(92% yield)
I
OtBu
0
H
2.14 (>98% yield;
67% overall)
36
Extremely low levels of radioactivity were found in the pyoluteorin
purified from Pf-5 cultures that had been fed [1300/140-j
u THDCP. Within the
parameters generally accepted for biosynthetic feeding studies, the permissible
error in determining radiochemical purity of the final product is + 3% of the
specific molar radioactivity over the course of three recrystallizations. The
total disintegrations per minute (dpm) that were counted for the purified
samples were in the range of 150-400 dpm. The specific activity of each
sample from the last three recrystallizations fell within the permissible error
range (969.39 ± 15.63 dpm mg-1 = 1.6% error) and corresponded to an observed
incorporation of 0.1%. However, due to the low levels of radioactivity for
each counted sample, the specific activities were recalculated taking into
account background radioactivity of -30 dpm. The standard deviation of the
adjusted specific activities was more than twice the acceptable range (768.01
± 59.52 dpm mg-1 = 7.5% error). It should also be noted that approximately
90% of the total radioactivity added to the culture remained in the supernatant
following extraction of the pyoluteorin and could not be recovered.
Despite the presence of a 13C label in THDCP administered to the Pf-5
culture, no evidence of enrichment was detected in the isolated pyoluteorin
sample. However, in light of the low incorporation of radioactivity, this
result was expected since the minimum detectable incorporation of ['CO]­
THDCP for this experiment was approximately 0.8%.
Developing definitive conclusions from these results is difficult due to
the inconsistency of the two interpretations of the radioisotope incorporation
data. On one hand, it appears that THDCP is, in fact, incorporated into
pyoluteorin, suggesting that it is a pathway intermediate. Furthermore, since
90% of the radiolabel remained in the culture supernatant, the cell membrane
may not be permeable to THDCP, thus preventing efficient utilization of
this putative intermediate. If this is the case, the actual incorporation of
THDCP into pyoluteorin was approximately 1%. Without a complete
characterization of the labelled compound remaining in the supernatant,
however, this point is merely speculation. The other possible interpretation
of the described result is that THDCP is not a pathway intermediate. While
this conclusion is supported by the lack of evidence implicating DCP as a
pathway intermediate, both of these statements are based on negative evidence
and, therefore, are suspect.
37
2.4 CONCLUSION
Although this study has demonstrated that proline is the primary
precursor leading to formation of the dichloropyrrole moiety of pyoluteorin,
the results regarding postulated advanced intermediates within the
pyoluteorin biosynthetic pathway are inconclusive. No evidence was obtained
for the involvement of either DCP or PCA within the pathway despite the
various approaches used. Although isotope labelling data suggests that THDCP
is a pathway intermediate, this evidence is tenuous at best since the extent of
incorporation is low, given the level of background radioactivity.
Furthermore, if THDCP is a pathway intermediate, DCP should have been
incorporated into pyoluteorin. The lack of evidence resulting from this study
has forestalled identification of the starter unit required for polyketide
assembly as well as the intermediate preceding halogenation. This is rather
unfortunate since these points were the basis of justification for this approach.
The limited success of these isotope feeding experiments brings into
question the usefulness of this approach for future work with this organism.
An isotopic labelling methodology will most likely be successful only if used
in conjunction with a cell free extract or purified recombinant proteins.
Because several genes within the pyoluteorin biosynthetic gene cluster have
been cloned and sequenced (see Chapter 3), purified recombinant proteins
can be obtained for use in future studies.
2.5 EXPERIMENTAL METHODS
Chromatographic analysis of pyoluteorin. Production of pyoluteorin
was routinely assayed from a crude extract preparation of bacterial cultures.
A 5-10 mL aliquot was removed from a bacterial culture and cells were
removed by centrifugation. The supernatant was then acidified to pH < 2
with 1 N HC1. The solution was subsequently extracted with ethyl acetate
(10% vol, 3x), the pooled organic layers back extracted with water (5% vol,
3x), and the solvent removed by rotary evaporation. The pyoluteorin
contained within the crude extract was assayed using either thin layer or
38
high performance liquid chromatography (TLC and HPLC, respectively).
TLC analysis: Efficient resolution of pyoluteorin from the culture extract
dissolved in Me0H was observed on aluminum backed, normal phase silica
gel plates (Merck Kieselgel 60 F254) developed using any one of the solvent
systems listed below. Pyoluteorin was visualized as an orange-brown
derivative upon treatment with aqueous diazosulfanilic acid (1 vol. of 5%
NaNO2, 2 vol. of 0.9% sulfanilate in 1M HC1, and 3 vol. of 20% K2CO3).
Solvent Mixture
Rf Value
CHC13 / Me0H
0.32
CHC13 / EtOAc
CHC13 / Acetone
0.34
Toluene/ Acetone
0.34
0.38
It was also possible to resolve pyoluteorin (Rf 0.32) on reverse phase C18
silica TLC plates (Whatman KC18F) developed with a mixture of 50% 0.5 M
NaCl and 50% Me0H.
HPLC analysis: Pyoluteorin was analyzed using a Waters C18 Nova-pak
column (8 x 100 mm, 4p) eluted with 45% H20/25% Me0H/30% ACN at 1.5
ml miri 1. Samples were dissolved in Me0H for injection onto the column
and the eluant was monitored by UV absorbance at 310 nm. The retention
time for pyoluteorin was 3.25 min.
Pyoluteorin could also be assayed by normal phase HPLC (Nova-pak, 8
x 100 mm, 414 with 75% hexane/25% EtOAc at 1.5 ml min-1 (retention time
6.3 min). This method was not routinely used, however.
Optimization of pyoluteorin production. Duplicate 5 mL cultures of
Pseudomonas fluorescens Pf-5 (JL4092) were grown in 523 media (Kado and
Heskett, 1970), King's medium B modified to contain 0.5% wt/vol glycerol
(King et al, 1954), B10 medium (Nowak-Thompson and Gould, 1994), Nutrient
Broth supplemented with 1% glycerol (Difco Laboratories, Detroit, MI), and
Bacto Peptone (Difco). Samples taken from each fermentation at 24 h, 48 h,
and 70 h, were assayed by TLC and HPLC for relative pyoluteorin production.
Peptone source: To investigate the effect of peptone sources on
pyoluteorin production, equal weights of Bacto Peptone (Difco Laboratories,
39
Detroit, MI), phytone peptone (Becton-Dickinson, Cockeysville, MD), soytone
peptone (Difco), neopeptone (Difco), and proteose peptone #3 (Difco) were
substituted in the modified King's medium B formulation. In addition, two
flasks containing proteose peptone #3 were supplemented with either Bacto
yeast extract (Difco) or Amberex yeast extract (Red Star Specialties, Milwaukee,
WI). Extractions and analysis for metabolites were performed as above.
Carbon source: Equal weights of glycerol, sucrose, glucose, fructose, and
gluconate were substituted as carbon sources into modified King's medium
B formulation containing phytone peptone. Filter-sterilized solutions of
glucose and fructose were added to the sterile medium aseptically. Extractions
and analysis for metabolites were performed as above.
Flask size: Medium to flask volume ratio effects on pyoluteorin
production were examined using cultures of a pyoluteorin over-producing
Tn5 mutant of Ps. fluorescens Pf-5 (JL4239). Cultures were grown in 2-500
mL flasks containing either 160 or 80 mL of modified King's medium B
containing phytone peptone, and in 2-250 mL flasks containing either 80 or
40 mL of the same medium at 20 °C for 48 h. Pyoluteorin production was
also examined under oxygen-limiting conditions. Cultures in 2-250 mL flasks
containing either 80 or 40 mL of the modified medium were incubated
without aeration at 20 °C for 48 h. Cultures maintained without shaking
grew poorly and were not analyzed.
Temperature effect: The temperature effect on pyoluteorin production
was investigated for both strains JL4092 and JL4239. Strains were grown in
modified King's medium B containing phytone peptone and either fructose
or glycerol at 18 °C, 22 °C, and 28 °C for 48 h. Extractions and analysis for
metabolites were performed as above.
Production of pyoluteorin by Pf-5 in culture. For feeding studies and
routine isolation of pyoluteorin, cultures of Ps. fluorescens Pf-5 were grown
in a modified King's medium B (referred to hereafter as phytone medium)
formulated as follows:
40
phytone peptone
Glycerol
K2HPO4
MgSO4
7 H2O
2.0%
0.5%
0.15%
0.15%
All percentages are mass/volume. Medium was made to volume with
deionized water and adjusted to between pH 7.0-7.2. Production was optimal
in 40 mL cultures contained in 250 mL Erlenmeyer flasks; however, large
scale cultures were grown in 100 mL of phytone medium contained in 1 L
Erlenmeyer flasks. All cultures were incubated at 19-22 °C at 150 RPM for
42-48 h.
Isolation of pyoluteorin. Cells were removed from a 1 L culture of Ps.
fluorescens Pf-5 (10-1 L flasks containing 100 mL phytone medium) by
centrifugation and the supernatant was acidified to pH < 2 with 6N HC1. The
solution was extracted with ethyl acetate (10% vol, 3x) and the pooled organic
layers back extracted with water (5% vol, 3x). The organic layer was dried
over anhydrous MgSO4 and the solvent was subsequently removed by rotary
evaporation. The crude extract was adsorbed onto a small amount of silica
gel and applied to a 2.5 x 15 cm silica gel (Silica Gel 60, 40-63 EM Science,
Gibbstown, NJ) column equilibrated in either 3:1 toluene/acetone or 4:1
CHC13/acetone and fractionated by flash chromatography (Still et al, 1978)
with the equilibrating solvent. The material thus isolated was further purified
by recrystallization from hot CHC13 and was spectroscopically identical to
pyoluteorin.
1H NMR (300 MHz; d6-acetone) 8 9.02 (br s, exchangeable), 7.15 (t, 1H, J =
8.2 Hz), 6.80 (s, 1H), 6.47 (d, 2H, J = 8.2 Hz), and 3.00 (br s, exchangeable). 13C
NMR (75 MHz; d6-acetone) 8 183.1, 157.2, 132.5, 130.9, 119.5, 117.3, 113.0, 110.5,
and 107.5.
L-14C1
feeding study. Phytone medium (40 mL in 250 mL
shake flask) was inoculated with Ps. fluorescens Pf-5 (over-producing mutant
JL4239). The culture was grown under standard conditions (see above) for 24
41
h. One milliliter of this culture was used to inoculate 100 mL phytone medium
in each of two, 1-L shake flasks. After 16 h, 9.99 ptCi (2.20 E 7 dpm) of [U -14C]­
L-proline (ICN, Irvine, CA) dissolved in 18 mL H2O was divided into two
equal portions and added aseptically to each 100 mL culture. Both cultures
were combined 44 h after inoculation and pyoluteorin was extracted from
the culture supernatant. Authentic, unlabelled pyoluteorin (20 mg) was added
to the crude extract, and the combined samples purified by flash
chromatography yielding 56 mg (207 1.unol) of pyoluteorin. The sample was
recrystallized to within 1% of constant specific radioactivity (1.18 E 5 ± 1.05 E
3), indicating a 19 % incorporation of proline into pyoluteorin.
[1-"a-L-Proline feeding study. A 40 mL seed culture of Ps. fluorescens
Pf-5 (wild-type strain JL4239) was grown under standard conditions (see above)
for 24 h. One milliliter of this culture was used to inoculate 100 mL phytone
medium in each of ten 1-L shake flasks. Sixteen milligrams of [1-13C] -L-proline
(CIL, Cambridge, MA) was dissolved in 10 mL H2O and equal portions added
aseptically to each of the culture flasks 13 h after inoculation. 48 h after
culture inoculation, pyoluteorin was isolated (47 mg; 173 innol) from the
combined cultures and recrystallized from hot CHC13. An enrichment of
5.7% over natural abundance of the pyoluteorin carbonyl resonance (8 183.0)
was observed in the 13C NMR spectrum (75 MHz; d6-acetone).
Synthesis of deschloropyoluteorin (2-(2',6'-dihydroxybenzoy1)-1H­
pyrrole; DCP; Scheme 2-12). 2,6-Dimethoxybenzoyl chloride (2.0 g; 9.97 mmol)
and pyrrole (1 mL; 14.4 mmol) were combined in 20 mL dichloroethane and
cooled to 5 °C in an ice/water bath. A slurry of A1C13 (1.5 g; 11.25 mmol)
suspended in 20 mL anhydrous CH2C12 was added to the mixture over 30
min with stirring under an inert atmosphere. The reaction was allowed to
warm to room temperature and stirred for 1 h. Work up of the reaction and
purification of the desired compound was as described (Rao and Reddy,
1990). Purification yielded 820 mg (40% yield) of 2,6-dimethoxy-DCP: mp
197-198 °C [lit. (Cue et al, 1981) mp 192-195 °C[;1H NMR (300 MHz; d6 -DMSO)
8 11.85 (br s, exchangeable), 7.34 (t, 1H, J = 8.5 Hz), 7.08 (m,41-1), 6.70 (d, 2H, J =
42
8.5 Hz), 6.29 (m, 1H), 6.10 (m, 1H), 3.65 (s, 6H); 13C NMR (75 MHz; d6-acetone)
8 184.5, 158.7, 136.9, 131.3, 125.6, 119.4, 118.6, 110.8, 105.1, and 56.2; EI HRMS
m/z 231.0896 (calc. exact mass for C13H13NO3 231.0895).
The 2,6-dimethoxy derivative (800 mg, 3.46 mmol) was subsequently
dissolved in 115 mL of benzene and added to a vigorously stirred solution of
A1Br3 (3.0 g; 11.28 mmol in 30 mL benzene). The bright orange reaction
mixture was stirred for 5 h at room temperature. The reaction was then
added to a 1 L flask containing 130 mL of 3 N HCl and 30 mL EtOAc and
stirred for 30 min. The aqueous layer was extracted with EtOAc (3 x 30 mL)
and the combined organic layers dried over anhydrous MgSO4. The crude
extract was concentrated to dryness and fractionated on a flash silica gel
column (5 x 15 cm) eluted in CHC13 and Me0H (9:1) yielding 510 mg of DCP
(72% yield): mp 143-145 °C [lit. (Takeda, 1958) mp 142-145 °C]; UV. (HPLC
solvent) 298 nm; 11-1 NMR (300 MHz; d6-DMS0) 8 11.76 (br s, 1H), 9.34 (s, 2H),
7.06 (m, 1H), 6.98 (t, 1H, J = 8.1 Hz), 6.39 (m, 1H), 6.35 (d, 2H, J= 8.2 Hz), and
6.11 (m, 1H); 13C NMR (100 MHz; d6 -DMSO) 8 183.6, 155.6, 133.3, 129.6, 125.1,
118.1, 116.1, 109.6, and 106.4; EI HRMS m/z 203.0583 (calc. exact mass for
CI IH9N03203.0582).
Deschloropyoluteorin "C-isotope trapping experiment. Two sets of
duplicate production cultures (a total of 4-40 mL cultures in 250 mL flasks)
were used in this experiment. One set was comprised of two flasks containing
phytone medium and the two flasks within the other set contained phytone
medium in which glucose was substituted for glycerol (phytone / glucose
medium). Each of the production cultures were inoculated with 400 JAL of a
Ps. fluorescens Pf-5 (JL4092) seed culture grown for 24 h. After 9.5 h, an
aqueous solution of [1-14C1-sodium acetate (10 liCi) was added to each of the
production cultures. The cells were collected by centrifugation from one
phytone and one phytone/glucose culture at 14.5 h and 18.5 h following
inoculation. The cells were then resuspended in 3-5 mL of the culture
supernatant and an equal weight of micro glass beads. The suspension was
sonicated on ice using a probe-type sonicator for 15 min at 90% full power.
The sample was then centrifuged to remove cell debris and glass beads.
Approximately 60 mg of DCP (in Me0H) was added to each lysate solution.
43
The DCP was isolated from the cell lysate using the methodology described
for pyoluteorin with one important addition. Since DCP and pyoluteorin
co-elute from the flash silica gel column, it was necessary to further purify
the sample by reverse phase chromatography. The DCP/pyoluteorin mixture
was applied to a 1.5 x 15 cm C18 reverse-phase silica gel column (Bakerbond;
40 gm; JT Baker, Phillipsburg, NJ) and eluted with 50% aqueous Me0H.
Repeated recrystallization from benzene of the recovered DCP resulted in
complete loss of detectable radioactivity.
The combined fractions containing pyoluteorin recovered from the C18
silica column were determined to contain approximately 2.8% of the total
radioactivity that had been added to the cultures. Unlabelled pyoluteorin
was added to these fractions, but it was not possible to recrystallize pyoluteorin
to determine accurate levels of incorporation. Therefore, a sample of the
recovered pyoluteorin was resolved from impurities by 2D-TLC (silica gel;
4:1 toluene-acetone; 9:1 CHC13-Me0H) and subjected to scintillation counting.
Deuterium exchange of deschloropyoluteorin with 21-1-trifluoroacetic acid
(Scheme 2-13). DCP (64 mg; 315 gmol) was dissolved in 1 mL of 2H­
trifluoroacetic acid and 700 gL of 21120. The solution was placed in a thick
walled, glass tube fitted with a telfon stopcock and frozen in a dry ice bath.
The tube was repeatedly evacuated and flushed with N2 (4 x). After the final
flush, the tube was sealed and heated in an oil bath at 75 °C overnight.
Solvent was removed under high vacuum and the crude product was eluted
on a flash silica gel column with 9:1 CHC13 -MeOH yielding 50 mg of purified
product (76% yield). The extent of labelling was estimated as >90% for the
pyrrole ring and -70% for the m eta-positions of the resorcinol ring, based on
the integration of resonances within the 1H NMR spectrum. Since the carbon
resonance corresponding to the pa ra-position within the resorcinol ring
exhibited limited isotopic splitting, it was presumed that limited deuterium
exchange occurred at this position. Therefore proton integrations were
normalized to the para-proton resonance.
44
2H- Deschloropyoluteorin feeding study. Phytone production medium
(5 x 100 mL in 1 L shake flasks) was inoculated with 800 'IL of an overnight
culture of Ps. fluorescens Pf-5 (JL4092). 400 piL of a 2H-DCP solution (50 mg in
2 mL EtOH) was added aseptically to each of the production cultures at 20 h
after inoculation. Cultures were incubated until 48 h after inoculation when
the supernatant was extracted as described above. The crude extract was
fractionated on a 2 x 20 cm C 18 reverse-phase silica column (Bakerbond; 40
gm; JT Baker, Phillipsburg, NJ) and eluted with a 3:2 Me0H-water mixture.
Both DCP and pyoluteorin were recovered (52.2 mg and 15.5 mg, respectively).
2H NMR analysis of pyoluteorin showed no evidence of 2H-DCP incorporation.
The 1H NMR spectrum for the recovered DCP was identical to authentic
material and indicated the exchange of the label had not occurred during the
course of the experiment.
Cell permeability study. Cultures (5 mL) of Ps. fluorescens Pf-5 (JL4092)
were grown in phytone medium for 15 h at which time individual cultures
received one of the following: 100 pit toluene, 100 pil DMSO, 50 mg mixed
alkyltrimethylammonium bromide (primarily C14), 50 mg cetylpyridinium
bromide, 50 mg methyltrioctylammonium chloride, PMBN to 1 pig mL-1 and
3 pig mL-1, or poly-L-lysine (MW 4000-15000) to 8 pig mL-1 and 16 lig mL-1.
Additional 5 mL cultures were prepared in which MgC127H20 was omitted
from the phytone medium and replaced with EDTA to a final concentration
of 6.5 pig mL-1. The phytone/EDTA cultures were amended with either PMBN
to 1 pig mL-1 and 3 pig mL-1 or poly-L-lysine to 8 pig mL-1 and 16 pig mL-1, 15 h
after inoculation. All of the cultures also received 10 pit of a stock DCP
solution (24 mg dissolved in 1.2 mL) at the same time point. Cultures were
allowed to grow for an additional 26 h. Pyoluteorin production and changes
in DCP concentration were analyzed qualitatively by HPLC as described above.
Synthesis of 2,6-dimethoxy-['4C0]-benzoyl chloride (Scheme 2-14). 1,3­
Dimethoxybenzene (261 piL; 276 mg; 2 mmol) and n-butyl lithium (1.25 mL
of a 1.6 M hexaneane solution; 2 mmol) were combined in a round bottom
flask (25 mL) containing a stir bar and 5 mL anhydrous THF. The solution
45
was refluxed for 4 h under Ar, after which time the condenser was removed
and the flask fitted with a rubber septum. A mixture of 220 mg Na213CO3
(CIL, Cambridge, MA) and 250 gCi Na214CO3 (ARC, St. Louis, MO) was placed
in a separate round bottom flask (10 mL) containing a stir bar and fitted with
a rubber septum. Both flasks were frozen in liquid N2, evacuated, and flushed
with Ar (3x). Upon final evacuation, the two flasks were connected via
candula needle and allowed to warm to room temperature. Concentrated
H2SO4 (5 mL) was added to the Na2CO3 mixture and the organolithium reagent
was again frozen in liquid N2 until CO2 evolution ceased. The candula needle
was removed, and the reaction was warmed to room temperature and stirred
for 24 h. The residue remaining in the reaction flask was dissolved in 10%
NaHCO3 and extracted with Et20. The aqueous phase was subsequently
acidified to pH 2 with 6 N HC1 and the reaction product extracted into Et20.
The organic layer was dried over anhydrous MgSO4 and evaporated, yielding
209 mg of [14C0] -2,6- dimethoxybenzoic acid (0.055 gCi mg-1; 78% yield): mp
183-185 °C [lit (Aldrich, 1997) mp 186-187 °C]; 1H NMR (300 MHz; CDC13)
7.33 (t, 1H, J = 8.4 Hz), 6.60 (d, 2H, J = 8.4 Hz), and 3.88 (s, 6H); 13C NMR (75
MHz; CDC13) 8 170.6, 157.8, 131.8, 111.5, 104.0, and 56.1; EI HRMS m/z 182.0579
(calc. exact mass for C9H1003 182.0579).
The entire sample of the labelled benzoic acid derivative was dissolved
in a minimal volume of SOC12 and 1 drop of dimethylformamide was then
added. The reaction was stirred overnight at room temperature under Ar.
The solvent was subsequently removed under high vacuum and the material
used directly in the Friedel-Crafts reaction as described above yielding 95.5
mg of labelled 2,6-dimethoxy-DCP, approximately half (48 mg) of which was
subsequently converted to labelled DCP (38 mg, sp. act. 0.982 tiCi
P4C01-deschloropyoluteorin feeding study. Ps. fluorescens Pf-5 (JL4092)
cultures (3 x 90 mL in 1 L shake flasks) grown in phytone medium lacking
MgC127H20 under standard conditions were started from one milliliter of
an overnight culture. Cultures were amended with PMBN (3 pg mL-1) and
EDTA (6.5 pg mL-1) 14 h after inoculation. The labelled DCP (14.73 mg in 3
mL DMSO) was simultaneously added to the cultures and growth continued
46
until 50 h after initial inoculation. Pyoluteorin was isolated using the method
described for the 2H-DCP feeding study. Following three recrystallizations of
the recovered pyoluteorin from hot CHC13, no radioactivity was detected.
rOD21-1)-Pyrrole-2-carboxylic acid synthesis (PCA; Scheme 2 -15). In a
round bottom flask fitted with a rubber septum and stir bar, 20 mL of Et20
was cooled to -10 °C followed by addition of 10 mL of EtMgBi (3.0 M ethereal
solution). A solution of 2.08 mL pyrrole (2.0 g; 29.9 mmol) in 15 mL Et20 was
added drop-wise and the reaction stirred for 15 min. The reaction mixture
was frozen in liquid N2, evacuated, and allowed to thaw in an ice bath (3x).
