AN ABSTRACT OF THE THESIS OF
Lorena I. Rangel for the degree of Master of Science in Botany and Plant Pathology
presented on July 31, 2012.
Title: Genomics-enabled Exploration of Insect Toxicity in the Pseudomonas
fluorescens Group
Abstract approved:
______________________________________________________________
Joyce E. Loper
Pseudomonas is a diverse genus of Gamma proteobacteria that are ubiquitous in
the natural environment, including soil, water, plant surfaces, and animals. The
Pseudomonas fluorescens group is a diverse collection of seven subgroups and more
than 50 named species. This group is known for their production of a variety of
secondary metabolites and antimicrobial compounds that contribute to biological
control of plant diseases. Recently, they also have been shown to relay both oral and
injectable insect lethality by mechanisms that are unknown. The first objective of this
study was to determine if ten fully sequenced strains in the P. fluorescens group are
lethal to insects, using larvae of the tobacco hornworm (Manduca sexta) as a model
system. A second objective was to characterize putative insect toxin complex (Tc)
genes in the genomes of the ten strains. Additionally, genes encoding putative insect
toxins (Tc) and possible modes of immunosuppression (the AprA metalloprotease and
GacS/GacA regulatory system) were examined for their contribution to lethality of one
strain of P. fluorescens.
I established that six of ten strains representing diverse lineages of the P.
fluorescens group exhibited injectable lethality to larvae of M. sexta, while four of the
strains exhibited no significant lethality. Seven strains of the P. fluorescens group had
Tc gene clusters. These clusters were categorized into six distinct types, based on the
organization, genomic context, G+C content, and phylogeny of the Tc genes. One of
the types included genes from strain A506, which was selected for further study
because of its injectable lethality towards M. sexta, and its importance as a model strain
in studies of phyllosphere microbial ecology and as a commercial biological control
agent. Of the genes (aprA, gacS and tcaABC-tccC) evaluated for their roles in insect
lethality, the tcaABC-tccC cluster had the greatest effect on insect lethality by A506.
This study provides the first evidence for a significant effect of a Tc cluster on insect
lethality by a Pseudomonas species. Nevertheless, none of the genes evaluated were
fully responsible for the injectable lethality caused by A506, indicating that loci other
than those evaluated here contribute to insect toxicity by this strain.
©Copyright by Lorena I. Rangel
July 31, 2012
All Rights Reserved
Genomics-enabled Exploration of Insect Toxicity in the Pseudomonas fluorescens
Group
by
Lorena I. Rangel
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented July 31, 2012
Commencement June 2013
Master of Science thesis of Lorena I. Rangel presented on July 31, 2012
APPROVED:
Major Professor, representing Botany and Plant Pathology
Chair of the Department of Botany and Plant Pathology
Dean of the Graduate 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.
Lorena I. Rangel, Author
ACKNOWLEDGEMENTS
I would like to thank Oregon State University for the Diversity Advancement
Pipeline Fellowship that funded my first year of work and the Agriculture and Food
Research Initiative Competitive Grants Program Grant no. 2008-35600-18770 from the
USDA National Institute of Food and Agriculture for a second year of support. I am
very grateful for the laboratory facilities I was able to use during this project that were
available at USDA-ARS Corvallis and the office and computer facilities they supplied.
I also would like to express my gratitude to the Department of Botany and Plant
Pathology who offered an excellent education and the opportunity to befriend others in
this line of work. I appreciate the relationships I have made with peers such as Rachel
Bomberger, Kat Sweeney and Alyssa Carey while here at Oregon State University. I
was very fortunate to work with Dr. Joyce Loper during my M.S. graduate work. She is
an excellent adviser and scientist. Her input was crucial in the completion and success
of this work. I enjoyed working with every single person in the Loper lab. I am
especially thankful for the help I received from Ed Davis during the bioinformatic
studies, Marcella Henkels during many of the bioassays, Dr. Virginia Stockwell with
statistics, Dr. Teresa Kidarsa for advice in mutant making, Dr. Sierra Hartney for a
variety of useful suggestions and Brenda Shaffer for her excellent lab managing
abilities and tips using the Geneious program. I would like to thank Dr. Denny Bruck
for allowing me to utilize his entomology lab and his technician, Amanda Lake, who
helped care for the hornworms. Lastly, I would like to thank Fred and Mick for being
there when I come home every day.