Upon final thawing, the reaction flask was connected via a candula needle
to a second flask containing 2.05 g Na213CO3 (CIL, Cambridge, MA) and a stir
bar under Ar. Concentrated H2SO4 (10 mL) was added to the Na213CO3 and the
Grignard reagent was immediately immersed in liquid N2 until CO2 evolution
ceased. The candula needle was removed, and the reaction warmed to room
temperature over 3 h. The mixture was then poured into an ice slurry of 3
M H2SO4 and the reaction product partitioned into Et20. The organic layer
was dried over anhydrous MgSO4, treated with activated charcoal, and filtered.
Crystallization was induced by partial evaporation of the solvent and
refrigeration, yielding 975 mg of while crystals: mp 204-207 °C (dec) [lit. (Letellier
and Bouthillier, 1957) mp 204-207 °C]; 1H NMR (400 MHz; d6-acetone) 8 10.89
(br s), 9.25 (br s), 7.06 (m, 1H), 6.87 (m, 1H), and 6.23 (m, 1H); 13C NMR (100
MHz; d6-acetone) 8 162.9, 124.5, 123.5, 116.2, and 110.6; EI HRMS for [12CO2H] -PCA
m/z 111.0320 (calc. exact mass for C5H5NO2 111.0320); EI LRMS for [13CO2F1] -PCA
m/z 112.1. Calculated 13C-enrichment of the synthetic sample was 96% based
on LRMS data.
FI313211]-Pyrrole-2-carboxylic acid feeding study. Phytone medium (3 x
100 mL in 1 L shake flasks) was inoculated with one milliliter of an overnight
culture ofPs. fluorescens Pf-5 (over-producing mutant JL4239). Cultures were
grown under standard conditions. Labelled PCA (75 mg) was dissolved in 18
mL of 0.1 M NaHCO3 and administered in equal portions to the cultures at
14 h and 21 h after inoculation. After 46 h of growth, pyoluteorin was isolated
47
from the culture supernatant as described above. The sample (47 mg) was
recrystallized from hot benzene and analyzed by 13C NMR. Comparison of
13C line intensities for the isolated sample to those of an identical, unlabelled
pyoluteorin sample did not reveal any evidence of isotopic enrichment.
(13C0]-Pyrrole-2-carboxylic acid N-acetylcysteamine thioester (NAC­
PC0]-PCA; Scheme 2-16). N,S-Diacetylcysteamine (2.4 g; 14.8 mmol) was
hydrolyzed in 80 mL aqueous KOH (2.8 g, 49.9 mmol) for 2 h. The reaction
pH was adjusted to 5.0 with 2 N HCl and the mixture was saturated with
NaCl. The brine solution was subsequently extracted with dichloromethane
yielding N-acetylcysteamine as a clear oily residue: 1I-1 NMR (300 MHz; CDC13)
8 7.03 (br s, 1H), 3.28 (q, 2H, J = 6.6 Hz), 2.53 (dt, 2H, J= 6.6 Hz and 8.4 Hz), 1.89
(s, 3H), and 1.36 (t, 1H, J = 8.4 Hz).
An aliquot of the N-acetylcysteamine preparation (800 A) was combined
with [13CO2H] -PCA (250 mg, 2.23 mmol) and 4-dimethylaminopyridine (27.5
mg, 0.23 mmol) in 15 mL of anhydrous dichloromethane. The mixture was
cooled to 0 °C and the flask flushed with Ar. 1,3-Dicyclohexaneylcarbodiimide
(460 mg, 2.71 mmol) was added and the reaction was maintained at 0 °C with
stirring. The reaction flask was subsequently removed from the ice bath,
allowed to warm to room temperature, and stirring continued for 4 h. The
reaction mixture was then filtered through celite and placed under high
vacuum overnight. Purification of the NAC-thioester was afforded by flash
silica chromatography (2.5 x 15 cm column, 4:1 EtOAc- hexane) followed by
recrystallization from 1,1,1-trichloroethane. A total of 195 mg of pure thioester
was obtained: mp 110-111 °C ; UVmax (Et0H) 296 nm; IR (neat) 3304, 2930,
2353, 1628, and 1598 cm-1;11 NMR (300 MHz; d6-acetone) 8 7.31 (br s), 7.11 (m,
1H), 6.91 (m, 1H), 6.19 (m, 1H), 3.32 (q, 2H J= 6.5 Hz), 3.06 (t, 2H, J = 6.5 Hz),
2.82 (s, 1H), and 1.82 (s, 3H); 13C NMR (75 MHz; d6-acetone) 8 180.1, 169.5,
130.2, 124.8, 115.2, 110.3, 39.5, 27.6, and 22.2; EI HRMS for NAC-P2CORCA
m/z 212.0619 (calc. exact mass for C9H12N202S 212.0619); EI LRMS for [13CO21-1]­
PCA m/z 213.1. Calculated 13C-enrichment of the synthetic sample was 96%
based on LRMS data.
48
(13C0]-Pyrrole-2-carboxylic acid N-acetylcysteamine thioester feeding
study. Ps. fluorescens Pf-5 (JL4092) cultures (10 x 100 mL in 1 L shake flasks)
grown in phytone medium under standard conditions were started from 800
!IL of an overnight culture. NAC-[13C0]-PCA (92.3 mg) was dissolved in 4
mL DMSO and equal portions were administered to the cultures at 13.5, 15.5,
18, and 20 h after inoculation. Pyoluteorin (15 mg) was isolated from the
combined culture supernatants 48 h after inoculation as described above. No
enrichment was observed for the 13C carbonyl resonance in the NMR spectrum
of the isolated pyoluteorin sample.
Synthesis of protected tetrahydrodeschloropyoluteorin (242-hydroxy­
6'-methoxybenzoy1)-1H-pyrrolidine; Scheme 3-17). 2,6-dimethoxy-DCP (203
mg, 0.88 mmol) potassium-tert-butoxide (100 mg, 0.89 mmol), and di-tert-butyl
dicarbonate (225
210.9 mg, 0.97 mmol) were refluxed in 30 mL anhydrous
THE under Ar for 2 h. The reaction was washed twice with saturated NaCI.
The reaction was fractionated on a silica gel flash column (1 x 15 cm) eluted
with 3:7 EtOAc- hexane, yielding 218 mg of the N-protected compound: mp
66-68 °C; UVmax (MeOH) 212, 248, and 286 nm; IR (CHC13) 2975, 2944, 1753,
1673, 1605, and 1475 cm-1; 1H NMR (300 MHz; CDC13) 8 7.40 (dd, 1H, J = 3.4
and 1.5 Hz), 7.29 (t, 1H, J = 8.4 Hz), 6.58 (dd, 1H, J = 3.4 and 1.5 Hz), 6.54 (d, 2H,
J = 8.4 Hz), 6.10 (dd, 1H J = 3.4 and 1.5 Hz), 3.74 (s, 6H), and 1.60 (s, 9H); 13C
NMR (75 MHz; CDC13) 6 182.6, 157.9, 149.3, 134.9, 130.7, 128.9, 124.3, 118.8,
109.8, 104.0, 84.5, 55.9, and 27.4; EI HRMS m/z 331.1418 (calc. exact mass for
C18H21N05 331.14197).
The N-protected pyrrole (164 mg, 0.495 mmol) was catalytically
hydrogenated over 10% Pd/C (77 mg) in MeOH (2 mL) containing 2 drops
conc. HC1 under 1 atm H2 for 36 h with vigorous stirring. The suspension
was then filtered through glass wool and the filtrate triturated with CHC13
forming a white solid. The precipitate was chromatographed on silica gel
with 1:1 EtOAc- hexane yielding 138 mg of the 2,6-dimethoxy-tBoc-pyrrolidine
as an oily residue: UV. (MeOH) 257 (shoulder) and 276 nm; IR (CHC13)
2970, 2933, 2884, 1726, 1707, 1605, and 1473 cm-1;1H NMR (300 MHz; d4 -MeOH)
8 7.37 (t, 1H, J = 8.4 Hz), 6.71 (d, 2H, J = 8.4 Hz), 4.94 (t, 1H, J= 5.7 Hz), 3.80 (s,
6H), 3.43 (m, 2H), 2.04 (m, 1H), 1.89 (m, 2H), and 1.41 (s, 9H); 13C NMR (75
49
MHz; CDC13) 8 200.8, 157.5, 154.5, 131.0, 117.9, 103.9, 79.1, 66.2, 55.7, 46.2, 28.5,
28.1, and 22.9; EI FIRMS m/z 336.1786 (calc. exact mass for Ci8H25N05336.18110).
The t-Boc protecting group was removed by treatment of the pyrrolidine
(138 mg, 0.412 mmol) with 1.5 mL trifluoroacetic acid under an inert
atmosphere at 0 °C for 40 min. Evaporation of the solvent under high vacuum
yielded the 2,6-dimethoxy-pyrrolidine derivative as an oily residue: UVmax
(MeOH) 214 and 274 nm; IR (CHC13) 2966, 2842, 2768, 1790, 1722, 1673, and
1592 cm-1; 11-1 NMR (300 MHz; CDC13) 8 9.59 (br s), 7.40 (t, 1H, J = 8.4 Hz), 6.60
(d, 2H, J = 8.4 Hz), 5.21 (br s, 1H), 3.80 (s, 6H), 3.53 (m, 2H), 2.13 (m, 1H), 2.11
(m, 2H), and 2.07 (m, 1H); 13C NMR (75 MHz; CDC13) 8 197.1, 158.1, 133.8,
113.4, 104.2, 66.9, 56.0, 47.0, 27.9, and 23.9; EI FIRMS m/z 236.1287 (calc. exact
mass for C13H18NO3 236.1287).
The 2,6-dimethoxy pyrrolidine derivative (18 mg, 0.52 mmol) was
dissolved in 2 mL anhydrous dichloromethane, was cooled to -70 °C and
BBr3 (510 !IL of 1.0 M solution) was added drop-wise. The reaction was removed
from the dry ice bath and allowed to warm to room temperature and stirred
for 2 h. The reaction mixture was poured into an ice slurry (20 mL), stirred
for an additional 20 min, saturated with NaC1, and subsequently extracted
into dichloromethane. Reaction work up yielded 6.5 mg of product. 1H NMR
analysis revealed that only a single methyl group was removed and therefore,
complete characterization of the compound was not undertaken. 1F1 NMR
(300 MHz; CDC13) 8 8.36 (br s), 7.45 (t, 1H, J = 8.4 Hz), 6.61 (d, 1H, J = 8.4 Hz),
6.42 (d, 1H, J = 8.4 Hz), 5.50 (br s, 1H), 3.95 (s, 3H), 3.63 (m, 2H), 2.64 (m, 1H),
2.12 (m, 2H), and 1.91 (m, 1H).
Synthesis of labelled tetrahydrodeschloropyoluteorin (2-(2',6'-dihydroxy­
13CO/4C01-benzoy1)-1H-pyrrolidine; [13C0114C0]-THDCP; Scheme 2-18).
Labelled DCP (63 mg, 0.310 mmol) was dissolved in 7 mL dimethylformamide
containing Na2CO3 (72 mg, 0.683 mmol) and benzyl bromide (81 III., 116 mg,
0.683 mmol). The reaction flask was charged with Ar and heated for 3 h at
60-65 °C. Solvent was removed overnight under vacuum and the residue
dissolved in 5-10 mL of EtOAc. The solution was subsequently washed with
1N HCl (2x) followed by saturated NaCl (2x). Benzylated product was purified
via flash silica chromatography (1 x 15 cm, 7:13 EtOAc- hexane) as an oily
50
residue: UV,.. (EtOH) 295 nm; IR (CHC13) 3393, 2965, 2365, 1604, and 1551
cm-1; 1H NMR (300 MHz; CDC13) 5 9.98 (br s), 7.28 (m, 4H), 7.17 (m, 2H), 7.01
(m, 2H), 6.67 (d, 1H, J = 8.0 Hz), 6.57 (d, 1H, J = 8.1 Hz), 6.24 (m, 1H), and 5.06
(s, 2H);13C NMR (75 MHz; CDC13) 8 184.7, 159.4, 157.6, 136.2, 133.4, 132.6, 128.3,
127.7, 126.9, 125.6, 120.7, 113.8, 111.0, 110.5, 104.3, and 70.6; EI HRMS m/z
293.1045 (calc. exact mass for C18H15NO3 293.1052).
O-Benzyl-DCP (98 mg, 0.334 mmol) was combined with potassium tert­
butoxide (93.8 mg, 0.836 mmol) and di- tert-butyl dicarbonate (192
182.4
mg, 0.836 mmol) and the mixture refluxed in 25 mL anhydrous THE under
Ar for 2.5 h. The reaction products were fractionated on a silica gel column
(2 x 15 cm, 1:4 EtOAc- hexane) and the fully protected product was recrystallized
from 1,1,1-trichloroethane and hexane yielding a white crystalline compound:
mp 119-120 °C; UVmax (EtOH) 254 and 287 nm; IR (CHC13) 2977, 2936, 2876,
2359, 1759, and 1720 cm-1; 1H NMR (300 MHz; CDC13) 8 7.42 (m, 1H), 7.35 (t,
1H, J = 8.2 Hz), 7.26 (m, 3H), 7.26 (m, 2H), 6.86 (d, 1H, J = 8.2 Hz), 6.84 (d, 1H, J
= 8.2 Hz), 6.58 (m, 1H), 6.11 (m, 1H), 5.03 (s, 2H), 1.49 (s, 9H), and 1.40 (s, 9H);
13C NMR (75 MHz; CDC13) 8180.5, 157.2, 151.4, 149.3, 149.0, 136.2, 134.6, 130.8,
128.4, 128.3, 127.7, 126.9, 124.1, 123.4, 115.5, 110.2, 110.0, 84.6, 83.4, 70.6, 27.4,
and 27.3; EI HRMS m/z 493.2098 (calc. exact mass for C28H31N07 493.2101);
Elemental analysis: found C 68.10, H 6.42, N 2.78, 0 22.70 (anal. calc. for
C28H31N07: C 68.18, H 6.34, N 2.84, 0 22.64).
Hydrogenation of the protected pyrrole derivative (114 mg, 0.231 mmol)
was accomplished over 10% Pd/C (20 mg) in 3 mL EtOH under 1 atm 112 for
5 h. The catalyst was removed by filtering the reaction through glass wool
and the products fractionated by flash silica chromatography (1.5 x 15 cm,
3:17 EtOAc- hexane) yielding 85 mg of N,O- t- Boc- pyrrolidine as an oily residue:
U Vmax (EtOH) 210 (shoulder), 254, and 312 nm; IR (CHC13) 3150, 2977, 2936,
2882, 2246, 1770, 1711, and 1693 cm-1; 1H NMR (300 MHz; CDC13) 8 12.44 (s,
1H), 11.80 (br s, 1H), 7.43 (t, 1H, J = 8.3 Hz), 7.36 (t, 1H, J= 8.3 Hz), 6.88 (dd, 1H,
J = 1.0 and 8.3 Hz), 6.85 (dd, 1H, J = 1.0 and 8.3 Hz), 6.73 (dd, 1H, J = 1.0 and 8.3
Hz), 6.70 (dd, 1H, J = 1.0 and 8.3 Hz), 5.13 (m, 2H), 3.67 (m, 2H), 3.47 (m, 2H),
2.38 (m, 1H), 2.24 (m, 1H), 1.93 (m, 6 H), 1.56 (s, 9H), 1.53 (s, 9H), 1.45 (s, 9H),
and 1.23 (s, 9H); 13C NMR (75 MHz; CDC13) 8 205.6, 203.6, 163.8, 161.5, 154.9,
153.6, 150.9, 150.8, 150.6, 150.0, 135.4, 134.2, 116.1, 116.0, 114.9, 113.5, 113.3,
112.4, 84.6, 84.1, 80.4, 79.7, 64.9, 64.7, 46.9, 46.6, 30.3, 29.7, 29.5, 28.3, 27.9, 27.6,
51
23.8, and 22.9; EI HRMS m/z 407.1944 (calc. exact mass for C211-129N07 407.1944).
Full deprotection of the N,O- t- Boc- pyrrolidine intermediate (84 mg, 0.206
mmol) occurred upon treatment with trifluoroacetic acid at 0 °C for 20 min.
Solvent was removed under high vacuum and the residue recrystallized
from EtOAc and pentane, yielding 35 mg of [13C0/14C0] -THDCP (sp. act. 0.982
m.Ci mg-1): mp 186-190 °C (dec); UVmax (EtOH) 278 and 356 nm; IR (CHC13)
2995, 2757, 2365, 1670, 1634, and 1610 cm-1; 111 NMR (300 MHz; d4-Me0H) 8
7.35 (t, 1H, J = 8.2 Hz), 6.43 (d, 2H, J = 8.2 Hz), 5.50 (dd, 1H, J= 5.5 and 9.3 Hz),
3.42 (m, 2H), 2.67 (m, 1H), 2.09 (m, 2H), and 1.98 (m, 1H); 13C NMR (75 MHz;
d4 -MeOH) 8 202.6, 166.3, 142.1, 111.2, 110.9, 70.1, 50.0, 33.2, and 26.9; EI HRMS
m/z 207.08950 (calc. exact mass for C11H13NO3 207.08954).
[13COP4C0]-Tetrahydrodeschloropyoluteorin feeding study.
Ps.
fluorescens Pf-5 (JL4092) cultures (7 x 100 mL in 1 L shake flasks) inoculated
from a common seed culture were grown in phytone medium under standard
conditions. Labelled THDCP (35 mg, 12.9 liCi) was dissolved in 14 mL H20, 1
mL of which was added to each culture 13 h and 16 h after inoculation.
Cultures were grown until 42 h after inoculation. Pyoluteorin was isolated
using standard methods and analyzed by 13C NMR. No enrichment above
natural abundance was detected; therefore, the sample was recrystallized to
constant specific radioactivity from hot CHC13. Specific activity was calculated
with and without background correction (31.6 dpm).
52
Chapter 3. Nucleotide Sequence Analysis of Loci Required
for Pyoluteorin Biosynthesis
3.1 INTRODUCTION
A genomic region was identified previously within Ps. fluorescens Pf-5
that is required for pyoluteorin production (Kraus and Loper, 1995; see Chapter
1). Based on the fact that this region spanned approximately 21-kb and that
transposon insertions within this region appeared to only affect pyoluteorin
production, it was suggested that the structural genes encoding the pyoluteorin
biosynthetic enzymes were localized within this 21-kb fragment. Because
pyoluteorin is a polyketide-derived metabolite (see Chapter 2), it was likely
that genes encoding a polyketide synthase could be identified via nucleotide
sequence analysis of the 21-kb genomic region. Although the principal
objective of this study was to identify the pyoluteorin polyketide synthase,
other loci also were identified that are likely responsible for chlorination of
a pyoluteorin biosynthetic intermediate(s). The identification of genes
encoding the polyketide synthase and the halogenases required for pyoluteorin
biosynthesis is intriguing in that the polyketide synthases have been
characterized extensively whereas halogenases are an almost unknown class
of enzymes.
3.1.1 Genetic organization of polyketide synthases
The biochemical pathways as well as the biosynthetic enzymes required
for fatty acid and polyketide biosynthesis share many similarities (Hopwood
and Sherman, 1990; Katz and Donadio, 1993). Both processes involve the
serial condensation of short chain CoA-thioesters (usually acetate-derived)
and the subsequent reductive modification of the extended carbon chain.
These reductive modifications involve the same types of transformations,
namely, carbonyl reduction, dehydration, and enoyl reduction. Whereas fatty
acid synthases (FASs) redundantly catalyze each one of these reactions and
53
generally recognize only malonyl-CoA as a substrate, PKSs exhibit remarkable
control over the extent to which each newly added carbon unit is modified.
Furthermore, PKSs can utilize a variety of carboxylic acid., (e.g. acetate,
propionate, or butyrate) as starter units (see Table 2.1) or for extending the
carbon chain. Therefore, polyketides generally possess an elaborate carbon
skeleton containing several oxidized functionalities.
The protein structures of fatty acid and polyketide synthases are also
similar, and these enzymes are classified as either Type I or Type II proteins.
Like the Type I FASs present in vertebrates, Type I PKSs are large
multifunctional proteins containing several catalytic domains required for
polyketide assembly. These domains are organized into one or more enzymatic
modules, each containing a minimal core set of domains: a ketosynthase
(KS), an acyltransferase (AT), and an acyl carrier protein (ACP). Additional
enzymatic domains, such as a ketoreductase (KR), may also be present within
an individual Type I PKS module and are located between the AT and the
ACP domains. In Type I PKSs possessing several modules, each module is
responsible for the addition and modification of a single extender unit. In
addition, these enzymes are processive and typically require f3-carbonyl
modification prior to addition of the next carbon unit.
module
6-Methylsalicylate Synthase
I
Penicillium patulutn
0
KS AT DH KR ACF>
(5.2 kb)
0
KR
SE
o
DH
SEnz
0
KS/AT
-1111,'
Figure 3-1. Genetic organization and part of the reaction pathway catalyzed
by a Type I PKS. For this example, a single enzymatic module is encoded by a
single ORF. Only the reduction, dehydration, and the final condensation
within the pathway are presented to illustrate the processive mechanism of
the Type I PKSs. Abbreviations are as follow: KS, ketosynthase; AT,
acyltransferase; DH, dehydrase; KR, ketoreductase; ACP, acyl carrier protein;
SEnz, thioester linkage to the enzyme.
54
The genes encoding several Type I PKSs have been characterized from
both bacterial and fungal sources. Fungal Type I PKSs generally are encoded
by a single ORF containing one module and are responsible for biosynthesis
of an aromatic metabolite, as exemplified by the 6-methyl salicylate synthase
(6MSAS) from Penicillium patulum (Figure 3-1; Beck et al, 1990). Bacterial
Type I PKSs, however, often contain multiple modules distributed among
several proteins and generally synthesize non-aromatic, macrolide antibiotics.
Examples of bacterial Type I PKSs exist in which 2, 4, 5, or 6 modules are
present within a single peptide (Cortes et al, 1990; Donadio et al, 1991; Aparicio
et al, 1996).
Actinorhodin PKS
Streptomyces coelicolor A3
KR
KS
AT
CLF
I
ACP
Cyclase
Cyclase
minimal PKS
PKS
10x Acetate --Yo
000
000
PKS/KR
1111"
sEnz
OH
HO
2
Actinorhodin
Figure 3-2. Genetic organization and part of the reaction pathway catalyzed
by a Type II PKS. Only the first reduction and cyclization within the pathway
are presented to illustrate the non-processive mechanism of the Type II
PKSs. ORFs are indicated by open boxes. Abbreviations are as follow: KS,
ketosynthase; AT, acyltransferase; CLF, chain length determining factor; KR,
ketoreductase; ACP, acyl carrier protein; SEnz, thioester linkage to the enzyme.
In contrast to the multifunctional protein Type I systems, the Type II
PKSs are multienzyme complexes comprised of a few, principally monofunctional proteins (Figure 3-2). Three essential proteins (a bifunctional
KS/AT, a chain length-determining factor (CLF), and an ACP) are present in
55
all of the Type II PKSs described to date (Hutchinson and Fujii, 1995). The
KA/AT, CLF, and ACP comprise a minimal PKS, which is necessary and
sufficient for polyketide biosynthesis (McDaniel et al, 1994). Unlike the
processive mechanism of the Type 1 PKSs, active sites within the minimal
Type II PKS are used iteratively to form an extended poly -(3- ketone chain
prior to any reductive modifications of the carbonyls (i.e. a non-processive
mechanism). The regiospecificity of the cyclization process is dependent upon
several cyclases and, in some instances a ketoreductase, that function in
concert with the minimal PKS (Hutchinson and Fujii, 1995). Other proteins
within the Type II PKSs are thought to be required for starter unit specificity
or post assembly modifications of functional groups present within the
polyketide structure.
Although the resveratrol and chalcone synthases are distinct from the
polyketide synthases, they nevertheless are responsible for the formation of
polyketide natural products. These synthases are single, homodimeric proteins
that do not require the 4'-phosphopantetheinyl prosthetic group necessary
for the PKSs (Kreuzaler et al, 1979; Schroder et al, 1988), and they possess a
single cysteine residue that is required for activity (Lanz et al, 1991).
Furthermore, the dissimilarity between the peptide sequences of the
resveratrol/chalcone synthases and the PKSs suggests that these two classes
of enzymes have convergently evolved to utilize a similar catalytic
mechanism (J. Schroder, personal communication).
Within the fluorescent pseudomonads, only two PKSs have been
previously reported. The PKS required for 2,4-diacetylphloroglucinol
biosynthesis in Ps. fluorescens Q2-87 exhibits similarity to the chalcone
synthases (Thomashow et al, 1996). A Type II PKS has been identified within
Ps. syringae and is responsible for biosynthesis of the coronafacic acid moiety
of the phytotoxin coronatine (Penfold et al, 1996).
3.1.2 Halogenating enzymes in microorganisms
Despite the existence of a few thousand halogenated secondary metabolites
(Gribble, 1996), practically nothing is known about the enzymes responsible
for the halogenation of these compounds. Historically, a non-specific enzyme
56
assay based on the halogenation of monochlorodimedone in the presence of
hydrogen peroxide and halide ion (Hewson and Hager, 1980) has been the
principal method used in the isolation of known haloperoxidases. Recently,
however, gene disruption experiments have demonstrated that several of
the proteins thus isolated are not required for halo-metabolite production in
the pathways to which they have been assigned (reviewed by van Pee, 1996).
Furthermore, halogenation by many of these halogenating proteins is non­
regiospecific, suggesting the protein has a negligible role in directing the
reaction. Sequence comparison of these haloperoxidases has revealed
structural similarities to several serine-hydrolases (Pelletier and
Altenbuchner, 1995). The halogenations catalyzed by these proteins are
presumed to involve hydrogen peroxide hydrolysis of the serine ester within
the hydrolase catalytic triad. The peracid thus formed reacts with halide ion
generating the reactive halogenating species (van Pee, 1996). In light of these
observations, it is quite possible that the halogenating activity of these proteins
is an experimental artifact and not representative of their biological function.
Genes encoding halogenases participating in secondary metabolic
pathways have been identified only recently. The encoded enzymatic activities
have been experimentally verified by either complementation or gene
disruption. Therefore, the discovery of several loci within the pyoluteorin
gene cluster that may encode halogenases is significant. Although data
concerning the catalytic mechanism of these enzymes is presently not
available, it is possible that alignment of the deduced amino acid sequences
will identify invariant residues that may be required for catalytic activity.
3.2 RESULTS AND DISCUSSION
3.2.1 Nucleotide sequencing of the pyoluteorin region
Nucleotide sequence analysis was initiated on genomic DNA of Pf-5
flanking each of the Tn5 insertions. One of the Tn5 inverted repeats and the
flanking Pf-5 genomic DNA was cloned from each of five Tn5 mutants
(Figure 3-3, insertions 4236, 4296, 4128, 4274, and 4175). The nucleotide sequence
57
of the genomic DNA flanking each Tn5 insertion was obtained via an
oligonucleotide primer complementary to nucleotides 18-37 of the Tn5
inverted repeat sequence (Gen Bank accession no. L19385). One of these
sequences (insertion 4274) showed significant similarity to genes encoding
the erythromycin polyketide synthase (PKS) of Saccharopolyspora erythraea.
Because pyoluteorin is a polyketide-derived metabolite (Cuppels et al, 1986;
see Chapter 2), a PKS should be required for pyoluteorin biosynthesis.
Therefore, initial sequence data was consistent with the hypothesis that
insertion 4274 was within a pyoluteorin biosynthesis gene. Sequence analysis
was continued with wild-type DNA of Pf-5 subdoned from cosmid pJEL1938,
which contained the 18.4 kb region depicted in Figure 3-3.
4296
4236
t
EcoR1 EcoRl
Hind111
EcoRl
I
orf-S
pitA
p/tB
4128
t
4274
4175
Hind!!!
4366
I
EcoRl
EcoRl
1
pltC
pltD
1.3 kb
Figure 3-3. Genomic region required for pyoluteorin production and the
biosynthetic genes identified by nucleotide analysis. All transposon insertions
are numerically labelled. Tn5 insertion locations are designated by black
diamonds. The orientation of a Tn3nice reporter insertion (4366) is is indicated
by the open flag. Loci names are below the corresponding ORFs.
Four complete ORFs in addition to a partial ORF were identified by
codon preference analysis (West and Iglewski, 1988). The inferred peptide
sequences of the two largest ORFs (pltB and pitC) exhibit extensive similarity
to several PKSs. The identification of several functional domains within
P1tB and P1tC that are required for polyketide assembly is convincing evidence
for the assigned function of these proteins. The inferred gene products of
pltA, pltD, and orf-S share sequence similarity to other halogenases. Because
halogenases are a poorly characterized class of enzymes, however, enzyme
function cannot yet be predicted from sequence similarity alone.
58
3.2.2 Identification and sequence analysis of the pyoluteorin PKS
Nucleotide sequence analysis of pltB and pltC. Two ORFs, pltB (7.4 kb)
and pltC (5.3 kb), which encode proteins 2458 and 1774 amino acids in length,
respectively, were identified within the pyoluteorin biosynthesis gene cluster
and share sequence similarity to known PKSs (nucleotide sequence data for
pltB and pltC are in Appendices 2 and 3, respectively). The translation initiation
sites for both pltB and pltC were assigned based on the existence of postulated
Shine-Dalgarno sequences 8 and 6 bp, respectively, upstream from the
identified start codons. It is unlikely that an alternative translation initiation
site exists upstream of the identified pltB start codon since it is immediately
preceded by an in-frame stop codon. Although it appears that pltB and pltC
are separated by 48 nucleotides (nt), a possible alternative pltC start codon
exists 12 by downstream of pltB. A pltC gene initiated from this alternative
start codon would encode 12 additional amino acid residues on the N-terminus
of P1tC. However, no convincing Shine-Dalgarno sequence exists immediately
preceding this alternative start site. Nevertheless it is possible that the two
ORFs may be translationally coupled, given the short distance between the
end of pltB and this possible pltC initiator codon (McCarthy and Gualerzi,
1990).
Comparison of pltB and pltC nucleotide sequences with entries in either
the EMBL or Gen Bank databases revealed that these ORFs exhibited
similarities to several known Type I PKSs. At both the nucleotide and amino
acid level, the most significant alignments of pltB and pltC were found with
the eryA gene of S. erythraea, which encodes the PKS required for erythromycin
production (Cortes et al, 1990; Donadio et al, 1991). Conserved amino acids
found in the catalytic sites of several PKSs are also present in the deduced
amino acid sequences of PltB and PltC and define catalytic domains within
these proteins. These results suggest that both PltB and PltC possess enzymatic
functions necessary for polyketide assembly.
59
Modular organization of the pyoluteorin PKS catalytic domains. The
genetic organization of the Pf-5 pyoluteorin PKS exhibits features common
to both bacterial and fungal Type I PKSs. Three KS, AT, and ACP catalytic
domains and a single functional KR domain are present in P1tB and PDC
(Figure 3-4). Two modules are present within P1tB. Of these, module 1 (1013
aa) is located at the N-terminus of P1tB and contains only a core set of
domains. Module 2 (1424 aa), located in the C-terminus of P1tB, contains an
apparently non-functional KR domain in addition to a core set of domains.
A single module (1774 aa) is present within PDC and contains the only
functional KR domain identified within the this PKS as well as a presumed
non-functional dehydrase (DH) domain. The simplified domain organization
of module 1 and the presence of inactive catalytic domains (i.e. KR in module
2 and DH in module 3) have precedence in bacterial systems (Aparicio et al,
1996; Donadio et al, 1991). Nevertheless, several features of the pyoluteorin
PKS such as the absence of a loading or thioesterase (TE) domain and a
single module within PltC, are attributes normally associated with fungal
PKSs.
ACP-2
ACP-1
KS-1
P1tB
KS-2
AT-1
AT-2
a
module 1
1
module 2
ACP-3
pltc Ili
KS-3
Naval
AT-3
I
1
400 aa
module 3
Figure 3-4. Modular organization of the functional domains identified in
P1tB and PltC. The individual domains within each of the three modules are
designated by open boxes and are labelled according to their assigned function.
Stippled boxes represent the non-functional KR and DH domains.
Interdomain regions are represented by solid lines. Abbreviations are as
follow: KS, ketosynthase; AT, acyltransferase; CLF, chain length determining
factor; KR, ketoreductase; ACP, acyl carrier protein.
60
Catalytic domains within the pyoluteorin PKS. Catalytic domains were
identified within the pyoluteorin PKS modules using ProfileGap analysis as
well as multiple sequence alignments for identified domains within known
PKS and FAS sequences (Figure 3-5). Well-defined modules such as KSs
could be identified by either method. However, poorly defined regions or
regions in which diagnostic sequence motifs were not found could be identified
only by using a statistical analysis of the Profile Gap alignments. Active-site
motifs and the invariant residues within the protein sequence could be
identified only by using multiple sequence alignments. The C-terminal and
N-terminal limits to any given domain also differed depending on whether
multiple sequence alignment or Profile Gap analysis was used to define the
domain (data not shown). It was not possible, therefore, to define the precise
length of each domain within the pyoluteorin PKS. Although the statistical
capability of Profile Gap analysis does not take into account all statistical
properties of biological sequences (Lipman et al, 1984), when used in
conjunction with multiple sequence alignments, this alignment tool was
invaluable in identifying regions of limited similarity within poorly
characterized domains.
A KS domain of approximately 415 as is present within each of the three
modules of the pyoluteorin PKS. An active-site Cys residue within the
sequence motif GPxxxxxxxCxSxL (Cortes et al, 1990) is present roughly 170
residues from the N-terminal start of each KS domain. Two His residues
found to be invariant in both Type I and Type II PKSs (Aparicio et al, 1996)
are also present within the KS of modules 1, 2, and 3. It has been suggested
that one of these His residues may increase the reactivity of the active-site
Cys by acting as a general base (Aparicio et al, 1996).
Active-site serine residues exist within the PxxxxGHSxGxxxA sequence
motif (Cortes et al, 1990) for each of the pyoluteorin PKS AT domains. The
AT domain of module 3 exhibits a Pro)Ala substitution within this sequence
motif. Also present within the AT domains of modules 1 and 3 are specific
His, Arg, and Gln residues that have been identified in all Type I PKSs and
FASs (Aparicio et al, 1996) and that are involved in the active-site structure
of the malonyl-CoA:acyl carrier protein transacylase of E. coli (Serre et al,
1995). In contrast to modules 1 and 3, the assigned AT region of module 2
exhibits limited similarity to the sequences of known AT domains and
61
Figure 3-5. Deduced peptide sequence alignment for the three PKS
modules encoded by pltB and pltC. Each functional domain is delimited by
a solid black bar above the aligned sequences. The dashed regions of this bar
designate the approximate boundaries for each domain. Profile sequences
surrounding active-site regions within each identified domain are also located
above the aligned sequences. The consensus sequence located below the
aligned pyoluteorin PKS modules was generated from conserved residues
(identified by uppercase text) within each domain. A 464 aa region from P1tC
has been eliminated for clarity and is designated by the double headed arrow.
Individual aa residues discussed in the text are in bold type.
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63
possesses several anomalous features. The Pro and His residues found in
the motif surrounding the active-site serine have been substituted by Ala
and Phe residues, respectively. Furthermore, of the His, Arg, and Gln residues
mentioned above, only the Arg is present in the deduced protein sequence.
Two sequence insertions of 8 and 64 amino acids C-terminal to the active-site
Ser are also apparent from the multiple sequence alignment with known
AT domains.
The enzymatic function of all ACP domains requires covalent attachment
of 4'-phosphopantetheine to the Ser residue within an LGxDS sequence motif
(Cortes et al, 1990). The Gly and Ser residues of this motif are present within
the C-terminal regions of modules 1, 2, and 3, but substitutions for the Leu
and Asp residues are present within the assigned ACP domains. In both
modules 1 and 2, an Asp -*Ser substitution has occurred. Additionally, a
Leu>Met substitution has occurred in module 2 as well as a Leu -*Tyr
substitution in module 3. Apparently these substitutions do not compromise
enzymatic function, because based on the model for polyketide biosynthesis,
functional ACP domains are an absolute requirement of the PKS. Consistent
with this conclusion, it is generally recognized that sequences flanking the
ACP active site motif do not exhibit noticeable sequence similarity among
PKSs.
A KR domain was identified within a 178 amino acid region between
the identified AT and ACP domains of module 3. The invariant GxGxxGxxxA
motif, which is found in all functional KR domains (Aparicio et al, 1996)
and is believed to form part of the binding site for the cofactor NADP(H)
(Scrutton et al, 1990), is present in the module 3 KR domain. In contrast, an
analogous region within module 2 of PltB exhibited very limited similarity
to the C-terminal region of a KR profile sequence. Extensive substitutions of
conserved KR amino acid residues within this module 2 region make it
unlikely that this domain is functional within the pyoluteorin PKS. Therefore,
the pyoluteorin PKS appears to contain only one functional KR domain
residing in module 3.
Within module 3 of the pyoluteorin PKS, a 558 as region between the
AT and KR domains was examined for the presence of DH domain. Statistical
analysis of the alignments between this region and a DH profile sequence
suggests that limited sequence similarity does exist. Although DH catalytic
64
domains are located typically between AT and KR catalytic domains in Type
I FASs (and some PKSs), DH domains are not well characterized, and it is
difficult to identify a DH domain on the basis of sequence homology alone.
No TE domain was identified within either P1tB or PltC. Although TE
domains are not strictly required within Type I PKSs, those that have been
identified are located at the C-terminal end of the last ORF (Donadio and
Katz, 1992; Swan et al, 1994; Yu and Leonard, 1995). It is unlikely that a TE
domain exists at the end of PltC since only 30 nt exist between the C-terminus
of the ACP and the translational stop codon.
Non-catalytic regions of the pyoluteorin PKS. The differences in lengths
of the interdomain regions (IDRs) within P1tB and P1tC accounts for the
variable module sizes within the pyoluteorin PKS. Whereas the IDR between
the KS and AT within modules 1 and 3 are approximately 100 residues in
length, the same region within module 2 contains 248 aa residues. The
KS/AT IDR of modules 1 and 3 align with the N-terminal portion of the
KS/AT IDR of module 2. The AT and KR domains of module 3 are separated
by 558 aa and the AT to ACP distances in modules 1 and 2 are 90 and 260
residues, respectively. A qualitative, composite alignment derived from the
individual pair-wise alignments for the C-terminal IDR of each module
suggests limited sequence similarity immediately preceding the ACP domains.
However, the disparity among the C-terminal IDR for modules 1, 2 and 3
precludes further comparison.
The pyoluteorin PKS does not possess a loading domain. Initiation of
polyketide assembly requires formation of an enzyme-substrate complex
between an activated starter unit (e.g. acetyl CoA) and the first KS domain.
Within bacteria, enzyme-substrate complex formation was attributed initially
to a loading module comprised of an extra AT and ACP domain located
between the N-terminus of module 1 and the first KS domain (Donadio et
al, 1991). Characterization of the rapamycin PKS indicates, however, that
N-terminal loading modules are not restricted to AT and ACP catalytic
domains (Aparicio et al, 1996). In the rapamycin PKS, three catalytic domains,
65
a carboxylic acid:CoA ligase, an enoyl reductase, and an ACP, are located at
the N-terminus of the first PKS module and are likely involved with activation
and reduction of the initial substrate (Lowden et al, 1996). The activated
starter unit is then transferred directly to the first KS domain for polyketide
assembly. In contrast to the bacterial PKSs, loading modules have not been
reported within fungal Type I enzymes. Furthermore, the heterologous
expression of 6MSAS in the bacterium Streptomyces coelicolor A3(2) (Bedford
et al, 1995) implies that polyketide initiation can occur through an alternative
mechanism within fungal Type I PKSs. Although no evidence for a loading
module within the pyoluteorin PKS was found, this enzyme may possess an
inherent, but as of yet unidentified, transferase activity required to initiate
polyketide biosynthesis. It is also possible that the initial substrate is directly
transferred from another protein functioning earlier in the pathway. A gene
encoding a CoA ligase within the pyoluteorin locus has tentatively been
identified (N.L. Chaney and J.E. Loper, unpublished) and may be involved
in the activation and subsequent transfer of the starter unit onto the PKS.
Alternatively, the pyoluteorin PKS may utilize the transferase activity from
a distinct, yet related pathway. The suggestion that a malonyltransferase is
required for both fatty acid and polyketide biosynthesis within two
Streptomyces species (Reville et al, 1995; Summers et al, 1995) are of particular
relevance to this possibility.
Polyketide assembly of the pyoluteorin resorcinol moiety. The presence
and organization of catalytic domains identified within the pyoluteorin PKS
correspond to resorcinol ring formation within pyoluteorin. The addition of
each C2 extender unit and modification of the resulting 13-carbonyl structure
could be catalyzed by the combined activities within the individual modules
of the pyoluteorin PKS (Figure 3-6). Although resorcinol ring synthesis within
pyoluteorin requires dehydration at the 3-position, no functional DH domain
was clearly identified. Therefore, dehydration may occur after release of the
fully extended polyketide from the PKS. Tautomerization and the subsequent
aromatization of I could very well be non-enzymatic processes due to the
resonance stabilization energy resulting from this structural rearrangement
(Armstrong and Patel, 1993). Release of the fully extended substrate from the
66
PKS apparently does not require a dedicated TE domain, as none was found
at the C-terminus of P1tC. However, a separate gene with similarity to several
TEs has been identified downstream of pltB and pltC and may terminate
polyketide biosynthesis (N.L. Chaney and J.E. Loper, unpublished).
Alternatively the PKS alone may terminate polyketide biosynthesis by
cyclization (perhaps catalyzed by the unusual AT domain of module 2) as
has been suggested for 6MSAS (Beck et al, 1990; Spencer and Jordan, 1992).
PItB
module 1
module 2
PltC
module 3
..........
OH 0
H
Pyoluteorin
Figure 3-6. Proposed route for formation of the pyoluteorin resorcinol
moiety. The structure of the starter unit derived from proline is unknown
and is designated as R. Condensation of each C2 extender unit and the
modification of the 0-carbonyl are catalyzed by each of the three modules
within the PKS. Release of the fully extended substrate is hypothesized to
occur by condensation between the two carbon centers identified by the arrow.
The aromatization of the resulting structure (conversion of I -4 II) is likely a
non-enzymatic reaction.
67
3.2.3 Identification and sequence analysis of the pyoluteorin halogenases
Nucleotide sequence analysis of pltA, pltD, and orf-S. Three ORFs in the
pyoluteorin biosynthetic gene cluster, designated pl t A, pltD , and orf-S exhibit
similarity to loci encoding chlorinating enzymes required for tetracycline
biosynthesis (cts4) within Streptomyces aureofaciens (Dairi et al, 1995) and
for pyrrolnitrin biosynthesis (prnC) within Ps. fluorescens (Hill et al, 1994).
pitA (1.3 kb) and pltD (1.6 kb) were sequenced completely; both genes are
transcribed in the same orientation as those encoding the pyoluteorin PKS
(nucleotide sequences for pitA, pltD, and orf-S are in Appendices 4, 5, and 6,
respectively). pitA and pltD are very closely linked to the PKS genes, and
putative Shine-Dalgarno sequences are present immediately upstream of
the start codon for each ORF. In contrast, a coding region exhibiting similarity
to both pltA and pltD is located at the extreme 5' end of the pyoluteorin
region (orf-S) and is transcribed divergently to the other genes within the
cluster. Because orf-S is at the terminal end of the pJEL1938 cosmid insert,
the complete nucleotide sequence was not determined. A Shine-Dalgarno
sequence is not present within the orf-S sequence; however, the first four
bases within this gene overlap the putative regulatory gene pltR (see Chapter
4) suggesting that translation of the gene products may be translationally
coupled. Transposon insertions within pitA and pltD inactivate pyoluteorin
production (Figure 3-1) indicating a role for their translated products in
pyoluteorin biosynthesis.
Comparison of PItA, PltD, and Orf-S to halogenases involved in
tetracycline and pyrrolnitrin biosynthesis. Dairi et al (1995) have identified a
1.4 kb ORF (cts4) encoding the chlorinating enzyme required for tetracycline
biosynthesis. An A+G rich region (AGAAGAG) between positions -20 to -14
upstream from the postulated start codon was identified as the putative
Shine - Dalgarno sequence of cts4. Several discrepancies between the nucleotide
and deduced peptide alignments for cts4 , pltA, and pltD prompted a closer
examination of the cts4 nucleotide sequence. In light of the unconvincing
placement and sequence of the Shine-Dalgarno ribosomal binding site
proposed for cts4 and because the Pf-5 sequences aligned to a region extending
68
approximately 300 nts 5' of cts4, it was considered likely that the beginning
of the cts4 ORF was incorrectly identified. Indeed, codon preference analysis
of the tetracycline biosynthetic gene cluster revealed that the reported sequence
contains a frame shift error (data not shown). Therefore, the Cts4 protein
has had an additional 100 amino acids appended to the N-terminal region
for all subsequent sequence alignments.