TABLE OF CONTENTS
Page
Chapter 1: INTRODUCTION ....................................................................................... 2
The Pseudomonas fluorescens group ........................................................................... 3
Toxicity of bacteria to insects ...................................................................................... 5
Counteracting insect immune response ........................................................................ 5
Insect toxins ................................................................................................................. 7
Toxin complex genes ................................................................................................... 8
Research objectives .................................................................................................... 10
Chapter 2: METHODS ................................................................................................ 14
BIOINFORMATIC ANALYSIS OF TC SEQUENCES IN GENOMES OF THE P. FLUORESCENS
GROUP ......................................................................................................................... 14
Identification of candidate Tc genes ...................................................................... 14
Domain analysis ..................................................................................................... 14
Organization and genomic context of Tc gene clusters ......................................... 14
G+C content of Tc genes ....................................................................................... 15
Alignment and Phylogenetic analysis of Tc sequences ......................................... 15
BIOASSAYS ASSESSING LETHALITY OF THE TOBACCO HORNWORM M. SEXTA ............... 16
Strains and conditions of bacterial growth ............................................................. 16
Constructing mutants of P. fluorescens strain A506.............................................. 16
Rearing M. sexta..................................................................................................... 17
Insect toxicity assays .............................................................................................. 18
Bacterial colonization of larvae ............................................................................. 18
TABLE OF CONTENTS (continued)
Page
Statistical analysis .................................................................................................. 19
Chapter 3: RESULTS .................................................................................................. 23
CHARACTERIZATION OF TC GENES IN P. FLUORESCENS ............................................... 23
Identification of Tc genes in genomes of the P. fluorescens group ....................... 23
Detection of conserved domains of Tc genes ........................................................ 23
Organization of Tc gene clusters............................................................................ 24
Phylogenetic analysis ............................................................................................. 25
Inference of Tc gene inheritance ............................................................................ 26
INSECT TOXICITY OF STRAINS IN THE P. FLUORESCENS GROUP ................................... 28
P. fluorescens A506, SS101 and P. sp. BG33R show insect toxicity .................... 28
Insect colonization by strains of P. fluorescens ..................................................... 29
Identification of genetic determinants of insect lethality in P. fluorescens ........... 30
Chapter 4: DISCUSSION ............................................................................................ 51
REFERENCES ............................................................................................................. 59
LIST OF FIGURES
Figures
Page
1.1 Phylogenetic relationships and genomic diversity of the Pseudomonas fluorescens
group ....................................................................................................................... 12
1.2 Characterized toxin complex gene distributions in various bacteria ...................... 13
3.1 Types I-VI of toxin complex gene clusters in Pseudomonas spp. .......................... 38
3.2 Phylogenetic analysis of component A amino acid sequences ............................... 39
3.3 Phylogenetic analysis of component B amino acid sequences ............................... 40
3.4 Phylogenetic analysis of component C amino acid sequences ............................... 41
3.5 Toxin complex genes from types II, III and IV share both genomic and
phylogenetic context ............................................................................................... 42
3.6A Hornworm lethality after injection of 105 CFU per larvae of P. fluorescens strains
on M. sexta .......................................................................................................... 43
3.6B Hornworm lethality after injection of 105 CFU per larvae of P. fluorescens strains
on M. sexta ......................................................................................................... 44
3.7 Hornworm lethality after injection of 106 CFU per larvae of P. fluorescens strains
on M. sexta .............................................................................................................. 45
3.8 Colonization of M. sexta by 105 CFU per larvae of strains SBW25, BG33R, SS101
and A506 ................................................................................................................. 46
3.9A Progress curves depicting mortality of larvae following injection of P.
fluorescens A506, A506∆tc and A506∆aprA ..................................................... 47
3.9B Progress curves depicting mortality of larvae following injection of P.
fluorescens A506, A506∆tc and A506∆aprA ...................................................... 48
LIST OF FIGURES (continued)
Figures
Page
3.10 Progress curves depicting mortality of larvae following injection of P. fluorescens
A506 and A506∆gacS ........................................................................................... 49
3.12 Colonization of M. sexta by 105 CFU per larvae of A506 and A506∆tc ............. 50
4.3 Exoenzymes and secretion systems found within the ten strains of the P.
fluorescens group .................................................................................................... 58