PrnC
megkspane. hclarthFDVII
10e3maGtql4 gaiLkIgAft
Cts4 mtdttadqtr hgdrpYDVVI
ialsGlaGtmL geiLAIChgfr
VliiEessbP RftICHtas/P WralmnriIa dRYgIrildh itsfystgry VasSt.Gittr
ImILDgahhP RfaV0ItstIr cOlvvLrlIs dRYgc.Xian lasfgdylan Vssah.GgSs
orf-S
angyIPVII
1.0aGiaGalt gavLAAmiln
PltD ...mndvqsg kapehYDIII
agnaisvimL eacLArnkyr
VULDeaghP RteVOSaatP eselLrIls WdIptiay lagpdkiigh VgaBacGiXl
VglArnrgptP pdltnatIP yTsmiFelIa dRYOOSikn iartrdiggk VapS.sGvIck
Pick
msdhdYDVVI iGg0paGstM asyLAXegvk
cavrEkelfe ROMAISaIVP aTtpviaeIg vmekI.Ekan fpkkfgaawt sadagpedItm
Consensus
nor-I -0-G--G- -- --- LAK- - -- V--L
P R--VOS--/P -T---L--I- - RY -I -t
V--S--G-I-
PrnC
nF.GFvFlikp ggehdpkeft QcvIpelpag pesSYYlopv DaYLAgakik
Cts4 nF.GFartird geepdpnets OfrIpsiv.g naalFFItgEt DsYWhaAvr
yOcledsOktt Vteyh.adol giraVttagge r...ftgrym IDcggprapL
yOcdarQyyr Venie.fddg OftesgadOs t...vraryl VDaragfrspL
orf-S
gP.arattlige napsspdhl. ...Vatclkv pesILFIcIU DyFaLaiAlk
heaesrgnik Irrpa.akl.
PltD nL.GFLYHdr sravdIggal GInVpash.. genILFItrEd
DaYLLaakig ygagIveidn spevl.veds glavatalOr w...vtadfm VOgaggggvL
PitA gFgILdhdfr saeilfnerk QegVdrdf.. .tfltvdttgkf
DriLLehkgs lOakvfOgve Iadveflapg nVienaklak rsveiWOanv VDasgrnvIL
Consensus - F -GP -PH
Q--V
SLFS-D- D-YLL--A­ -0- --0­
V-v- --0­
VD
PrnC
atkfIcLreep crfktheral ythmlgvkpf ddifkvlopqr w. awhegt
IhheFeggta wvipFretapr atnnIvIlegl gldprvIp.k tdisagdeFd
Cts4 argIgLreep arlkhhersi ftlattvgvdsi ddhvdmpael
rppypandst mhhiFergwm wiipFnobsg atnplcSVgi glderrYpar la:1143.0,Fr
orf-S
P1tD argagLvaga stqktrtlef athmlgvvpf decvggd...
.fpggtehggt lhhvFdpgwv gvipPlalx$1 arnplv0V1v alredICp.. .strtigdqvla
PitA grrIgLrokdtvfngfaihs wfdnfdrksa cgs
pdk vdyiribflp mantWvwgip itetitaVgv vtqkgnYt.n sdatyeefFw
Consensus
P - - - -­
BY
Y
F-
PrnC eflarfpeig aq?rdavpVR dWyktdrIgd asnacygetrY cLmlhangfi
Cts4
shydrfpavg rgLkgarsVA eWvrtdtmgv ssartvgerW cLmshaagti
DpIFsrglen
DpIFIrglsn
orf-S
PltD glielypglg rhLagarrvIt eterlrqpprg vyrtalerrC
IMfdegaaan DIIFsrklan
PitA eavkcrenlh daLkasegVI prldceadysy gmkevcgdeF vLigdaartv
DpiFsagvav
Consensus
VI -H
L
D--F
tavtihalaa rlikkIrdiD FsperFeyie rlgqicIldhn
tceiinalme rbashlredD FaverFayve elegglIdea
aaelvlalah rlikAahsgD YrapaLndfv Itgdsiials
alnsariasg diiekvknnD FslosaFtbye gmirngiknw
A- -D F-
-F
PrnC ddfveccyta FsdfrkWdaf hrlw...avg tiltmarlvg aharfrasrn
Cts4
dklvnnsfis FahypLWnsa fri4...asa eviggkriin altrtketgd
egdldhLdnd ppylOylcsal meeyyglind skaevEaysa grkpadeaaa
dahcgaLd.d npypOlwcp. ldfykeafde ItelcEavda ghttaeeaar
orf-S
P1tD drialaayva FrdpeLWnaf arvwllgsia atitarkind afakdldpry
fdeidgLaed ....0fwega yrgykdilnt tIgIcDdyks akvsaahaaa
PitA yefit...ly YrIniLFtaf vg
dpry rldilgLIgg dvysOkrlev lammaiiaa vesdpEhlwh kylgdmgypt
Consensus
F - - - -LW
L­
E
PrnC
rihaliderd fatertafgfgy citgdkpqln nskyslItam rbaywtqtra
paevkkyfdy nianfallkay
ittriglalk
Cts4
llegrvread wmIpalgin. ..dsdthhin ptadkmi... riaewatghh rpeir
..... ....elaasa eevraamrvk p
orf-S
PltD sifaelanas fvtpifdfa.
nphar vycattIrkl kalwtvplmqv nsevgrlify refrkpaIrk es.
PitA akpaf
Consensus
Figure 3-7. The deduced peptide sequence alignments for the putative
halogenases PitA, PltD, and Orf-S with PrnC and Cts4. Residues in UPPER
CASE letters signify conserved substitutions. Those in BOLD UPPER CASE
letters are invariant among the available sequences (only the N-terminal
151 residues are available for Orf-S). The consensus sequence was calculated
with a plurality of 4. Residues involved in forming necessary secondary
structure for binding NAD co-factors are indicated by asterisks (*) within the
the boxed region.
69
Multiple sequence alignment of the Cts4 and PrnC halogenases with the
three putative halogenases of the pyoluteorin gene cluster show a considerable
amount of similarity throughout the protein sequences (Figure 3-7). Of the
78 conserved residues among the deduced proteins, the majority are
concentrated in the N-terminal region. The most intriguing aspect of these
sequences is that, with the exception of P1tD, they each possess a core
GxGxx(G/A) sequence believed to form the 13a--f3 secondary structure
required for NAD co-factor binding within many oxidoreductases (Scrutton
et al, 1990). The NAD co-factor binding site had not been recognized previously
within the reported halogenase sequences. This result suggests that the
halogenases are a subclass of the oxidoreductase enzymes.
Speculation on the parameters of biological halogenations. While there
appear to be no obvious mechanisms that would account for biological
halogenations, it is possible to consider some of the chemical parameters for
the enzyme substrates that may guide future research. These points are merely
speculative at this point; although the genes have been identified, subcloned,
and theoretically can be easily expressed, it is virtually impossible to undertake
any mechanistic studies without knowing the substrate requirements of the
enzyme.
The solvation of chloride (i.e. C1) within protic solvents generally makes
it a fairly poor nucleophile. Therefore, chlorination is likely to occur via an
electrophilic addition or substitution mechanism. Such a reaction would
require the electrophilic equivalent of a chloronium species (i.e. Cl'). Although
results from previous research involving the haloperoxidases suggest this
species might be HOC1, this conclusion is probably not biologically relevant
because in vitro halogenation of a susbtrate requires the use of high H202
concentrations. Within biological systems, reactive oxygen compounds like
1-1202 are efficiently eliminated; therefore, it is unlikely that H202 could reach
sufficient concentrations in vivo to effect halogenation. Alternatively, a
chloronium electrophile may arise directly from metal-catalyzed reaction
with molecular oxygen.
If the halogenation reaction does involve an electrophilic addition or
substitution, the substrate necessarily must be a good nucleophile. As a ic­
70
excessive heterocycle, pyrrole is quite facile at undergoing electrophilic attack.
Furthermore, the aromaticity of the pyrrole carbon skeleton allows charge
delocalization throughout the ring and could conceivably assist in stabilizing
reactive intermediates and accounting for adding of chlorine atoms at the l­
and the 3-positions on the ring. Within the pyoluteorin pathway, therefore,
halogenation is likely to occur after the pyrrolidine moiety has been oxidized
to the pyrrole.
3.3 CONCLUSIONS
Several loci have been identified within the Pf-5 pyoluteorin biosynthetic
gene cluster by sequence analysis, and the function for each of the encoded
gene products serves either in a biosynthetic or a regulatory capacity. PltB
and PltC encode a Type I PKS required for assembly of the pyoluteorin
resorcinol moiety. Because many PKS sequences have been analyzed and the
catalytic mechanisms of these enzymes are well understood, there is little
doubt as to the assigned function of PltB and PltC. Nevertheless, the absence
of a loading domain and the unusual AT domain make this PKS worthy of
further study.
It appears that three loci within the pyoluteorin gene cluster potentially
encode halogenases. Furthermore, sequence alignment of PltA, PltD, and
Orf-S with two known halogenases has identified a potential NAD co-factor
binding region that may play a role in catalytic activity. The presence of
three halogenases in the pyoluteorin region is quite surprising because only
two chlorine atoms are present within pyoluteorin. Several possible
explanations might account for this observation: i) Pf-5 may produce several
(but as of yet unidentified) pyoluteorin homologues that differ only in the
extent of halogenation. ii) One of the halogenases may be required for the
biosynthesis of the presumed P1tR co-inducer. iii) One of the halogenases
may be non-functional. iv) There may be a cryptic chlorination involved in
pyoluteorin biosynthesis. v) Several alternative routes to pyoluteorin may
exist requiring chlorination of various precursors. vi) orfS may not be
involved in pyoluteorin biosynthesis, as no mutants in this region have
been derived. In the midst of this speculation however, it should be stressed
71
that the catalytic functions for Orf-S, PltA, and PltD have been assigned
tentatively since this class of enzymes is relatively unknown (nucleotide
sequence data has been reported for only two other halogenases). In light of
the novelty of these enzymes, characterization of the halogenation reaction
required for pyoluteorin biosynthesis promises to be very exciting.
3.4 EXPERIMENTAL METHODS
Nucleic Acid Manipulations. Six DNA fragments from the cosmid
pJEL1938, which contains the genomic pyoluteorin biosynthetic region of Ps.
fluorescens Pf-5 (Kraus and Loper, 1995), were subcloned into pUC19 using
the restriction sites identified in Figue 3-3 and transformed into Escherichia
coli DH5a using standard methods (Sambrook et al, 1989; Ausubel et al,
1992). E. coli strains were routinely cultured in Luria-Bertani medium at 37
°C (Sambrook et al, 1989). Double stranded DNA was isolated for automated
sequencing by either QlAprep -Spin plasmid mini-prep kits (Qiagen,
Chatsworth, CA) or by Promega Wizard maxi-prep kits (Promega, Madison,
WI) and quantified by UV spectrophotometry.
DNA Sequence Analysis. Sequence analysis of each subcloned region
was initiated from the pUC primer sites and subsequently completed by
"primer walking" in each direction across the entire length of the subclone.
Automated DNA sequence analysis and primer synthesis were performed by
the Center for Gene Research Central Services Laboratory at Oregon State
University and by Macromolecular Resources Sequi-net Division at Colorado
State University using dideoxynucleotide chain-termination (Sanger et al,
1977) on Applied Biosystems models 373A and 377 sequencers. Compilation,
manual editing, and analysis of the sequence data were done using the
University of Wisconsin Genetics Computer Group (Genetics Computer
Group, 1991) programs (GCG). Open reading frames were identified within
the DNA sequences by codon usage analysis utilizing the codon preference
72
frequencies compiled for Ps. aeruginosa (West and Iglewski, 1988). Protein
functions were initially assigned on the basis of sequence similarity detected
upon comparison to the Gen Bank or EMBL databases using BLAST (Altschul
et al, 1990) and FASTA (Pearson, 1990) algorithms, respectively.
Deduced protein sequence analysis. The catalytic domains within P1tB
and P1tC were identified by multiple sequence alignments (Pile Up) and by
profile sequence alignments (Profile Gap) using standard parameters. The
alignment analysis used various domain sequences from the following
representative Type I polyketide and fatty acid synthases (Gen Bank accession
numbers are in parenthesis): the erythromycin PKS of Saccharopolyspora
erythraea (M63676 and M63677), the rapamycin PKS of Streptomyces
hygroscopicus (X86780), the soraphen A PKS of Sorangium cellulosum
(U24241), the mycocerosic acid synthase of Mycobacterium tuberculosis
(M95808), the 6-methylsalicylate synthase of Penicillium patulum (X55776),
and chicken fatty acid synthase of Gallus gallus (J04485). Interdomain sequences
within the modules of P1tB and P1tC were manually aligned after comparison
of several Gap generated alignments for these regions.
Statistical analysis of the alignment between the domains in either P1tB
or P1tC and the profile sequences above was performed using the Best Fit
algorithm with the RANDOMIZATION command option. Scores were
calculated for each alignment and compared to the average alignment score
for ten random sequences that maintained base composition and length.
Scores that were 5 standard deviations above the mean were considered
indicative of significant similarity. Those scores that were 1-5 standard
deviations above the mean were considered of limited similarity.
Conserved residues were identified within PltA, P1tD, and Orf-S by
multiple sequence alignment with Cts4 of St. aureofaciens (D38214) and PrnC
of Ps. fluorescens (U74493). The putative NAD co-factor binding site was
identified manually.
73
Chapter 4. Nucleotide Sequence Analysis of a Putative Transcriptional
Activator Regulating Pyoluteorin Biosynthesis
4.1 INTRODUCTION
Secondary metabolite production is regulated by many factors including
cell growth rate, extracellular signalling metabolites, and nutritional factors
such as carbon, nitrogen, and phosphate sources (Demain et al, 1983; Grafe,
1989; Liras et al, 1990; Horinouchi and Beppu, 1992; Vining, 1995). These
regulatory factors do not necessarily operate independently, nor does each
factor elicit an identical response for each antibiotic pathway that may exist
within a single organism. For example, S. coelicolor A3(2) cultures containing
nitrate as the sole nitrogen source exhibit differential production of the
pigments undecylprodigiosin and actinorhodin in late log phase and
stationary phase, respectively (Hobbs et al, 1990). The complex regulatory
networks affecting antibiotic production appear to operate on two distinct
levels. Global regulatory loci have pleiotropic effects that can either directly
or indirectly regulate antibiotic production, whereas regulatory loci directly
linked to biosynthetic genes generally mediate gene transcription within
that gene cluster (Martin, 1992; Hutchinson et al, 1993).
Antibiotic production within Pf-5 is globally regulated, in part, by the
sigma factor RpoS (as), which controls the transcription of many genes in
response to starvation or the onset of stationary phase (Kolter et al, 1993;
Lange and Hengge-Aronis, 1994; Hengge-Arronis, 1996). Inactivation of the
rpoS locus within Pf-5 results in the over-production of both pyoluteorin
and the antifungal metabolite 2,4-diacetylphloroglucinol (Sarniguet, et al,
1995). Concurrent research involving a Ps. fluorescens strain that is strikingly
similar to Pf-5, has demonstrated that increasing the gene dosage of the
essential "housekeeping" sigma factor RpoD (a') also resulted in the over­
production of both antibiotics (Schnider et al, 1995). Both results support the
hypothesis that RpoS and RpoD act in concert to regulate secondary
metabolism possibly via competition for RNA polymerase.
Antibiotic production within Pf-5 is also globally regulated by a twocomponent regulatory system involving the sensor kinase ApdA (Corbell
74
and Loper, 1995) and the response regulator GacA (Laville et al, 1992). Within
the two-component paradigm, an ATP-dependent auto-phosphorylation of
ApdA (the sensor kinase) occurs in response to an environmental stimulus.
Subsequently, ApdA phosphorylates GacA (the response regulator) which in
its activated form, is capable of interacting with the target gene sequences to
induce gene transcription (for general reviews of two-component regulatory
systems, see Albright et al, 1989; Stock et al, 1990; Parkinson and Kofoid,
1992). Mutations within either apdA or gacA result in several pleiotropic
effects including complete loss of antibiotic production. Although both ApdA
and GacA are necessary for gene expression in Pf-5, neither the environmental
stimulus nor the genes targeted by GacA have been identified.
Antibiotic pathways within other Pseudomonas spp. are locally regulated
by gene products that are translated concurrently with the required biosynthetic
genes (see Chapter 1, section 1.2). Recently, the gene cluster responsible for
2,4-diacetylphloroglucinol production in Ps. fluorescens Q2-87 has been
characterized (Bangera and Thomashow, 1996), and a 6.5 kb DNA fragment
was shown to confer 2,4-diacetylphloroglucinol production within
heterologous hosts. However, larger fragments containing flanking DNA
sequence were unable to transfer antibiotic production. A linked gene encoding
a 23 kDa repressor protein has since been identified via sequence analysis
and accounts for the lack of expression in heterologous hosts (Thomashow
et al, 1996).
In the course of sequencing the pyoluteorin biosynthetic gene cluster,
an ORF was identified that is divergently transcribed from and located
upstream from the pltABCD loci. The similarity of this gene to the LysR
family of transcriptional activators (Henikoff et al, 1988; Schell, 1993) and the
presence of a putative operator region upstream of pltA provides compelling
evidence at this time for the role of the translated protein in the regulation
of the pyoluteorin biosynthetic genes. Unfortunately, there is no experimental
evidence that inactivation of pltR negatively affects pyoluteorin production
within Pf-5. Therefore, the conclusions discussed below are tentative pending
further genetic analysis of pltR.
75
4.2 RESULTS AND DISCUSSION
4.2.1 Nucleotide sequence analysis of pltR
A 1.0 kb ORF (pltR), divergently transcribed from p1tABCD (Figure 4-1),
was identified within the Pf-5 pyoluteorin gene cluster (nucleotide sequence
of pltR is in Appendix 7). The translation initiation site as well as a possible
Shine-Dalgarno sequence (positions -11 to -8 relative to the start codon) were
identified 766 nt upstream of pltA. An alternative start codon and ShineDalgarno sequence exist approximately 60 nt upstream of the identified
initiation site, but it is unlikely that this is the site of translation initiation
due to the presence of an in-frame stop codon 12 nt downstream. No
alternative start sites are present within 100 nt downstream of the identified
initiation site. Codon preference analysis detected an atypical codon usage
and low G+C bias patterns within the first 500 nt of pitR. Whereas other
genes within the pyoluteorin cluster exhibit G+C bias in the range of 75-85%,
G+C bias for the pltR 5' region was approximately 35-45%. The low G+C
content within pltR may have a regulatory consequence for pyoluteorin
production (via infrequently used tRNAs) or it may simply reflect a residual
structural motif that is required for regulatory function.
Hinal
1
EcoRl
4296
tEcoRI
EcoRI
4236
t
I
1
4128
4274
I t
4175
HindlIl
f
4366
I
EcoRI
EcoRI
I
1
sa mat-sunamimssimummounemulm,minammangsansm..
pltR
pitA
pltB
pltC
pltD
1.3 kb
Figure 4-1. Genomic region required for pyoluteorin production and the
regulatory gene identified by nucleotide analysis. All transposon insertions
are numerically labelled. Tn5 insertion locations are designated by black
diamonds. The orientation of a Tn3nice reporter insertion (4366) is is indicated
by the open flag. Shaded ORFs were described in Chapter 3.
76
4.2.2 Similarity of P1tR to LysR-type transcriptional activators
The deduced P1tR peptide sequence (344 aa) exhibits significant similarity
to amino acid sequences of more than 20 members of the LysR family of
transcriptional regulators (Henikoff et al, 1988; Schell, 1993). LysR-type
regulators influence an extremely wide range of phenotypes and have several
common features: i) They generally act as transcriptional activators and require
a co-inducer for activity. ii) The gene encoding a LysR homolog is often
closely linked to and transcribed divergently from the regulated target gene(s).
iii) A highly conserved N-terminal domain containing a helix-turn-helix
(HTH) motif, a somewhat variable co-inducer binding domain, and a
conserved C-terminal domain are present within all LysR-type regulators.
iv) LysR regulators generally recognize target promoters containing an
inverted repeat within the -75 to -55 region (relative to the transcription
initiation site). v) Expression of lysR- homologs is often negatively autoregulated. Despite these similarities, there are no consistent biochemical
relationships between either the required co-inducers or the proteins encoded
by the regulated target genes.
Based on the initial database search results, the highly conserved Nterminal domain that is characteristic of all LysR-type proteins was readily
apparent within P1tR (Figure 4-2). Further analysis using a calibrated weight
matrix (Dodd and Egan, 1990), predicted with absolute certainty that a HTH
motif exists between residues 20-41 within the N-terminal region of P1tR. In
addition to the N-terminal domain, a co-inducer binding and a C-terminal
domain were assigned via comparison of the P1tR sequence with a 70%
consensus sequence for each of the three domains common to LysR-type
proteins (Schell, 1993). Furthermore, many amino acid residues along the
entire length of a profile sequence compiled from ten LysR-type proteins
representing several phylogenetic groupings (Schlaman et al, 1992) also were
conserved within P1tR.
77
Figure 4-2. Gap alignment of P1tR and a LysR profile sequence. Identified
domains are indicated by bars above the sequence alignment. Domains were
identified from the 70% consensus sequences described by Schell (1993) as
shown. The helix-turn-helix motif (residues 20-41) within the N-terminal
region was calculated using the method of Dodd and Egan (1990). The
consensus sequence was calculated from the PltR-LysR profile alignment
and adheres to the following conventions: Amino acid residues in UPPER
CASE are identical within both sequences. Conservative substitutions are
designated as follows: g, hydrophobic residues (F, W, Y, V, I, L, M); f,
hydrophilic residues (N, D, E).
78
helix-turn-
Sfrnflr.
P1tR
Mk aLgvvndidl YisVtktgSF setgrlLgIp
LysR profile mpevqtdhpe taeasmoqMr hLd.lrqlry FeaVaeenSL traaeeLhVs
Consensus
M- L
S--V
-SS
L S-
helix
PltR
LysR profile
Consensus
ofASstrocSff lef.1g..lf .r.fr.f... to
pssVmRrIns LEkELetcLF nRstkcliLT EtG11F1Eha knisrcighA
qpaVsRqVrr LEdDLgvpLF eRtgrgvkLT EaGeeLwEev qralqaldnA
--V-R-S-- LE-fL---LF -R
LT E-G- S-E­
A
Co-inducer binding
1.i g
1p
p
1
rsEVkEhtas tlGvLkVsaP vaFgr..rhv ap.LLsriLn hhPglkieFs
LysR profile mdEVeElgpg qsGrLwIayP tsLastiywf 1pqLLdafMq qyPdvelsLt
Consensus --EV-E---G- L -13 -P
LL -S
-P
S-
P1tR
domain
P1tR
LysR profile
Consensus
lf..fSdSS
lnD.kaLdps idnvdIcikL gilP..dsnL IptkLadmrr
VicasPeyir
tgDseeLvea lragrIdlaL sgvpprciagL VaveLlqedm VVvvaPnlph
-D -L
I
-L
-P
S -L
V
P
P1tR
LysR profile
Consensus
q4Gcpqt1E. .DLyqbacli hstcsnfSlt WqfkVDgL1K klmpssRlSv
plGpqkavEl aDLqeeefim 1prggrsS..
WrdaVErFwK qagiqpRiSm
-G
E- -DL
W- Vf -S-K
R-S­
C -term. domain
vP
L
P1tR
LysR profile
Consensus
nssEL.1vDg alq.GIGIih aPtwiVhEqI asgqLVsLld
EYceadpqqg
eveDLeavVg lvaaGVGVav vPesvVeDeV epvrLVkLpf
EWmldkraif
--fL---V­ ----GSGS-- P---V-f-S ----LV-L-- ES
P1tR
LysR profile
Consensus
aLYalRar.. ...SsvVpAk trLFiNE1kR sigStpyWdl pfekeipqtl
1LFvaRrhmk rrvSplVqAf weLLaBEavR
11sSldqFq
-LS--R---- ---S--V-A- -LS-fE- R
-S
S
P1tR
LysR profile
Consensus
atlhfdtamh salSrATTlq dksqts
SsATTa.
S-ATT
Figure 4-2
79
4.2.3 A putative PltR binding site within the pyoluteorin gene cluster
Promoters targeted by LysR regulators contain an inverted repeat
sequence centered near the -65 promoter region that exhibits a conserved
TNTNA-N7-TNANA motif (Schell, 1993) and is essential for transcription
mediated by LysR-type activator proteins (Ebright, 1986). An inverted repeat
containing the TNTNA-N7-TNANA motif was identified within the
pyoluteorin gene cluster and is centered 45 nts 5' to the translation start
codon of pltR (Ebright box; Figure 4-3). It should also be noted that sequences
flanking the identified Ebright box are particularly A+T rich, an attribute of
some LysR-regulated promoters (Vale et al, 1991 and references therein).
The proximity of the putative PltR binding region to the presumed
pltR promoter region may serve to auto-regulate expression of PltR, as is
characteristic of LysR regulons. Other potential PltR binding regions within
the pyoluteorin gene cluster were identified by partial similarity to the
TNTNA-N7-TNANA motif; however, these sequences did not possess the
symmetrical inverted repeat characteristic (data not shown).
ATCAATTTTCCCAAAGCCAACTAATCATGGCCCCAGCACTGCTTGACAAACAAAAAGCCT
TAGTTAAAAGGGTTTCGGTTGATTAGTACCGGGGTCGTGACGINACTGTIrTGTTTTTCGGA
pl to
-10
AGCCGTACTAAGGAGGCTGTCATGATCTATTT GTAAT TT GC CTT TACK-1AAATAAGACAC
TCGGClaiTGATTtCTCCGACAGTACTAGATAAACATTAAACGGAAATGTTTTTATTCTGTG
-35
RB S
TAAAAATTCTAAAAGGATTTAGGAATGAAGGCGCTAGGCGTTGTCAATGACATAGACCTT
ATTTTTAAGATTTTCCTAAATCCTTACTTCCGCGATCCGCAACAGTTACTGTATCTGGAA
PltR
MKA
L
GVVN
D
I DL
Figure 4-3. The putative promoter region of pltR. The identified Ebright
box containing the conserved motif TNTNA-N7-TNANA is in bold type.
The ribosomal binding site (RBS) and the N-terminal sequence of PltR are
also shown. Tentative -35 and -10 promoter regions for the pitA transcript
are also indicated by the boxed nucleotides.
80
The presence of the Ebright inverted repeat sequence has tentatively
allowed identification of the pltA promoter region (Figure 4-3). Although
the -35 and -10 RNA polymerase recognition sites within the pltA promoter
region do not conform strictly to known Pseudomonas promoter regions
(Deretic et al, 1989), the identity and positioning for 7 out of 12 nts within
the pitA promoter regions are identical within three a"-dependent promoters.
Nevertheless, it must be stressed that in the absence of functional
characterization and identification of the pitA promoter sequences, presumed
similarities within the tentatively assigned -35 and -10 regions are speculative.
4.2.4 Models for PltR-dependent regulation of pyoluteorin biosynthesis
Identification of P1tR as a putative transcriptional activator is a significant
contribution when viewed in the context of the global regulatory loci known
to influence pyoluteorin production. In addition to the global regulators
ApdA, GacA, RpoS and RpoD described above, a La protease has been identified
recently as a negative regulator of pyoluteorin production (C. Whistler and
I.E. Loper, unpublished). La protease induction is often associated with the
heat shock response (Neidhardt and VanBogelen, 1987) and is involved in
the proteolytic regulation of cellular development (Gottesman and Maurizi,
1992). Although secondary metabolism is not formally viewed as a
developmental stage within bacterial metabolism, it is, nevertheless, closely
coupled with transition into stationary phase. It is possible that the identified
Pf-5 La protease may regulate pyoluteorin biosynthesis via P1tR degradation.
Recent studies also suggest that ApdA and GacA influence the
accumulation of RpoS (C. Whistler and J.E. Loper, unpublished); however,
it is unlikely that this two-component system regulates pyoluteorin
biosynthesis via RpoS because both ApdA- and GacA- mutants fail to produce
pyoluteorin. Alternatively, ApdA and GacA may be required for production
of the P1tR- binding co-inducer. Because pyoluteorin biosynthesis is also
affected significantly by available carbon sources (Nowak-Thompson et al,
1994), ApdA and GacA may exert their control either by sensing carbon
sources or by regulating co-inducer biosynthesis in response to an unknown
81
signal. In light of the number of regulatory loci required for pyoluteorin
biosynthesis and the many possible interactions between their gene products,
it is apparent that pyoluteorin biosynthesis is controlled by a complex
regulatory network, an understanding of which is in its infancy.
4.3 CONCLUSIONS
The divergent orientation of PltR in relationship to the other pyoluteorin
biosynthetic genes, the identified LysR-type domains (especially the Nterminal HTH motif), and the presence of the inverted repeat promoter
binding region strongly suggest that PltR is a LysR-type regulator. Because
pltR is also closely linked to p1tABCD, it is likely that pyoluteorin biosynthesis
is regulated by PltR.
Whereas several global regulatory loci controlling pyoluteorin
production have been identified, this is the first report of a possible pathwayspecific regulatory mechanism. In keeping with the LysR regulatory model,
PltR most likely requires a co-inducer to effect transcription of the pyoluteorin
gene cluster. The absence of this co-inducer within other Ps eudomonas species
could account for low transcription levels of the gene cluster within
heterologous hosts (B. N.-T., J. Kraus, and J.E. Loper, unpublished results).
Because the biocontrol properties of Pf-5 are, at least in part, dependent upon
pyoluteorin production and since transcription of the pyoluteorin gene cluster
varies with respect to the plant host, the discovery of the pltR locus holds
great promise for improvement of biocontrol activity.
4.4 EXPERIMENTAL METHODS
The methods used for nucleic acid manipulations and DNA sequence
analysis of pltR were identical to those described in Chapter 3, section 3.4.
Deduced protein sequence analysis. The described domains within PltR
(see section 2.3) were identified via Profile Gap using standard parameters.
The LysR-profile used was constructed from the following representative
82
LysR-type regulators (Gen Bank accession numbers are in parenthesis): PtxR
(U35068) and TrpI (X51868) of Ps. aeruginosa , TcbR of an unidentified Ps.
species (M80212), RcbR of Chromatium vinosum (M64032), NahR (J04233)
and CatR (U12557) of Ps. putida , LysR (J01614), LeuO (J03862), and IlvY
(M14492) of E. coli, and G1tC of Bacillus subtilis (M28509). The presence of a
helix-turn-helix motif was confirmed using the program Helix-turn-helix v.
1.0.5 (December, 1993) available via anonymous ftp from the ebi.ac.uk server.
The putative P1tR promoter sequence was identified using the search routine
in the GCG sequence editor (SeqEd).
83
Chapter 5. Concluding Summary
5.1 PYOLUTEORIN BIOSYNTHESIS
The results of the work described herein have demonstrated that proline
is the primary precursor leading to formation of the dichloropyrrole moiety
of pyoluteorin. Although other putative intermediates were tested, the lack
of incorporation of these labelled compounds into pyoluteorin have precluded
any assessment of the biosynthetic pathway. Nevertheless, identification and
characterization of several genes residing within a genomic region that was
previously found to be required for pyoluteorin production (Kraus and Loper,
1995) has provided some insight as to the assembly of pyoluteorin, as well as
establishing this region as the biosynthetic gene cluster.
The Type I polyketide synthase (PKS) encoded by pltB and pltC contains
several functional domains that each catalyze a single reaction required for
forming the resorcinol moiety of pyoluteorin. Some structural features of
the pyoluteorin PKS were unexpected; namely the absence of both a loading
and a thioesterase domain. With the exception of the rapamycin PKS (Aparicio,
1996), most Type I PKSs possess each of these domains, which are responsible
for the initiation and termination, respectively, of polyketide assembly.
Although these domains are not absolutely required for PKS activity, their
absence makes it difficult to otherwise reconcile polyketide chain initiation
and termination. Concurrent work in our lab has identified and partially
characterized genes encoding a CoA-ligase and a thioesterase within the
pyoluteorin gene cluster (N. Chaney and J. Loper, unpublished). The CoA­
ligase exhibits similarity to many amino acid activating synthetases that are
required for antibiotic biosynthesis in a variety of organisms and may function
in the initiation of polyketide biosynthesis by transferring the activated starter
unit (perhaps prolyl-CoA) to the pyoluteorin PKS. Likewise, the thioesterase
also shares similarity to a variety of thioesterases found within several
biosynthetic gene clusters and may catalyze the release and cyclization of the
terminal PKS substrate. Nevertheless, future research must be directed at
verifying their presumed functions, because the proteins encoded by these
84
genes were identified based on sequence similarity alone.
In contrast to the polyketide portion of pyoluteorin, the transformation
of proline to the dichloropyrrole moiety is not easily discerned from gene
sequences within the biosynthetic gene cluster. Three loci have been identified
that encode halogenases that are likely responsible for the chlorination of
one or more pyoluteorin pathway intermediates. It is not immediately
obvious, however, why three halogenases exist within a pathway that resolves
to a dichioro- functionality. Nevertheless, chlorination is required for the
biological activity of pyoluteorin (B.N.-T., unpublished result), suggesting
that these genes are required for the antifungal properties of pyoluteorin.
Prior to this study, only three other halogenases had been identified, making
this a poorly characterized class of enzymes. An NAD co-factor binding site
was identified tentatively via protein sequence alignment of the halogenases
described herein, suggesting that a formal oxidation or reduction may be
required for halogenation. In light of the fact that chloride ion is a poor
nucleophile, halogenation most likely occurs electrophilically via covalent
bond formation between a chloronium equivalent (i.e. CI+) at an electron
rich carbon center. Therefore, oxidation of the proline-derived pyrrolidine
moiety to the pyrrole may be a prerequisite to chlorination due to the ability
of pyrrole to undergo electrophilic reactions.
One chemically feasible mechanism that accounts for the formation of
the pyrrole and the subsequent halogenation is based on the identification of
a gene encoding an acyl CoA-dehydrogenase within the pyoluteorin gene
cluster (N. Chaney and J. Loper, unpublished). It is possible that this enzyme
may catalyze the initial oxidation of the pyrrolidine ring to the A2-pyrroline
derivative, 5.1 (Scheme 5-1). In a subsequent NAD+-dependent reaction
catalyzed by either PltA or Orf-S, the A2-pyrroline would be further oxidized
to 5.2, introducing the chlorine atom at C5 via proton abstraction from C4.
Addition of the C4 chlorine atom would occur analogously via C5 proton
abstraction mediated by PltD. Removal of the second proton from C4 would
afford the 4,5-dichloropyrrole moiety of pyoluteorin, 5.3.
85
Scheme 5-1
PItA
N
H
0
NAD+
5.1
PltD
fyFX4 a
AIT,E7X
N
O
N
0
a
a
H
5.3
5.2 REGULATION OF PYOLUTEORIN BIOSYNTHESIS
The translated gene production of the pltR locus, which has been identified
within the pyoluteorin biosynthetic gene cluster, exhibits sequence similarity
with other LysR transcriptional regulators. The divergent orientation of pltR
from the pltABCD gene cluster, the identification of a helix-turn-helix motif
within the N-terminal region of P1tR, and an inverted repeat sequence within
the presumed promoter region of the pltA locus also suggest that P1tR is a
LysR-type regulatory protein. The proximity of pltR to several pyoluteorin
biosynthetic genes further implies that P1tR specifically regulates the
transcription of the biosynthetic gene cluster. Because most LysR-type proteins
regulate transcription only in response to a co-inducer molecule, it is likely
that pyoluteorin production also requires an as of yet unknown, co-inducer.
This is possibly the first report of a LysR-type regulatory gene controlling
antibiotic biosynthesis (Doull and Vining, 1995). Future studies will be directed
towards demonstrating the function of the gene product as in relationship
to pyoluteorin production.
86
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APPENDICES
111
Appendix 1. Production of 2,4-diacetylphlorogiucinol by the biocontrol
agent Pseudomonas fluorescens Pf-5
NOTE: The material presented within this appendix was written under
joint authorship with Drs. J. Kraus, J.E. Loper, and S.J. Gould and was published
in Can. J. Microbiol. (1994) 40:1064-1066. B.N.-T. was the principal author of
this work and responsible for the isolation and structural confirmation of
2,4-diacetylphloroglucinol and assisted in the quantitative analysis of
antibiotic production. Dr. J. Kraus identified bioactivity attributed to 2,4­
diacetylphloroglucinol production and was responsible for the quantitative
analysis of antibiotic production within this strain.
Antibiosis has been established as an important mechanism of biological
control against plant pathogens through the application of modern genetic
techniques (Lugtenberg et al. 1991; O'Sullivan and O'Gara 1992; Weller and
Thomashow 1993). Current research has focused on metabolites produced by
fluorescent pseudomonads and has implicated oomycin A (Howie and Suslow
1991), phenazine-l-carboxylate (Pierson and Thomashow 1992; Thomashow
and Weller 1991), 2,4-diacetylphloroglucinol (Keel et al. 1992; Vincent et al.
1991), pyoluteorin (Maurhofer et al. 1992), and hydrogen cyanide (Voisard et
al. 1989) as factors contributing to the suppression of plant disease.
Pseudomonas fluorescens strain Pf-5, originally isolated from a cotton
rhizosphere, is an effective biocontrol agent against Pythium damping-off of
cotton (Howell and Stipanovic 1980) and cucumber (Kraus and Loper 1992)
and Rhizoctonia damping-off of cotton (Howell and Stipanovic 1979). Pf-5
also inhibits ascocarp formation by Pyrenophora tritici-repentis on wheat
straw residue (Pfender et al. 1993). The antibiotics pyrrolnitrin and pyoluteorin
have been isolated from cultures of Pf-5 and, when these metabolites were
applied to seed surfaces, seedling damping-off caused by Rhizoctonia solani
and Pythium ultimurn was suppressed (Howell and Stipanovic 1979, 1980).
In addition to these metabolites, Pf-5 produces hydrogen cyanide, ammonia,
a pyoverdine siderophore, and a previously uncharacterized antibiotic (A3),
112
which inhibits growth of R. solani and P. ultimurn (Kraus and Loper 1992).
We report herein the isolation and identification of A3, the effect of various
carbon sources on A3 production, and its antibiotic activity against several
phytopathogenic organisms.
Antagonism of P. ultimum by Pf-5 in culture was attributed earlier to
the production of pyoluteorin (Howell and Stipanovic 1980). Pf-5 mutants
deficient in pyoluteorin production, however, inhibit growth of P. ultimum
on glucose-supplemented nutrient agar (NAG1c) (Kraus and Loper 1992), on
which Pf-5 produces A3. Cultures of Pf-5 grown on NAG1c were extracted
with aqueous acetone (80% v/v acetone), and A3 was partially purified by
thin layer chromatography (TLC) (silica gel developed in chloroform/acetone,
9/1, v/v). Because 2,4-diacetylphloroglucinol (Phl) is produced by many strains
of P.fluorescens that exhibit biocontrol activity (Defago 1993; Harrison et al.
1993; Levy et al. 1992; Shanahan et al. 1992) and inhibits mycelial growth of
P. ultimum (Keel et al. 1992), we considered it a likely candidate for the
identity of A3. A synthetic sample of Phl co-migrated with A3 on TLC and
both compounds formed a brilliant yellow derivative when TLC plates were
visualized with diazosulfanilic acid. Furthermore, analysis by C18 reverse
phase HPLC (0.8 x 10 cm Waters Nova-pak radial compression cartridge
eluted with H20/MeCN/Me0H, 45:30:25 at 1.5 mL/min) revealed that both
compounds exhibited identical retention times (6.4 min) and possessed
identical UV spectra (UV., 270 nm).
To identify the structure of A3 unequivocally, we isolated a sufficient
quantity of the compound for spectroscopic analysis from a Tn5 mutant of
Pf-5 (JL3985) that over-produced A3 regardless of the carbon source present
in culture media (Pfender et al. 1993; Sarniguet et al. 1994). The bacterium
was cultured in 1.5 L of glycerol-Phytone medium (5.0 g/L glycerol, 20 g/L
Phytone Peptone (Becton and Dickinson, Cockeysville, MD), 1.5 g/L K2HPO4,
1.5 g/L MgSO4 7 H2O, pH 7.0) at 20 °C with agitation (200 rpm). After 48 h,
the culture supernatant was acidified to pH 2 with 3M HC1, extracted with
ethyl acetate (3 x 150 mL), and the crude extract (0.5 g) was fractionated on a
flash-silica gel column (5 x 15 cm) eluted with toluene/acetone (3/1, v/v).
The fractions containing A3 (250 mg) were combined and re-chromatographed
on a flash-silica gel column (2.5 x 15 cm) eluted with chloroform/methanol
(9/1, v/v). Fractions containing pure A3 (180 mg) were combined and analyzed
113
by standard 1H and 13C NMR spectroscopy. The chemical shifts in both the 1H
and the 13C NMR spectra of A3 were identical with those observed for authentic
Phl. (A3 was recrystallized from acetone/water: mp 173-174°C (lit. Strunz et
al. 1978, 168-172°C); 1H NMR (400 MHz, acetone-d6) 8 16.30 (br s, 1H), 5.85 (s,
1H), and 2.60 (s, 6H); 13C NMR (100 MHz, acetone-d6) 8 204.56, 172.69, 170.06,
104.67, 95.62, and 32.76.)
The spectrum of antibiotics produced by Pf-5 was affected by the carbon
source composition of the culture medium (Table 1).When Pf-5 cultures
were grown in nutrient broth (Difco Laboratories, Detroit, MI) supplemented
with glucose (20 g/L), Phl was produced almost exclusively. This was consistent
with the earlier observation that antibiotic A3 is produced on NAG1c. This
result contrasts with the report by Shanahan et al. (1992) that Phl production
by P. fluorescens F113 was reduced in response to glucose in culture media,
and may reflect either differences in medium formulation or a fundamental
difference in the regulation of Phl production between the two Pfluorescens
strains. In contrast to Phl, pyoluteorin and pyrrolnitrin production by Pf-5
was diminished if nutrient broth was supplemented with glucose but
enhanced if the medium was supplemented with glycerol (Table 1). The
differential regulation of antibiotic production within a given strain of
Pseudomonas sp. is not without precedent and has been described for P.
fluorescens strain HV37a (James and Gutterson, 1986).
Table 1. Concentrations of antibiotics produced by P. fluorescens
Pf-5 in culture.
Antibiotic concentration
(ggimL culture)
Culture medium
Nutrient broth
Nutrient broth + 2% glycerol
Nutrient broth + 2% glucose
2,4-diacetyl-
phloroglucinol
<0.02
<0.02
16.60
pyoluteorin
pyrrolnitrin
0.50
5.75
0.19
<0.09
2.07
<0.09
NOTE: Five mL cultures were incubated at 20°C for 48 h with shaking, acidified with
HC1 to pH=2 and extracted twice with 5 mL ethyl acetate. The ethyl acetate fractions
were concentrated and analyzed by photo-diode array HPLC in comparison with authentic
standards. The values presented are an average of three replicate cultures.
114
The profound effect of carbon source on Phl production by Pf-5 in culture
media suggests that the chemical composition of habitats that the bacterium
occupies in the spermosphere or rhizosphere will also effect Phl production.
Phl has been detected in the rhizosphere of gnotobiotic plants inoculated
with P. fluorescens CHAO (Keel et al. 1992), indicating that the rhizosphere is
conducive to the production of Phl, at least under certain conditions. However,
not all occupied sites or environmental conditions may be conducive to the
in situ production of this metabolite at the time or in the concentrations
needed to suppress a fungal pathogen. Although the role of Phl in disease
suppression by Pf-5 has not yet been investigated, isolates of plant pathogens
that are suppressed by Pf-5 are sensitive to Phl in culture. Inhibition of P.
ultimum N1 (Kraus and Loper 1992), R. solani J1 (Howell and Stipanovic
1979), and P. tritici-repentis 6R180 (Pfender et al. 1993) were observed at Phl
concentrations of 128 ug/mL in potato dextrose agar (Difco), and Erwinia
carotovora subsp. carotovora W3C105 and E. carotovora subsp. atroceptica
W3C37 (Xu and Gross 1986) were inhibited at 250 ug/mL of Phl in nutrient
yeast broth (Stanisich and Holloway 1972). These data are in agreement with
previously reported inhibitory activities of Phl (Keel et al. 1992; Levy et al.
1992).
Indirect evidence for the in situ production of antibiotics by Pseudomonas
spp. and evaluation of these metabolites in disease suppression is usually
obtained by comparing the biological control activities of antibiotic-deficient
mutants to that of a parental strain. Identification of mutants defective in
antibiotic production has commonly been attempted by screening mutants
for loss of inhibition in culture. The array of inhibitory metabolites produced
by Pf-5, however, necessitates the use of chemical analysis to screen mutants
because the presence of an unidentified but biologically-active compound,
such as Phl, could mask the existence of a biosynthetic mutant blocked in a
single antibiotic pathway. The case-in-point is a study conducted in our
laboratory (Kraus and Loper 1992) in which individual culture extracts from
several thousand Tn5 insertion mutant cultures were assayed by TLC for
pyoluteorin and pyrrolnitrin production. Pyoluteorin-deficient mutants did
not inhibit P. ultimum on certain media but were inhibitory against the
fungus in other culture media, including NAGlc, which we now know to be
conducive to Phl production by Pf-5. The ability of Pf-5 to produce Phl may
115
explain our initial failure to obtain pyoluteorin or pyrrolnitrin non-producing
mutants by screening for loss of inhibition of P.ultimum or R. solani. Indeed,
the common approach of screening mutants by inhibition assays can not
successfully detect biosynthetic mutants if the phenotypes of such mutants
are masked by the production of an unsuspected antibiotic(s).
116
Appendix 2. Nucleotide Sequence and Translated Product of pltB
1
CCCAGTCGAAGGAGATGACTGAATGGATGCTCGTGCGCCCATGGATTTTGAACCCATTGC
+ 60
MDARAPMDFEPIA
61
AATCATCGGGAGTGGATGCCGCTTTGCCAAAGGTGCATCGACCCCCGAGGCATTCTGGGA
+
+
+
+
+
+ 120
121
GCTCCTTCGTGCTGGGACCGAiiiiGTCGGACCGGTTCCCGCAGAGCGCTGGGACACAGC
+
+
+
+
+
+ 180
181
GGCGATCTACGATGAGAGCGCTGCTGAAACCGGTACGACCTACAGCAAGGTGGGGGCGTT
+
+
+
+
+
+ 240
241
CCTCGAGCACATCGATCGCTTCGATGCGCATTACTTTGGCATCTCGGCCTCTGAAGCCAA
+
+
+
+
+
+ 300
301
GGAAATGGACCCCCAGCAGCGCCTGCTGCTGGAGGTCGCCTGTGAAAGCGTGGCCCGTGC
+
+
+
+
+
+ 360
361
CGGCCTGACCCGCGAGCAGCTCAAAGGCAGCAGGACCGCGGTCTATGTCGGCATGCTGGG
+
+
+
+
+
+ 420
421
CATGGATTACCTGGCACTGCACTCCCGCGAGGCCGGCATCGAACAGATCAACCCCTACTA
+
+
+
+
+
+ 480
481
TGCCGCCGGCAAGGAATTCAGCTTCGCCGCCGGGCGTATTGCCTACCACCTGGGTGTTCA
+
+
+
+
+
+ 540
541
TGGGCCGGCAATGACCGTGACCACTGCATGCTCTTCTTCCCTGGTGGCCATGCACCTGGC
+
+
+
+
+
+ 600
601
CTGTCGCGCCTTGCAGGCAGGGGAGGCGGACATGGCCCTGGCCGGAGGGGTGAACCTGAT
+
+
+
+
+
+ 660
661
GCTGGCGCCGGACCTGACGATCTACATGAGCCAGATCAGGGCGATCTCGCCCAGTGGTCG
+
+
+
+
+
+ 720
IIGSGCRFAKGASTPEAFWE
L LRAGTDFVGPVPAERWDTA
AIYDESAAETGTTYSKVGAF
L EHIDRFDAHYFGISASEAK
E MDPQQRLLLEVACESVARA
G LTREQLKGSRTAVYVGMLG
MDYLALHSREAGIEQINPYY
AAGKEFSFAAGRIAYHLGVH
G PAMTVTTACSSSLVAMHLA
CRALQAGEADMALAGGVNLM
L APDLTIYMSQIRAISPSGR
117
721
CTGCCGGGTATTCGATGCGGCGGCCGACGGGATCGTCCGGGGCGAAGGCTGCGGGGTCAC
+
+
+
+
+
+ 780
C R V F D A A A D G I V R G E G C G V T
781
GGTGCTCAAGCGCCTGGCAGATGCCCTACGCGATGGCGATCCGATCCAGGCGGTGATCAG
+
+
+
+
+
+ 840
L K R L A D A L R D
Q A V I R
G DP I
841
901
961
GGGCTCGGCGATCAACCAGGATGGCGCCAGTGCCGGCCAGACCGTGCCCAACGCCAATGC
+
+
+
+
+
+ 900
G S
S
A INQ DG A
A GQ T V PN AN A
CCAGGCTGCAGTGATCAGCCAGGCCCTGAAAGTGGCCGGCTTGAGCGTGGACGACATCGA
+
+
+
+
+
+ 960
S Q A
L
D
Q AAV I
K V A G LSVDD I
CTATGTCGAGGCCCACGGCACCGGTACGCCCCTGGGCGATCCGATCGAACTCTCTTCGCT
+
+
+
+
+
+ 1020
Y V E A H G T G T P L G D P
I IEL S SL
1021
GGACAGTGCCTTCCAGGGGCGTGAGCGGCCGCTGTGGGTGGGCTCGGTCAAGGCCAACAT
+
+
+
+
+
+ 1080
D S A F Q G R E R P L W V G S V K A N M
1081
GGGCCATCTGGATGCGGCCGCCGGAATGGCCAGCGTGATCAAGACCATGATGGTTCTCAA
+
+
+
+
+
+ 1140
G H L D A A A G M A S V I
K
K TMMV L
1141
GCACGCCGAGGTGCCTGCGCAACTGCATCTTGCACAGTTGAACCCACTGGTGGACTGGAA
+
+
+
+
+
+ 1200
H A E V P A Q L H L A Q L N
1201
GCGTTCCCGGCTGGCGGTGCCCACGGCGATCGAGTCGTTGCCCGACCGGCCGCGCCTGGC
+
+
+
+
+
+ 1260
R S R L A V P
P LVDWK
T A IESL PDR PR L A
1261
TGGAATCAGTGGTTTCGGGCTCAGTGGCACCAACGTCCACATGATTCTCGAAGATGCCAG
+
+
+
+
+
+ 1320
G I S G F G L S G T N V H M I L E D A S
1321
CGTCTATCGGCAGGCACAGCCGCAGCAGGAACGATCGGCACAGGGCCGGCCCTGGGTCCT
+
+
+
+
+
+ 1380
Y R Q A Q P Q Q E R S A Q G R P W V L
1381
GCCGGTATCGGCCAGGTCCGCGCAGGCCGTGGTGGAGCAGGCCAGGGCCTATGCCGTTCA
+
+
+
+
+
+ 1440
P V
H
S AR S A Q A V V EQ AR A Y A V
1441
TCTGCCGCAGCAGGACGATGGGCAATTGCAAGCCTTTGTCGCCAGCGCCATTCATCGTCG
+
+
+
+
+
+ 1500
L PQQDDGQLQAF VA S A IHRR
118
1501
TGATCACTTTCCCTACCGGTCGGCCGTGGTGGGTGCGAACGCCGGGCAATTGAAGAGCCA
+
+
+
+
+
+ 1560
DH F P Y R S
Q L
K
S Q
1561
GCTGGAGCAGCTGCCGGCCCCGACCTTGGCCTGCACGACGGACGAAGAGGATCGGCGCGG
+
+
+
+
+
+ 1620
1621
GCCCGTGCTGGTGTTTACCGGGCAGGGCGCCCAGTGGGTGGGAATGGGCCGCGACCTGCT
+
+
+
+
+
+
1681
1741
1801
AVVGANAG
L EQL PAP T L AC T TDEEDRR G
PVLVF
1921
1981
GQGAQWVGMGRDL
RE PAF LAM
D Q A LAQW
RR
1680
L
TGAACGGGAGCCGGCCTTCCTGGCGATGATCCGGCGTTGCGACCAGGCGCTGGCCCAGTG
+
+
+
+
+
+
E
I
C
1740
GGCAAGCTGGTCGGTGGAGGCCGAGCTGCGCAGCGACGCCAGTGGGTCCCGCCTGCACCT
+
+
+
+
+
+ 1800
A SW
S
S G S R L H L
SVEAE LR
DA
GACCGAATTTGCCCAGCCCTGCATCTTTGCCATCCAGGTGGCGATCAGTGAATGCCTGCG
+
T
1861
T
E
F
AQ
+
P
C
I
+
F
A
+
I QVA
I SECLR+ 1860
+
CCAGTGGGGTGTGATCCCGGCGGCGGTGGTCGGCCACTCCATGGGGGAAGTGGCCGCGGC
+
+
+
+
++
QWGV I PAAVVG
H
S
MG
EVAAA
1920
CTATTGCGCCGGAGCACTGGACCTGGAGTCGGCGGTGCGGGTCATCCACCATCGGGCCCA
+
+
+
+
+
+ 1980
YCAGAL
D
LE SAVRV I
H
H
RAQ
GGCCATGAAGGACACCCTGGGGCAGGGGCGGATGCTGGTGGTGGGCTTGCCCGCGCCGAC
+
+
+
+
+
+
AMKDT LGQGRMLVVG
L
PA
P
2041
GCTCCAGTCGCGTCTGGCGAACAACCCGCAACTGGAACTGTCGGTGGTCAACAGTCGAAA
+
+
+
+
+
+
S
P Q L E L
2101
CAGCTGTGTGGTGTCCGGTAGCCCGCAGGCTGTACAGGCTCTGGATCAGCAACTGCGTGA
+
+
+
+
+
+
LQ
RLANN
S V V N S RN
S CVVSGS PQAVQA LDQQLR
CGAAGGCATCTTCACTTACCTGATGCCGGCGGAATATGCCTTCCACTCCTGCCAGATGGA
+
+
+
+
+
+
2221
TGAGTGCCTGACGCAGATCCGCGCGGGGCTGGAGGATTTGCCGGTGGTTGCCGCGCACAC
+
+
+
+
+
+
E C L
L E D L
T
E G IF T YLMPAEY AFHSCQMD
PVVAAH
2100
2160
D
2161
RAG
2040
T
2220
2280
119
2281
GCCCTGGATTTCCACCAGCGCGATGCCCGAGGAGCCGATCCTGGCAGATGCCGACTACTG
+
+
+
+
+
+ 2340
PW
S T S AM P E E P I
D YW
I
2341
2401
2461
LA DA
GGCGAGGAATGCCCGTGGCATCGTGCGCTTCGATCGCGCCATCGAACAGTTGATCGAGCA
ARNARG IVRF DR A IEQL I
+ 2400
E
Q
GGGGCACCGGCTGTTCGTCGAGATCGGCCCGCACACCGTGTTGGCGGCGTCGATCAACCA
+
+
+
+
+
+ 2460
GHRL FVE I
G
P
HTVLAA
S INQ
GGCCCTGGCCGACAAGGG'AACCCAGGGACTGGTGTGCGGCGCCTTGCACAAGCAGGGCGA
+
+
+
+
+
+ 2520
G
T Q G
L
D
ALADK
LVC GA
HKQG
2521
CGCCGCCCTGGAGCTGGCCAGTATTGTTGCCCGCCTGTACGAGTGGGGCGCAGGTCCCGA
+
+
+
+
+
+ 2580
S I
Y
P D
2581
CTGGCAAGCCTTCCAACCCAGGGAGGCGGCGCTGGAGCTGCCGGCCTATCCCTGGCAGCA
+
+
+
+
+
+
WQ A F Q P
E L P
2640
GGAGCGTTTCTGGTTTGCCCCGGCGCCGCGGCCGCAGCCGGCAGGGCTCGTGTCCCAGCT
+
+
+
+
+
+
P A P R P Q
S Q L
2700
2641
AALE LA
VARL
EWGAG
REAAL
AY
ER FWFA
P WQQ
PAG LV
2701
GCGGGCGCAGGTACTGGTGTATGACGCCCAGGGCAATCTCTGCGCCCAGGCCAACGATGT
+
+
+
+
+
+
2761
GGCGCTGAGCGTGCCGCAGCTGGCACAAGTCGCTGTGCCGGCACCGGCCAAGGTGTCCGC
+
+
+
+
+
+
S V P Q
2821
R A Q V L V Y D A Q G N L C A Q A N D V
LAQVAVPA PAKVSA
AL
2760
2820
AGCGGCGCAGCCGGTGGGGGATGTGCGGGCGCAGATTGGTGCACTGCTGACGCAGATCAT
+
+
+
+
+
+ 2880
I
L L T Q I I
AAQ PVG DVRAQ
GA
Tn5 insertion
mutant 4296
2881
2941
CGGCGTGGCCTGTGCC GACCCGGACCCGGATCGAGGCTTCTTCGAACTGGGCCTGAGCTC
GVACA
D
P
D
P
D
R
G
F
F
E
+2940
L GL S
S
GATTTCCCTGGTTGAATTCAAGCGCATGCTGGAGCGCCAGTTCGCCCTCAAGCTGTCGGC
+
+
+
+
+
+
S
LVE FKRMLERQ FAL
K
L
S
AA
3000
120
3001
3061
GACCGTGGGCTTCGACTATCCCACCATCAACCGGCTGGGCCAGTACCTGGAAGGGCTGCT
+
+
+
+
+
+
TVG
3181
D
Y
T INRLGQYLEGL L
P
GTCCCGGGAGCCGGCCAGCACCCCGGTAACGGTCGATGCCGGGGCGACCGACGCCGCGGG
+
+
+
+
+
+
S
3121
F
RE PA
S
T
PVTVDAGAT DAAG
3060
3120
GAGCGTCGCCGTGGTGGCCATGGCGTGCCGGTTTCCCCAGGCCGACAGCCCCGAGGCACT
+
+
+
+
+
+ 3180
SVAVVAMAC RF PQAD
S
P
EAL
CTGGAAGCTGATGCTGGAACAGACGGACACCGTGGGGCCGGTACCGCCGTCCCGGCTCGC
+
+
+
+
+
+
WKLMLEQT DTVG PVP
P
S
RLA
3240
CGGCGCTAAGCCGGAGGAAACCTTCCCGCGGTTTGCCAGCCTTATCCAGCGCCCCGAAGG
+
+
+
+
+
+
P E E T F P R
S L I Q R P E G
3300
3301
GTTCGACGAAGCGTTCTTCCGCATTTCCCCCAAGGAAGCCCGGAGCATGGAC.CCCCAGCA
+
+
+
+
+
+
F D
F F R I S P K E A R S
3360
3361
ACGGCTGTTGCTGATGGTGGCCTGGGAGGCTCTGGAACGGGCCGGTATCCCTCAGGAGAA
+
+
+
+
+
+
L
3420
3421
GCTGCTGGAACAGAGGGTCGGGGTGTTTGTCGGGGCCAACTCCCACGACTACGAAACCCG
+
+
+
+
+
+
L
S H D
R
3480
3481
GGTCCTGGGTAGCGCGCAAGGCGTGGATGCCCACTACGGCACTGGCAGTTCGTTTTCGGC
+
+
+
+
+
+
L G S
T G
A
3540
3541
GATCTGCGGGCGCCTCTCGCA1 rri CTCGGCGTGCGTGGGCCGAGCCTGACGGTGGACAC
+
+
+
+
+
+
1 C
S H F
P S L
T
3600
CGCATGCTCGTCATCGCTCACGGCCATCCATCTGGCGTGCAACAGCTTGCGTGCCGCGGA
+
+
+
+
+
+
S S S L
I H
N S L
E
3660
3661
GTGCGACATCGCGATTGTGGGTGGGGTGAATGTCATCGCCTCGGCGTCGATCTTTCAATC
+
+
+
+
+
+
C
D
A I VG
I A S A
3720
3721
CATGGGGCAGGCCGGCGCATTGGCCCCGGACGGCATCAGCAAGGCCTTCGACGACAGTGC
+
+
+
+
+
+
3241
3601
GAK
FA
M DPQQ
EA
RL
LMVAWEAL ERAG
LE QRVGVFVGAN
AQGVDAHYG
GRL
AC
TA
I
MG
LGVRG
LAC
P
DG I
YET
S SF S
TVD
RAA
S IF QS
GVNV
QAGALA
I PQEK
S
KAF DD SA
3780
121
3781
3841
3901
3961
4021
CGACGGTTACGGTCGAGGGGAGGGCTGCGGCGTGGTCATTCTCAAGCGCCAGGCCCAGGC
+
+
+
+
+
+
D G Y
E G C G
I L
AA
GRG
KRQAQ
VV
3840
CGAGCGGGAGCGTGACCCGATTGTCGCGACGATTCTCGGTTCGGCGGTCAATCACGACGG
+
ERE RD
+
+
+
+ 3900
P IVA T IL G +S AVNHDG
TGCCTGTGCCGGGCTGACAGTGCCCAATGGTCCGGCGCAAGAGGCGCTGATCAGCGAAGC
+
+
+
+
+
+
L
P NG P
I
AC AG
TV
S EA
EAL
AQ
3960
ACTGGCCAACGCCGGCGTGCATCCGGGGCAAGTCAGCTACGTGGAGGCCCATGGCACCGG
+
+
LANAGVH
+
P
G
QV
+
S
YVEA
+ 4020
+
H
G
T
G
CACGGTGCTGGGTGACCCCATCGAGCTCAACGCCCTGCACAACGCCTATCGCCAGGCCAG
+
+
+
+
+
+
TVL
G
D
P
I
E
LNA
L
H
NAYRQAA
4080
S
4081
CCCCGACAGTCCGCCGCTGACGGTGGCCTCGGTCAAGGCCAATATCGGGCACCTCGAGGC
+
+
+
+
+
+
P D S P
4140
4141
GGCAGCGGGCATCGCTTCGCTGATCAAGGCCTGCCTGGTGGTGGAGCACGGCCGGATTGC
+
+
+
+
+
+
I A S L I K
R
4200
4201
CCCGCAAGCCCATCTGCAGCGGGCCAACACCCGTGTCGACTGGGCGGCCATGAACCTCAA
+
+
+
+
+
+
P
L
K
4260
4261
GCTGGCGCATCAGGCCATGGACTGGCCGGGCCGGCCGGAGTCGCGGGTGGCCGGGGTCAG
+
+
+
+
+
+
DW P G R P E S
S
4320
TGCCTTTGGTTTCACCGGGACCAACGTGCATGTGCTGCTCAAGGGCTATACGGCGCCCGC
+
+
+
+
+
+
G
F T G
L
4380
GACAGCGCCTTTGCCACCCGCCACAGCACCAGTGGCCTTGTGCCTGTCCGCGGCCACCCC
+
+
+
+
+
+
P L P
A
L C L S
P
4440
GGCGGCATTGGCGGAGCTGGCCCAGCGCTATGTGTCCTTCCTGGGCGCTACCGAGCACTG
+
+
+
+
+
+
E
S F L
E H C
4500
CCCACAGACCATCTGCTACAACGCGCTGATGCGGCGCACGGCG1 i CAAGGAACGCCTGGT
+
+
+
+
+
+
P Q T
C
L
F K E R
4560
4321
4381
4441
4501
P LTVA A S V K A N IGH L E A
AAG
I IA
QAH
QRANT RVDWAAMNL
LAH QAM
AF
RVA GV
TNVHVL
PAT
TA
AALA
I
KGY TA PA
PVA
LAQRYV
YNA
MRRTA
AAT
GAT
LV
122
4561
CGTCCACGGCCAGGATTGCCGCGAGCTGGCTCAGGCACTACAGGCCTGGCTGGCCGGTAG
+
+
+
+
+
+
H
G
Q
D C
R E L A
L
G
S
QA
4621
CCCCATCGCCAATGACCGCAAACCCGCGGCCGGCGAACCCTGGGCAACCCTGGCCGAGGC
+
I AN
P
4681
4741
4801
4861
4921
4981
4620
QAW LA
+
+
+
D R K PAAGE P W A T
+
LA EA
+ 4680
CTTTGGCCGCGGCGCCCAGTCGCCGGGCCCCGAGCGCTTGCCGGACGGCTGTCAGGCCAT
+
+
+
+
+
+
F
S P G P
P D G C
I
4740
CGGGTTGCCCACCTATCCCTGGCAGCTCAACGACTACTGGATCGATGCTGGCCAGCCGGC
+
+
+
+
+
+
G L
P T
Q
4800
GRGAQ
ERL
QA
Y PWQLNDYW IDA G
PA
TACGGCGGTGCAGCCCGCCCGGGCAGCCTCGGGCCATCCTTGCCTGCAGGGGCTGGTACG
+
+
+
+
+
+ 4860
S G H P C
TAVQ PARAA
LQG LVR
GCCGGCCGGGCAGCTCTGGTACTGGTCGGGGGCTCTCGCTCCCCAGGCCGGGCATTACGA
+
+
+
+
+
+
PAG
Q
LJ
WYWS GALA
P
QAG
H
Y
4920
D
CCCGCTGGGCGAGCAGGGGTACAGGGTCAAGACTCACCTGTTGCTGGATGCCGTGCTCCA
+
+
+
+
+
+ 4980
P L G E Q G Y
T H L L L
Q
RVK
DAVL
GGCGGTGCGGGAAACACCACGGGGCGTGCAGCAGATCCGCGACTTGCAGATTGCCCAGCT
+
+
+
+
+
+
A V R E T PRGVQQIRDLQ I
AQ
5040
L
GCGCCTGCGCGGCGAACAGCACCTCACTTCGCACCTGAGCATCCACCTCACCCAGGCGCC
+
+
+
+
+
+
R L
E Q H L T S H L S
H
L T
P
5100
5101
TGACGCCTGCTTCGAACTGGCCTTGCAAGGGGCCGGCGACGAGCGCCGCCAGGTCTGCAT
+
+
+
+
+
+
DA C F
M
5160
5161
GAGCGGAACCCTGGTTGACTGTGCAGCGCAGTTGCAGGAGGAAACCCTGTGCGGGGTGTC
+
+
+
+
+
+
S
L C
5220
GATGCTCGATGAGCCTTCACCGCCGGCGCCGGACGTTGGCCTGTGCCCATGGTCCGGGTG
+
+
+
+
+
+
ML
P S P P A P
L
5280
TGCGGCCACGGGCGGGCAGCGGGCCTTGTACCGTTTTGCCCACTCCCTGACGGCGGCCGA
+
+
+
+
+
+
T G G
L
S L
E
5340
5041
RG
5221
5281
I
QA
LALQGAGG DERRQVC
GT
LVDCAAQLQEET
DE
AA
DVG
QRA
YR FAH
GVS
C PWSGC
TAA
123
5341
5401
5461
GCGCCAGACCCAGTTGCTGGCCAGTGTGGTCGAGCTGTTTGAAGGCGCCCACGCCGCCGC
+
+
+
+
+
+
RQTQ
L VGF SGLQVWADL PAQVW
5641
5701
5761
5821
5881
5941
6001
6061
5400
5460
GCTGGCCGGCCATGACGCCGACAAACCCGACAGCCTGCAGGTAGTGGATGCCCGGGGTTG
+
DA
+
D
+
K
P
+
+
LQVVDARGC GC
D
+ 5520
CCAGCTGGCGCTGTTCGAAGGTCCGCAGTTTGGCCATCCTGGTTCGTGGTACTTGCCCGA
+
+
+
+
+
+ 5580
Q
5581
LASVVE LFEGAHAAA
GCTGGTCGGGTTCTCCGGGCTCCAGGTATGGGCAGACCTGCCGGCGCAGGTATGGATTGT
+
+
+
+
+
+
I V
L
5521
L
LA
L
F
E
G
P
Q
F
G
H
P
G
SWY
L PD
CCTCCAGACCGCACCCCTCGACCTGCCGATGATCGCTCGCCAATGGCAGGACTATCCGAT
+
+
+
+
+
+
5640
GCCGGGCGAGGGCGCTCGGCAACGCGAGGGCTACTGGATGGTGCTGGCCTGGAGCACTGC
+
+
+
+
+
+
5700
L QT A PLDL PMIARQWQDY
P GEGARQREGYWMVLAWS
PM
TA
CGAGGTCCAGCCACTGGCTGCGGCGTTTGCGGCTGAACAGCGTCCGGTTGAAGTCATCGA
+
+
+
+
+ 5760
+
EVQ
P
LAAA
F
AAE
Q
R
P
VEV
II
E
GCTGCACGCCGGGCAACAACCTCTGGCCCGCAAACTGTCCTCGGCGTTGCGCGGGGCAGT
+
+
+
+
+
+
L
P
S S A L
5820
GGCCGATCCGTCCTGCCTGGGGGTGATCGTTGCCGGCGTTGAGGCTCAGGAGGCCGACGG
+
+
+
+
+
+
P S C L
I
5880
HAGQQ
AD
LARKL
GV
RGAV
V A G V E A Q E A DG
CCTCGGTATATCCCTGGTGGCCTCTGCGGCGCTGGTACAGGCCTTCGCCGGTGCGATTGC
+
+
+
+
+
+ 5940
LG
S
LVASAALVQAFAGAA I
A
CAGCGTCGGAACACCGGCAAAGCCGGTCTGGTTCGCCCTTCATGCCAGTGATGCTGCGTC
+
+
+
+
+
+
T
P
A L H A
S
SVG
AK PVWF
S DA A
GCCGGCCATGGCCGCTGTCCAGGCCACCTGGCAGGGCGCCGCGCATATCTTCGCCCTGGA
+
+
+
+
+­
+
P A MA AVQ A T WQG A A H IF
A
L
LVT
QG
DRR
6060
E
GCATCCTGCCTGGTGGGGCGGTCTGGTGACTTTGCAGGGCAGCGACAGAAGAAGCTACGC
+
+
+
+
+
+
H
G
L
S
S
PAWWG
6000
YA
6120
124
6121
CAGCCTCTGCCGGCTGTTGCACGGCCAACCTGGCCATGATCACTTTGCGATCAGCGGCGC
+
+
+
+
+
+ 6180
S LCRLLHGQPGHDHF A
I SG A
6181
CCGGGTCGAGGTGCAATACCTGGTGGAGGATCAAGCCGACCCGCTACAGCGTCTGGAGCC
+
+
+
+
+
+ 6240
6241
GCCGGCGCTGAACGGAACCGTTGTGCTGCATGCCGTCCCGGGATCTGACCTGGAGACTGT
+
+
+
+
+
+ 6300
P A L N G T V V L H A V P G S D L
T V
6301
GCTGACGGCGCTCGGGCAGCGGGGTGTGCAGCGTGTGCTGCTGCTCTGCGAGGCCCCCGG
+
+
+
+
+
+ 6360
L T A L G Q R G V Q R V L L L C E A P G
6361
GCAACTGCACATGCCTGAGCGGATGCCCGAAGCGATGGCGATCAGCAGCCTCTCGGACCT
+
+
+
+
+
+ 6420
6421
GAGCCGGGAAAACCTGGCTGACACCTTCGCGACGCTGCGAGCGCAAGACCGCATCGCCGG
+
+
+
+
+
+ 6480
L A D T F A T L R A Q D R
A
G
6481
CTTTATCCACCTCGATCTTGACTGGCGCACGGTTGCGCTCAAGGAGCCAGAGTTTGTCGT
+
+
+
+
+ 6540
+
F
H L D L D W R T V A L K E P E F V V V
RVEVQYLVEDQADPLQR LE P
Q LHMPERMPEAMA IS SL SDL
AQDR I
Tn5 insertion
mutant 4128
6541
GCGAATGCAGGAAGGCGTTCGGCC GCTTGAGGTCTTGCAGCAGGTTCACCAGCTGATCGA
+
+
+
+
+
+6600
R M Q E G V R P L E V L Q Q V H Q
L ID
6601
6661
TGACCCTGAAGCGTTCTTCCTGATCCTGGGCAGCGTGTCTTCCCTGCTTGGCGGGGCCGG
+
4+
+
+
+ 6660
D PEAFF L ILGSVSSLL G
G A G
CTTCGCCCGCTCGGCGATTGCCGATGCATATGCCTTGTGGGTGCATGCACAGCGCCGGCG
+
+
+
+
+
+ 6720
F A R
S
A
I
A D A Y A L W V H A Q R R R
6721
CCAGGGGTTGAACTGCCAGCTGCTGCACCTGACGCAGAGCGAACAGGAGCTTGAGCAGGA
+
+
+
+
+ 6780
+
6781
CGCCGCAGCCCGCACGGCCATGCAAGGCAGCGGCCTGCAGCCTTTGCAGCGCTCGCAGAT
+
+
+
+
+
+ 6840
A A A R T A M Q G S G L Q P L Q R S Q I
Q GLNCQLLHL TQSEQELEQD
125
6841
AGTGCAGGCCATAGCCCGCGTGCTGGGTGGCCAGGGCCAGTGCGGGCTGCTCAATGTCGA
+
+
+
+
+
+ 6900
Q A I A R V L G G Q G Q C G L L N V D
6901
CTGGCAACAACTCAAAGGGCTGTACCTGAGCGTGCTGCCCTGGCCCTTGCTCGAACACCT
+
+
+
+
+
+ 6960
6961
GGGTGCAGCGGATAGCGCAGCGGATCAGCGTCTGGCCGAGCTGATTGGCCTGCCACCGCT
+
+
+
+
+
+ 7020
G A A D S A A D Q R L A E L I G
W QQLKGL YLSVLPWPLLEHL
L PPL
GCAGCAGCGCCGGGCCATGCAGGCGCTGGTCTGCGAGGTAGTGGGGCAGGTGTTCGGCGT
+
+
+
+
+
+ 7080
7021
Q Q R R A M Q A L V C E V V G Q V F G V
7081
TGCCGATGGCCTGGAGCTCGATGTCAGGAAGGGCTTCTTCGACATGGGCATGTCCTCGGT
+
+
+
+
+
+ 7140
A D G L E L D V R K G F F D M G M S S V
7141
CATGTCGCTGGACCTGCGCTCACGGCTGGGCCGGGCGTTGAGCATCGACCTGCCTTCGAC
+
+
+
+
+
+ 7200
S
T
7201
CTTCGGCTTCGAATACACCTCCATCGAACAGGTCACGGACTACCTCATGGGCCAGCTCCT
+
+
+
+
+
+ 7260
F G F E Y T
7261
GGCGCCTGAAACCCGGGAGCCGGTGGCGGCGCCCGAACCTGTGTCGCCAGCGTCGCGGCA
+
+
+
+
+
+ 7320
A P E T R E P V A A P E
MSLDLRSRLGR AL SIDL P
TSIEQV TDYLMGQL L
P V S PASRH
7321
TCAGGACCTGCATGAACTGTCACGGGCCGAGCTGATTGGTGCTCTCGAAGACGAGCTGCG
+
+
+
+
+
+ 7380
Q D L H E L
7381
CGATATCGCCAATTACTAGCCCTGGGGCTGGATGACAACTGCCAGAGAATCACTGATCGA
+
+
+
+
+
+ 7440
D I A N Y *
R A E EL IGALEDELR
126
Appendix 3. Nucleotide Sequence and Translated Product of pltC
1
CCrGGGGCTGGATGACAACTGCCAGAGAATCACTGATCGAGGCCTTTATGGATAACGATG
+ 60
W GWMT T ARES L IEAF MDN
D V
61
TCCGCGATGTAAGCAAGGAACAGCTGCAAGAGAGTCTTGCTCAGGCAATCACTACCATCC
+
+
+
+
+
+ 120
R D V S K E Q L Q E S L A Q A I T T I R
121
GTGCGCTCAAGGAAAAGGTGGCCGGCAAGAGCTCGGCGCCTGTCGAACCGATCGCCGTAG
+
+
+
+
+
+ 180
A L K E K V A G K
A V V
181
TGGGCCTGGGGTGCCGGCTACCGGGCAGTGCCGACACACCGAAGCGGCTGTGGAGCCTGC
+
+
+
+
+
+ 240
G L G C R L P G S A D T P K R L W S L L
241
TGAAACATGCCACCGATGCGGTGGGCGACATGCCCAGCGACCGTCTGTACGGCACCGACT
+
+
+
+
+
+ 300
K H A T D A V G D M P S D R L Y G T D Y
S SA PVEP I
Tn5 insertion
mutant 4274
301
ATTACCATCCTGATCCCCAGGCACCCGGCAAGG CCTACGTCATGCGCGGTGGCTTCATCG
+
+ 360
Y H P D P Q A P G K A
Y V M R G G F I E
361
AGGGGGTGGATCAGTTCGACCCGGGCTTCTTCGGCATTTCGCCCAAGGAAGCCGAAGGCA
+
+
+
+
+
+ 420
G V D Q F D P G F F G I S P K E A E G M
421
TGGACCCCCAGCAGCGCCTGGCCCTGGAGGTTGCCTGGGAGGCCCTGGAGAACGCCGCGA
+
+
+
+
+
+ 480
D P Q Q R L A L E V A W E A L E N A A I
481
TCGCCCCCGACAGCCTGCATGGCAAGAAGCTCGGCGTGTTCATGGGGGTCAGTACCAATG
+
+
+
+
+
+ 540
A P D S L H G K K L
G V F M G V
S TN D
541
ATTACGTGCGCCTGCGCCAGCAGTTGGGCGCGGTCGAGGACGTCAACGCCTACCAGTTCT
+
+
+
+
+
+ 600
Y V R L R Q Q L G A V E D V N A Y Q F
Y Y
601
ATGGCGAAACCAGCTTCGTGGCCGGGCGCATTGCCTACACCCTGGGCTCCAGGGGCCCGG
+
+
+
+
+
+ 660
G E T S F V A G R I A Y T L G S R G P A
661
CGGTGGTGCTCGACACCTCCTGCTCCTCATCCCTGGTGGCCCTGCACCAGGCCTGCAACA
+
+
+
+
+
+ 720
V L D T S C S S S L V A L H Q A C N S
127
721
781
GCCTGCGCAGCCGCGAGAGCGAGCTGGCGCTGGCCGGTGGGGTCAACCTGATCCTGTCGC
+
+
+
+
+
+
L R S
S E
L
I L S P
LALAG GVN
RE
780
CCTACGGTTTCATCCTGGTCAGCAAGCTGCGGGCCGTGGCCCCCGATGGCCGCTGCAAGA
+
Y
G
I LV
F
+
+
S
K
+
+
L R A V A PDGRC
K
+ 840
T
841
CCTTCGACGCGGCGGCCGATGGCTACGGGCGCGCCGAAGGCTGCGTGATCCTTGCGCTCA
+
+
+
+
+­
+
F
K
900
901
AGCGGCTGAGCGATGCGGTACGCGACCAGGACCCGGTGCTGGCCGTGATCGAGGGTAGTG
+
+
+
+
+
+
R L S
D
I E G S A
960
961
CGGTCAACAACGACGGCGCCAGCAGCGGCATCACCGTGCCCAACATCCACGCCCAGGAAG
+
+
+
+
+
+
1020
DAAADG YGRAEG CV I LAL
DAVRDQ
NNDGA
S
S
G
PVLAV
I
TVPN I HAQEE
1021
AGGTGATCAGGCTGGCGCTCGGCCAGGCCGGGCTCCAGGGCAGCGAGGTCGACTATGTCG
+
+
+
+
+
+
L G
L Q G S
1080
1081
AGGCCCATGGCACCGGCACCGCGCTGGGCGACCCGATCGAACTGCACGCCCTGCATGCGG
+
+
+
+
+
+
E L
L
1140
1141
TACTGGGCAAGCAGCGGCCCGTCGATGCACCGCTGCTGGTGGGCTCGATCAAGGCCAACA
+
+
+
+
+
+
L G
P
P L
S I
1200
1201
TGGGGCATCTCGAACCGGTCGCCGGGGTGACCGGGCTGGCCAAGGTCCTGCTGTGCCTGC
+
+
+
+
+
+
G
L L C L Q
1260
1261
AACAAGAGGCCCTGGTGCCCCAGGTGCACTTCAACACGCCCAACCCGCGGATCGAATGGG
+
+
+
+
+
+
Q
F
EW D
1320
1321
ATCGCCTGGCCCTGAAGGTGGTCACCGAATCCACGCCCTGGCCACGCCAGGGCAAGGCGC
+
+
+
+
+
+
R
L
E S T PW P
G
1380
GGCACGCCGGTGTCAGTTCGTTTGGTGTCACCGGCACCAACGCCCATGTGCTAGTGGGCG
+
+
+
+
+
+
1440
1381
1441
1501
I RLA
EVDYVE
QAG
A H G T G T A L G DP I
KQR
VDA
EA LVP QVH
L AK V
N T PNPR I
KVVT
LA
HAGVS
S
F
K ANM
LVG
G HL E PV A G V T
HAV
HA
KAR
RQ
GVT GTNAHVLVG
D
ACGCGCCGCTGCGCGAACGTGCCCAGGGGCGCGACAACCCCTGGCAGCTGATCACTCTGT
+
+
+
+
+
+
A P
G
R DN
L
T L S
LRE RAQ
PWQ
I
CGGCCAAGGGCGAGACGCCCCGGCGCCAGATTGCCGGACGCTATGAACGTTTTATCGCCG
+
+
+
+
+
+
AK
G
E
T
P
RRQ
I AG
RYERF
I AD
1500
1560
128
1561
1621
ACAACAGCCAGCTCGAACTCAAGGACCTGTGCTACACGGCGAACGTCGGGCGGGCGCACT
+
+
+
+
+
+
Q L
D L C
H
F
NS
ELK
YTANVG RA
TTGGCCATCGTTTCGCGGCCGTGGCCGATAGCCGTGAGGGGCTGCGCGAGCAACTGGCAG
+
+
+
+
+
+
GHRFAAVAD
S
REG LREQ LAA
1620
1680
CCTATGCGTCACGCAAGGTCGTGGGGCATGTATTCGAAGGGCGCTGCCAGGGAGCGGCGG
+
+
+
+
+
+
1740
1741
CGCCGCTGGTGATGCTCTTTCCGGGGCAGGGCTGCCAGTACCGGGCAATGGCCCAGGCAC
+
+
+
+
+
+
P
F P
C
Q
L
1800
1801
TGTATGACAGCGAACCATTCTTCAAGGCGCAGATCGATGAATGCCGCGCCCTGTTGCAGC
+
+
+
+
+
+
1681
Y A SRK VVGHVF EGRCQG
LVML
Y
D
S
E
P
GQG
F
F
AAA
YRAMAQA
IDECRALLQ LQ P
K
1861
CGCTGATGGACGTGGACCTGCTGACCCTGGTGCTGGACGCGGGTGCGGCCAGTGACAGCT
+
+
+
+
+
+
L L T
L
S D S Y
1921
ACCTGCAACAGACCCGCTATGCCCAGCCGGCGATATTCGCGGTCGAATATGCCCTGGCGC
+
+
+
+
+
+
1981
2041
2101
2161
2221
2281
2341
LMDVD
DAGAA
LV
LQQTRYAQ PA
I
FAVEYALAR
1860
1920
1980
GGTTGTGGATGCATTGGGGGGTCGCTGCCGATGCGCTGTTCGGACACAGTTTCGGCGAGA
+
+
+
+
+
+
DA L F G H S F
I
2040
TCAGTGCAATCTGTGTAGCCGGGGCGGTATCCCTGGCTGACGCGCTGCGTATGGTGGAGG
+
+
+
+
+
+
S A
C
SS
DA L
2100
CGCGTGGGCGCCTGGCCCAGCAACTGATGACGGCCGGCGGCGCGATGTACGCACTGGGCA
+
+
+
+
+
+
R L
Q
G
GM
2160
TGAGCGAGGCGCAACTGCTGGAGCTGCTCAAGGACCGGCCCGGCAGCGCGATCGAACTGG
+
+
+
+
+
+
S
L L E L L K
P G S A I E
2220
CGGCGGTCAACAGCCCGCAGGACGTGGTGGTGGCCGGGCCGCAAGCCGAGGTCCAGGCGC
+
+
+
+
+
+
L
2280
LWMHWGVAA
VAGAV
AQ
EAQ
GE
LA
LMTAG
RMVEA
A M Y A L GM
DR
LA
A V N S PQDVVV A G PQA E
VQA
TGGCCGAAGCGGCGCTCGCCAGCGGTTGCAAGGTCAAGAAGCTTGCGGTTTCCCATGCCT
+
+
+
+
+
+
A E A A L A SGCK VKK L A V
S
H AF
TCCATACCGCAGCCACCGAGCCGATGCTTGAAGCGTTCCGCCAGACGGTGGCGCAGATCA
+
+
+
+
+
+
H
E P M L E A F
QQ
I T
TAAT
RQTVA
2340
2400
129
2401
CCTTCAGCGAGCCGCGCTTGCCGGTCATCAGCAGTGTCACTGGCCGGGTGCATACGCTCT
+
+
+
+
+
+ 2460
L S
2461
CCAGCCTGAGCTCGCCCGATTACTGGTGTACGCACACCCGCCAGGCCGTGAGGTTCTGCG
+
+
+
+
+
+ 2520
F SEPRLPVISSVTGRVHT
S
2521
2581
L SS PDYWC THTRQAVRF CE
AAGGGGTCAACACCCTGATCGCAGAGCTGGGGGTGAAAACCTTCCTTGAAGTGGCCTCCG
+
+
+
+
+
+ 2580
S
D
G VNTLIAELGVK T
F LEV A
ATGCTGTGTTGACGCCGCTGATCGGCCGTCACCCCCTGGCGGACGGCAGCCTGGTCCTGG
+
+
+
+
+
+ 2640
A V L T P L
L A ADG SLVL A
2641
CCAGCCTGCGCCGGGCCGGCGATCCGTCCAGGGACCTGCGCCTGGCCGCCGCGCAGCTCT
+
+
+
+
+
+ 2700
S L R R A G D P S R D L R L A A A Q
L Y
2701
ACGTGGGCGGCCATAACCTCGACTGGGCCCGGCTGCACGAGCATGACGGGGCCTTGCGCC
+
+
+
+
+
+ 2760
G G H N L D W A R L
2761
H EHDG A LRQ
AGGCCTTGCCCGGGTATGCGTTTCAGCGCCAGCGCTACTGGTTCGACAACGCCAGCGGCT
+
+
+
+
+
+ 2820
A L
P
G Y A F Q R Q R Y W F DNA SG S
2821
CGCCCCTGCAACAGGTCGCTGCGGGAGCGGTCGGCCGGCTGCTGGGGCATTC.C4GTCAATG
+
+
+
+
+
+ 2880
P L Q Q V A A G A V G R L L G H S V N A
2881
CACCCACTCCGGCATTCGAGTCGACGCTCGATAGCGCGCTCCTGCAGGTTGTGGGCGGCG
+
+
+
+
+
+ 2940
P
T P A F E S T L D S A L L Q V V G G E
2941
AGATCCGCGATGACCTGGCGCTGCTGCGTCCTGACCGGTTGCTGGCAGCCCTGAGCGATG
+
+
+
+
+
+ 3000
3001
AGCTGGCCGGCCACTTGCAGCTGGACACCTATGGGGTGAGCCTCGCCAGCATCACCCCGG
+
+
+
+
+
+ 3060
L A G H L Q L D T Y G V S L A S I
RDDLALLRP P DRL LA AL SDE
T PA
3061
CCCTGGCGTTCCATGTCGATGACCAGTTGCATCTGTTCACCGAACTCAAGCCGCTGTCCG
+
+
+
+
+
+ 3120
L A F H V D D Q L H L F T E L K P L S G
3121
GGGCGGCCTGGGAGGTCAACTGTTCGGCCCTGAGCGCAGCGGCCAAGGTTGCCGGTGCCG
+
+
+
+
+
+ 3180
A A W E V N C S A L S
A A A K V AG AD
3181
ATTGGCAGCCGGTCCTGTCGCTGACCCTGGAAGGCCTGCCGGCGGCTGTCCGGGGCGGCC
+
+
+
+
+
+ 3240
W Q P V L S L T L E G L P A A V R G G L
130
3241
TGCCGGACCCGGGCCATGCCGCCACCCAGGACGCAGGCTTTGTCTACCACATGCAGTTGC
+
+
+
+
+
+ 3300
P DPGHA A TQDAGF VYHMQL
P
3301
CGGCAGAACCCGATCCGCACGGCAATCACCTTGGGCAGGTCCTCGAGTACCTGGGGCAGG
+
+
+
+
+
+ 3360
A E P D P H G N H L G Q V L E Y L G Q A
3361
CGGTGCCGGGCGATGCGCCGGGCTCGATAAGCGGCATCCGCCGCTGGGTCGCCAGCCGGT
+
+
+
+
+
+ 3420
3421
CCGCTTCGCCCCGGGCACAAAGCCTGGTGATTGCCACGAGCCAGGCACATCCGCAGCAGT
+
+
+
+
+
+ 3480
A
S P R A Q S L V I A T S Q A
3481
TCGACTGCGCCTTGTACGATGCGCACGGCCAGTGTGTAGGCAGCCTGGAAGGGCTGACCC
+
+
+
+
+
+ 3540
D C A L Y D A H G Q
L
3541
TGGCTGCCGCCCCGAGTGAGGAGGCGCTGCGTGGCATGCTTTACCAGCCCGACGTGCTCT
+
+
+
+
+
+ 3600
A A A P S E E A L R G M L Y Q P D V L Y
3601
ACAGCCTGGATTGGCTCGAACGCCCTCGCCAGCGCGCCGAAGTGCCGCAGCAGGACCGCT
+
+
+
+
+
+ 3660
PGDAPGSISGIRRWV A SR S
H PQQF
C V G SLEGL T
S LDWLER PRQRAEVPQQDRL
Tn5 insertion
mutant 4175
3661
TGTTCACCCTGGTCAGCCGCTCGCCGGAGACCGCCGCTCCCCT GGTCGAACACCTGCAGC
+3720
F T L V S R S P
3721
GTGGCGGGCATCGTGCCCGGGTCCTGTGCCCGCAGCAGTTGCTCGATAGCGCGCGGAGGG
+
+
+
+
+
+ 3780
G G H R A R V L C P Q Q L L D S A R R V
3781
TGCTGGCCCAGGACCCGAGCCAGGCATTGACCGATGAAGGCGAGGCCTTGGCCGCCGATA
+
+
+
+
+
+ 3840
E T A A PL VEHLQR
L AQDPSQAL T DEGEAL
A A DI
3841
TCATCGTGCTCGATGGCAAAGACATCGAGGATGCCGGCTCGACCACGCTGCAGAGCCTGT
+
+
+
+
+
+ 3900
3901
CGAGCCTGCAGGCGACACTGTTCCACCCGTTGCTGGAGATGGTCCAGGCGCTCATCGAAC
+
+
+
+
+
+ 3960
3961
TGGGCCCGCGCGGTGGCCGGTTGTGGCTGGTGACCCAAGGGGCAAACCCTGTTGGCCTGG
+
+
+
+
+
+ 4020
G P R G G R L W L V T Q G A N
D
IVLDGKDIEDAGS T T LQSLS
S LQA TLFHPLLEMVQAL IEL
P VG L
131
4021
ATCGCGACCAGCCCCTGCAAGTGGCGACCGGGCCGTTGTGGGGGCTGGGCAAGACGCTCG
+
+
+
+
+
+ 4080
RDQPLQVA TGPLWGLGK T
L A
4081
CCCTGGAGCACCCCGAACACTGGCGCGGGCTGATCGACCTGGCCCCCGACGATCCTCACT
+
+
+
+
+
+ 4140
L I
D
4141
GGGCAAGGGCGCTGGCCGAAGAAGTCAGCGACTCCGATGGCGACGACAAGATCTGCCTGC
+
+
+
+
+
+ 4200
A R A L A E
R
4201
GCCCGGGCAAGCGTTATGTCCAGCGCCTGAACCATTTCAGCGCTGCGCAGTTGCCAGCAC
+
+
+
+
+
+ 4260
IDL AP DDPHW
E V S DSDGDDK IC L
P GKRYVQRLNHFSAAQL P
A Q
4261
AGGCCTATGCACCTTGCCCGCAGGGCAGTTACCTGATTACCGGCGGCATGGGTGGAATTG
+
+
+
+
+
+ 4320
4321
GCCTGGCCATGGCCCAGTGGCTTCTGGATAAAGGCGCGGGCGCTGTGCTGATCACCGGCC
+
+
+
+
+
+ 4380
L A M A Q W L L D K
A Y A PC PQGSYL I
T GGMGGIG
G A G A V L ITGR
4381
GGCGGCCACTGGAGGATGTCGCCACGGGCCTGGAGCGCTTCGGCGCTGCGGCATCGCGGG
+
+
+
+
+
+ 4440
R P L E D V A T G L E R F G A A A S R V
4441
TGAGTTACGTCCAGGCCGACATCACCTCGCCCCAGGACATGCAGCGGCTGTTCTGCGGTC
+
+
+
+
+
+ 4500
G
L
4501
TGGAGGCGTCGGGCGCGAGCCTGAAGGGCATCTTTCATGCCGCCGGGATTTCAATTCCGC
+
+
+
+
+
+ 4560
E A S G A
L K
4561
AGGATCTCAAGGACGTCGACCGTGACAGTTTCGACCAGGTGATGCGGCCCAAGGTCGAGG
+
+
+
+
+
+ 4620
D L K D V D R D S F D Q V M R P K V E G
4621
GCACCTGGTTGTTGCACGAACTGTCCCTGGGGCTGGACCTGGACTTCTTTGTCCTGTGTT
+
+
+
+
+
+ 4680
4681
CGAGCATCGCCAGTGTCTGGGGTTCGCAGCATGTCGCCAGCTACGCAGCCGCCAATCAGT
+
+
+
+
+
+ 4740
S IA S V W G S Q H V A S Y A A A N Q F
4741
TCCTCGACAGCCTGGCGTGGCAGCGTCGGGCCATGGGCCTGAGTGCCCTGGTGATCGACT
+
+
+
+
+
+ 4800
4801
GGGGGCTGTGGGCCGGCGGCAGTCACCTGTTCGACGAGCAGGTGCTGAATTTCCTCACCA
+
+
+
+
+
+ 4860
G L W A G G S H L F D E Q V L N F L T S
S YVQADI T SPQDMQRLF C
G GIF HAAG IS I PQ
TWLLHELSLGLDLDFF V LC S
L DS L AWQRRAMGL SA L V I DW
132
4861
4921
GCGTTGGCTTGAAACAGATCGCCCCGGTACAGAACGTCGGCCTGCTGTCGCGGATTCTGG
+
+
+
+
+
+
G
L K Q I A
L L S
PVQNVG
5161
5341
S
R G PQPLLQY IR S QA P
+ 5040
T
A
R
G DS SNVEILQQL A G A DEA A+ 5100
ALA
YVWE
YAQ
LGVK
5160
AGACCGAACAGGTGCGGGCCAAGCTCGAGGATGGCGGCAGCCTGATGGACTACGGCCTGG
E
QVRAK
L
E
D
G
G SLMDY GL D+ 5220
ACTCGCTGCTGGTGATGGACATGGTCGCCCGCTGCCGGCGCGATCTGAAGCTGGAGATCA
+
+
+
+
+
+
S
5281
+ 4980
P Q M V V S G V D W N R F K PL L
CTGCCCTGGCATTGCTGGATGACTACGTGTGGGAGCAGTACGCGCAGTTGCTCGGGGTCA
+
+
+
+
+
+
L L D D
Q
L
T
5221
L
GGGCCGGCGACAGCAGCAACGTGGAGATCCTGCAGCAACTGGCGGGCGCCGATGAAGCCG
A
5101
E
TCGAATCCCGCGGCCCGCAACCGCTGCTGCAGTACATCCGCAGCCAGGCTCCGACCGCCA
E
5041
4920
CCAGCGAGCTGCCCCAGATGGTGGTGTCAGGGGTGGACTGGAATCGCTTCAAGCCACTGC
S
4981
R IL A
L L V M D M V A R C RRDLK L
E
I
AGGCCCGCGAGTTTCTTGAGTGTCCGGGCCTGATGTGGCCGGACTTCCTGGCCCGTTCGA
+
+
+
+
+
+
A REF LECPGLMWPDF L AR S
TAAAGGAACAGGGCTGCGTGGCCGAGGCCTGACGGGCCGCGGGCAGTGCAA
+
+
+
+
+- 5391
KEQ
G
C
VA EA
*
5280
K
I
5340
133
Appendix 4. Nucleotide Sequence and Translated Product of pltA
AAGACCTGAATGCAATATCCAGGACTGTCGAGCAACTAAAGGCCGAGTGCGCCTAACAGG
1
+ 60
GAGTGGGCAATGAGCGATCATGATTATGATGTAGTGATTATCGGTGGCGGGCCGGCGGGT
+
+
+
+
+
+ 120
M S D H D Y D V V I
61
I GGG PA G
121
TCGACCATGGCCTCCTACCTGGCAAAAGCCGGTGTCAAATGCGCGGTGTTCGAAAAAGAA
+
+
+
+
+
+ 180
S T M A S Y L A K A G V K C A V F E K E
181
CTGTTCGAGCGCGAGCATGTTGGCGAGTCGCTGGTACCGGCCACCACTCCGGTGCTGCTG
+
+
+
+
+
+ 240
L F E R E H V G E S
L V P A T T PVL L
241
GAAATCGGGGTGATGGAAAAGATCGAGAAAGCCAACTTCCCGAAGAAGTTCGGCGCTGCC
+
+
+
+
+
+ 300
E I G V M E K I E K A N F P K K F G A A
301
TGGACCTCGGCAGATTCCGGCCCCGAAGACAAGATGGGCTTCCAGGGGCTGGACCACGAT
+
+
+
+
+
+ 360
W T S A D S G P E D K M G F Q G L D H D
361
TTCCGTTCGGCGGAAATCCTCTTCAACGAGCGCAAGCAGGAAGGGGTCGATCGCGACTTC
+
+
+
+
+
+ 420
F R S A E
L F N E R K Q E G V D R D
F F
421
ACGTTCCACGTCGACCGCGGCAAGTTCGACCGCATTCTTCTGGAGCACGCAGGTTCGCTG
+
+
+
+
+
+ 480
T F H V D R G K F D R I L L E H A G S L
481
GGGGCCAAGGTCTTCCAGGGCGTGGAGATCGCTGACGTCGAGTTTCTCAGCCCGGGCAAT
+
+
+
+
+
+ 540
G A K V F Q G V E I A D V E F L S
P GN
541
GTCATTGTCAATGCCAAGCTGGGCAAGCGCAGCGTGGAGATCAAGGCCAAGATGGTGGTG
+
+
+
+
+
+ 600
I
V N A K L G K R S V E I K A K M V V
601
GATGCCAGCGGTCGCAACGTGCTGCTGGGCCGCCGGCTGGGCTTGCGAGAAAAGGACCCG
+
+
+
+
+
+ 660
D A S G R N V L L G R R L
L R E K D DP
661
GTCTTCAACCAGTTCGCGATTCACTCCTGGTTCGACAACTTCGACCGCAAGTCGGCGACG
+
+
+
+
+
+ 720
F N Q F A I H S W F D N F D R K S A T
134
721
CAAAGCCCGGACAAGGTCGACTACATCTTCATTCACTTCCTGCCGATGACCAATACCTGG
+
+
+
+
+
+ 780
Q S P D K V D
H F
DY IF I
781
L PM TNT W
GTCTGGCAGATCCCGATCACCGAAACCATTACCAGCGTGGGCGTGGTTACGCAGAAGCAG
+
+
+
+
+
+ 840
W Q I P I T E T I T S V G V V T Q K Q
AACTACACCAACTCCGACCTCACCTATGAAGAGTTCTTCTGGGAAGCGGTGAAGACCCGG
841
+
N Y T N
901
+
S
+
+
+ 900
+
D L T Y E E F F W E A V K T
R
GAAAACCTGCATGACGCGCTGAAGGCATCGGAGCAGGTCCGCCCGTTCAAGAAAGAGGCG
+
+
+
+
+ 960
+
E N L H D A L K A S E Q V R PF K K E A
961
GACTACAGCTACGGCATGAAAGAAGTCTGTGGCGACAGCTTCGTGCTGATCGGCGATGCC
+
+
+
+
+
+ 1020
D Y S
I
G D A
Y G M K E V C GDSF V L
Tn5 insertion
mutant 4236
1021
GCACGGTTCGTCGACCCGATCTTCTCCAGCGGCGTCAGCGTTGCACTCAAC AGTGCGCGC
A R F V D
1081
1141
1201
S
G V
S
S
+ 1080
V A L N
+
+
+
+
T HYEGMIRNG IKNWYEF
S A R
+ 1200
+
I
T
L
TATTACCGCCTGAACATCCTCTTCACCGCGTTCGTTCAAGACCCACGCTACCGCCTGGAC
+
R
+
L N
L
+
F
+
T A F V Q QDPR Y
+ 1260
+
R
L
D
ATCCTGCAATTGCTGCAAGGGGACGTCTACAGCGGCAAGCGCCTGGAAGTGCTGGACAAG
L
Q
+
L
+
L
+
Q G D V Y
+
S
G
K
R
+
L
E V
+ 1320
L
D
K
ATGCGCGAAATCATCGCTGCGGTTGAAAGCGACCCGGAACACCTCTGGCACAAGTACCTG
+
+
+
+
+
+ 1380
M R E
1381
F
ACTCACTACGAAGGCATGATCAGGAATGGCATCAAGAACTGGTATGAGTTCATCACGCTC
I
1321
I
ATCGCCAGCGGCGACATCATCGAGGCGGTGAAGAACAACGACTTTAGCAAGTCCAGTTTC
+
+
+
+
+
+ 1140
I
A S G D I I E A V K N N D F S K S S F
Y Y
1261
+P
I IAAVE S DPEHLWHK Y
L
GGCGACATGCAGGTTCCTACCGCCAAACCCGCGTTCTAAACACTAAACACCCAGTCGAAG
+
+
+
+
+
+ 1440
DMQVPTAK K
P A F*
135
Appendix 5. Nucleotide Sequence and Translated Product of pltD
CGCGGGCAGTGCAACCCAGACCCATGATCTGTGATTGAGGTGGTTATGAACGATGTGCAG
+
+
+
+
+
1+
MN DV Q
60
TCTGGCAAGGCGCCAGAGCATTACGACATTCTCTTGGCGGGCAACAGCATCAGCGTGATC
+
+
+
+
+
S V I
120
ATGCTCGCCGCCTGCCTGGCCCGGAACAAGGTCCGGGTCGGTTTGTTGCGCAACCGGCAG
+
+
+
+
+
L
180
61 +
S GK A PEHY DI L L AGNS I
121 +
MLAAC LARNKVRVG
L RNR Q
ATGCCCCCCGACCTTACCGGTGAGGCGACGATTCCCTATACCTCGATGATTTTCGAGCTG
+
+
+
+
+
181 +
MP
P
DLTGEAT I
P
YT
S M IF EL
ATTGCCGACCGCTATGGCGTGCCGGAAATAAAGAATATCGCCCGCACCCGGGATATCCAG
+
+
+
+
+
A
P E I
I
R D I Q
241 +
I
DRY GV
KN
ART
240
300
Tn3-nice insertion
mutant 4366
CAGAAGGTGATGCCGTCTTCCGGGGTCAAGAAGAACCTCGGGTTC ATCTATCACCAGCGC
+
+
+
+ - -+
Q
P S S
L G
301 +
KVM
F IYHQR
GVKKN
AGCCGGGCGGTGGACCTGGGCCAGGCGCTGCAATTCAACGTGCCCTCCGAGCATGGCGAG
+
+
+
+
+
361 +
S
RAVDLGQALQ
F
N V P SEHGE
AACCATCTGTTCAGGCCCGATATCGATGCCTATCTGCTGGCGGCGGCCATCGGTTATGGC
+
+
+
+
+
421 +
N HL F R PD I DA Y
L
LAAA I GY
AQLVE I
DN
S
PEVLVE
D
420
480
G
GCGCAGCTGGTGGAGATCGATAACAGCCCAGAGGTGCTGGTCGAGGACAGCGGGGTCAAG
+
+
+
+
+
481 +
360
S CVK
540
GTAGCTACGGCACTGGGGCGCTGGGTCACTGCCGATTTCATGGTTGATGGCAGCCAGGGC
+
+
+
+
+
L
600
GGCCAGGTGCTGGCGCGGCAGGCTGGCCTGGTCAGCCAGGCTTCGACGCAGAAGACCCGG
+
+
+
+
+
G
S T Q K T R
660
541 +
601 +
ATA
G R W V T A D F M V D G SQG
QVLARQAG LVS
QA
136
ACCCTGGAATTCTCCACTCATATGCTCGGGGTGGTGCCGTTCGATGAGTGCGTGCAGGGC
661 +
+
+
+
+
+
720
T L E F S T H M L G V V P
F DEC VQG
GATTTTCCCGGCCAGTGGCATGGCGGCACTCTGCATCACGTGTTCGATGGGGGCTGGGTG
721 +
+
+
+
+
+
780
D
F
P GQWHGG T LHHVF DG GW
V
GGGGTCATCCCGTTCAACAACCATCAGCACTCGCGCAACCCTTTGGTCAGCGTGCTGGTT
781 +
+
+
+
+
+
840
G V
I
P
F N N H Q H
S
R N
P
L V S V L V
TCACTGCGTGAGGACCTCTGCCCGAGCATGGACGGCGACCAGGTCCTGGCCGGCCTGATC
841 +
+
+
+
+
+
900
S L R E D L
C PSMDGDQVLAGL I
GAGCTGTACCCCGGCCTGGGGCGGCACCTGTCCGGCGCCCGGCGGGTGCGCGAGTGGGTG
901 +
+
+
+
+
+
960
E L Y P G L G R H
L SGARRVREWV
CTGCGCCAGCCGCCCCGGCAGGTCTATCGCACGGCGCTCGAACGCCGCTGCCTGATGTTC
961 +
+
+
+
+
+
1020
L R Q P P R Q V Y R T A L
E R R C LMF
GACGAGGGCGCCGCGAGCAACGATCTGTTGTTCTCGCGCAAGCTGTCCAATGCTGCGGAA
1021 +
+
+
+
+
+
1080
D E G A A S N D L L F S R K L S N A A E
CTGGTTCTGGCCCTGGCGCACCGGCTGATCAAGGCGGCGCACAGCGGTGACTACCGCAGC
1081 +
+
+
+
+
+
1140
L V L A
K A A H S G D Y R S
L AHRL I
CCGGCCCTGAATGATTTTGTCCTGACCCAGGACAGCATCATCAGCTTGAGTGACCGGATC
1141 +
+
+
+
+
+
1200
P A L N D F V L T Q D S I
I
S L
S DR I
GCCTTAGCGGCTTATGTGTCGTTTCGCGACCCCGAGTTGTGGAATGCCTTCGCCCGTGTC
1201 +
+
+
+
+
+
1260
A L A A Y V S F R D P E L W N A F A R V
TGGCTGCTGCAGTCGATTGCCGCCACCATCACCGCGCGCAAGATCAACGATGCCTTTGCC
1261 +
+
+
+
+
+
1320
W L LQS IAA T
I
T ARK INDAF A
AAGGACCTGGACCCGCGAGTGTTCGATGAAATCGACCAGCTCGCAGAGGACGGTTTCTGG
1321 +
+
+
+
+
+
1380
K DLDPRVF DE IDQL A E DGF W
ATGCCTCTGTATCGGGGGTACAAGGATATTCTCAACACTACGCTGGGCCTTTGTGATGAC
1381 +
+
+
+
+
+
1440
M P L Y R G Y K D I L N T T L G L
C DD
137
GTCAAAAGCGCCAAGGTCTCTGCTGCGCACGCGGCGAGCAGCATCTTTGCGGAGCTTGCC
1441 +
+
+
+
+
+
1500
K S A K V S A A H A A S S
F A E L A A
AACGCCAGTTTTGTTCCGCCTATTTTTGATTTTGCTAATCCTCACGCTCGTGTCTATCAA
1501 +
N A
+
S
F V
+
P
P
+
I
F
D F A N
+
P
1560
+
H A R V Y
Q
CTGACCACCTTGAGAAAGCTCAAGGCGCTCTGGTGGGGCCTGATGCAAGTGCCCTCAGAG
1561 +
+
+
+
+
+
1620
L T T L R K L K A L W W G L M Q V P S E
GTCGGACGGCTGATTTTCTATCGATCCTTCAGAAAACCTTCCCTGCGCAAGGAGAGTTGA
1621 +
+
+
+
+
+
1680
G R L I F Y R S F R K P S L R K E S *
AATGGACTTCAACTACGACGATACCCAGAAAAAACATGCGGCCATGATCGCCCAGGTGTG
1681 +
+
+
+
+
+
1741
138
Appendix 6. Nucleotide Sequence and Translated Product of orf-S
GCGACCACCTTGCAAGACAAGAGCCAGACATCATGAATCAGTACGACGTCATTATCATCG
1
MNQYDV I
+ 60
I
I
G
61
GTAGTGGTATCGCCGGCGCGCTGACCGGCGCCGTCCTCGCGAAGTCCGGGCTGAACGTTC
+
+
+
+
+
+ 120
S G I A G A L T G A V L A K S G L N V L
121
TGATCCTCGACTCGGCCCAGCACCCACGATTCTCCGTCGGCGAAGCGGCGACACCGGAAA
+
+
+
+
+
+ 180
L D S A Q H P R F S V G E A A T PE S
I
GCGGTTTTCTGCTGCGTTTGCTCTCAAAGCGCTTCGACATCCCTGAAATCGCCTACCTCT
181
+
G
F
L
+
L
R
L
+
L
S
+
K R F
D
+
P
I
+ 240
E IAYL S
CGCACCCCGACAAGATCATCCAGCACGTCGGTTCGAGCGCCTGCGGGATCAAGCTGGGCT
241
+
H
P
D K
+
I
I
Q
+
H V G
+
S
S A C
+
+
300
G IK LGF
301
TCAGTTTTGCCTGGCATCAAGAGAACGCGCCGTCGTCCCCCGACCACCTTGTGGCCCCGC
+
+
+
+
+
+ 360
S
A
P P
361
CGCTGAAGGTGCCGGAAGCCCATC ii ri CCGGCAGGACATCGACTATTTCGCCCTGATGA
+
+
+
+
+
+ 420
L K V P E A H L F R Q D I D Y F A L M I
421
TTGCCCTGAAACACGGCGCCGAATCCAGACAGAACATCAAGATCCGTCGACCTGCAGCCA
+
+
+
+
+
+ 480
A L K H G A E S R Q N I K I
F A WHQENA PSS PDHL 4
R R PAAK
AGCTT
481
485
L
139
Appendix 7. Nucleotide Sequence and Translated Product of pltR
1
AGCCGTACTAAGGAGGCTGTCATGATCTATTTGTAATTTGCCTTTACAAAAATAAGACAC
+ 60
TAAAAATTCTAAAAGGATTTAGGAATGAAGGCGCTAGGCGTTGTCAATGACATAGACCTT
+
+
+
+
+
+ 120
61
M K A LGVVN D
I
D L
121
TATATATCCGTAACTAAAACGGGTAGTTTCTCCGAAACAGGAAGACTACTTGGCATCCCA
+
+
+
+
+ -+ 180
Y I S V T K T G S F S E T G R L L
G I P
181
CCTTCTTCGGTGATGCGCAGAATCAATAGCCTCGAAAAAGAACTTGAAACCTGTCTATTC
+
+
+
+
+
+ 240
P
L E T
S SVMRR IN S LEK E
C LF
AATCGATCGACAAAATGCTTGATACTTACCGAAACAGGCCTGCTCTTTCTCGAACATGCA
241
+
N R
S
T
+
K
C
L
+
L
T
+
E
T
+
+ 300
G GLLF L EH A
301
AAGAACATCTCGAGATGCATTGGGCATGCTAGATCAGAAGTAAAAGAACACACTGCATCG
+
+
+
+
+
+ 360
K N I S R C I G H A R S E V K E H T A S
361
ACCCTTGGCGTTCTCAAGGTTTCAGCACCCGTGGCCTTTGGACGGCGCCATGTAGCGCCT
+
+
+
+
+
+ 420
T L G V L K V S A P V A F G
R R H V A P
421
TTATTGAGCAGAATACTGAACCATCATCCCGGTTTGAAAATTGAATTCTCGCTTAACGAC
+
+
+
+
+
+ 480
L L S R
481
AAAGCTCTCGACCCCAGCATCGATAACGTCGATATCTGTATCAAGCTGGGCATATTGCCG
+
+
+
+
+
+ 540
K A L D P S I D N V D I C I K L
I LNHHPGLK IEF S LND
G IL P
541
GACAGTAATCTGATTCCAACGAAACTTGCGGATATGCGGCGCGTGCTCTGCGCAAGCCCG
+
+
+
+
+
+ 600
D S N L I P T K L A D M R R V L C A S P
601
GAATACATCCGGCAACACGGCTGTCCGCAAACTCTTGAAGACCTGTACCAACATGCCTGC
+
+
+
+
+
+ 660
E Y I R Q H G C P Q T L E D L Y Q H A C
661
CTGATCCACAGCACCTGCAGCAACTTCTCACTGACCTGGCAATTCAAGGTCGATGGCCTG
+
+
+
+
+
+ 720
L I H S T C S N F S L T W Q
F KVDG L
140
721
CTCAAGAAGCTCATGCCCAGCAGCCGGCTATCGGTCAACAGTTCCGAGTTGCTGGTGGAC
+
+
+
+
+
+ 780
L K K L M
P S S R L S V N S S E L L V D
781
GGCGCCCTTCAGGGCATAGGAATCATCCACGCGCCCACCTGGCTGGTGCATGAACAGATC
+
+
+
+
+
+ 840
G I I H A P T W L V H EQ I
G ALQG I
841
GCCAGCGGCCAACTGGTGTCGCTGCTCGACGAATATTGCGAGGCCGATCCGCAACAGGGA
+
+
+
+
+
+ 900
A S G Q L V S L L D E Y C E A D
P QQG
901
GCGATCTACGCACTGAGGGCACGCAGCAGCGTTGTCCCGGCCAAGACCCGCCTGTTCATC
+
+
+
+
+
+ 960
A
961
I
Y A L R A R
S
S V V
P
A K T R L
F
I
AATGAGTTGAAACGCTCGATCGGCAGCACGCCTTACTGGGACTTGCCGTTTGAAAAGGAA
+
+
+
+
+
+ 1020
N E L K R S I G S T P Y W D L P F E K E
1021
ATACCGCAGACCCTGGCCACCCTGCATTTCGATACCGCAATGCATTCAGCGC'i 11 CCAGA
+
+
+
+
+
+ 1080
I
P Q T L A T L H F D T A M H S A IJ S R
1081
GCGACCACCTTGCAAGACAAGAGCCAGACATCATGAATCAGTACGACGTCATTATCATCG
+
+
+
+
+
+ 1140
A T T L Q D K S Q T S *
1141
GTAGTGGTATCGCCGGCGCGCTGACCGGCGCCGTCCTCGCGAAGTCCGGGCTGAACGTTC
+ 1200