LIST OF TABLES
Table
Page
1.1. Strains of the Pseudomonas fluorescens group ..................................................... 11
2.1 Bacterial strains and plasmids used in this study .................................................... 20
2.2. Primers used in the construction of the toxin complex knock-out mutant of P.
fluorescens A506................................................................................................... 21
3.1 BLASTp analysis of Tc genes in strains of the P. fluorescens group ..................... 33
3.2. All tcaA/tcdA-like gene products possessing the VRP1 (PF03538) domain ......... 35
3.3. All tcaC/tcdB-like gene products possessing the SpvB (PF03534) and MidN/MidC
(PF12255) domains ............................................................................................... 35
3.4. Comparison of G+C content between toxin complex genes and respective overall
genomes................................................................................................................. 36
3.5. Lethality of strain A506 and derivatives against larvae of M. sexta at 48hrs ........ 37
3.6. Relative area under the mortality curve (RAUMC) of M. sexta inoculated with
strain A506 and derivatives ................................................................................... 37
Genomics-enabled Exploration of Insect Toxicity in the Pseudomonas fluorescens
Group
2
Genomics-enabled Exploration of Insect Toxicity in the Pseudomonas fluorescens
Group
Chapter 1: INTRODUCTION
Plant diseases and pests have been battled against since the dawn of
agriculture. The invention of chemical pesticides and their deployment greatly
increased yields of many agricultural crops during the 20th century but the need for
other disease management strategies persists. Chemicals are not available for
management of all plant pests and diseases and many instances of pathogen or pest
resistance to pesticides have been reported (29, 54, 60, 84-85, 89, 100, 111). There is
also public pressure to reduce or eliminate the use of these products due to
environmental health and safety concerns. Biological control agents could be a
promising alternative to synthetic compounds for managing plant diseases and pests.
Biological control is the intentional introduction of living organisms into a
given environment to suppress the activity or population of a target organism. This can
be accomplished through one or more organisms, including the host plant but
excluding man (5, 19). This concept was first described in relation to plant pathogens
in 1914 by C.F. von Tubeuf and to insects in 1919 by H.S. Smith (5). There are many
mechanisms of biological control, including induction of host resistance, parasitism,
predation, competition, and antibiosis. Biocontrol is a field of research in both
entomology and plant pathology, as it is used to manage insect pests as well as
pathogens, but has been more widely employed for the management of insects. In
entomology, biological control most often relies on beneficial insects or parasitoids
that control an agricultural pest, but microbial pathogens of insect pests are also
important. In plant pathology, biological control is achieved most commonly by
microorganisms that are antagonistic towards plant-pathogenic bacteria, fungi or
Oomycetes, or those that induce resistance responses in the plant host.
3
The objectives of this study were to 1) determine if ten strains in the
Pseudomonas fluorescens group, which are known for biological of plant diseases, are
lethal to insects, using larvae of the tobacco hornworm (Manduca sexta) as a model
system, 2) characterize putative insect toxin complex (Tc) genes in the genomes of the
ten strains, and 3) determine the roles of Tc genes and other loci in the insect lethality
of one strain of P. fluorescens.
The Pseudomonas fluorescens group
One group of microorganisms that has been a focus of research for biological
control of plant diseases is the genus Pseudomonas. This genus encompasses a diverse
group of Gamma proteobacteria that are Gram-negative, rod-shaped and possess polar
flagella. These bacteria are ubiquitous in the environment, including soil, water, plant
surfaces, and animals (39). They are globally abundant and well known for their
production of a variety of secondary metabolites and antimicrobial compounds that
contribute to biological control of plant diseases. The ecological and physiological
diversity of the genus extends to the genomic level (106). Pseudomonas species
typically have genome sizes that vary from 4.6 to 7.1 megabases with 4,237 to 6,396
predicted genes, and G+C contents ranging from 57.8 to 66.6% (39). Genomic
diversity is particularly apparent from the relatively small size of the core genome
(1,491 genes) that is shared among all Pseudomonas species and the large size of the
pangenome (25,907 genes) for the genus (80). The genus Pseudomonas currently
comprises more than 100 named species that have been divided into groups and
subgroups based on multilocus sequence analysis (86).
Many biological control strains fall into the diverse P. fluorescens group,
which is made up of seven subgroups and more than 50 named species. The ten strains
chosen for this study are members of the P. fluorescens group whose genomes have
4
been sequenced (80, 92, 106) (Table 1.1). Phylogenetic analysis of strains of
Pseudomonas spp. having fully sequenced genomes indicated that these ten strains fall
into a single large clade composed of three sub-clades (80) (Figure 1.1A). The
genomic diversity of the ten strains was revealed by a ten-way best-match BLASTp
search, which identified a core genome containing 2,789 predicted protein-coding
genes, representing only 45% to 52% of the predicted proteome of each strain (Figure
1.1B). Each of the ten strains has 313 to 930 genes that are not present in any of the
other genomes. These unique genes are likely to contribute to the distinctive biological
control properties of the strains (Table 1.1). Most of the strains produce one or more
antibiotics known to contribute to biological control (39, 42, 98). These antibiotics
include hydrogen cyanide, phenazines, phlorolucinols, pyoluteorin, pyrrolnitrin and
lipopeptides (42, 98), and genes for their biosynthesis reside in regions of the genome
present in only a sub-set of strains (80).
The ten strains evaluated in this study fit into at least four species within the P.
fluorescens group: P. chlororaphis, P. protegens, P. brassicacearum, and P.
fluorescens (Table 1.1). P. chlororaphis subsp. aureofaciens 30-84, P. fluorescens Q287, and P. brassicacearum Q8r1-96 can suppress take-all of wheat caused by the
soilborne fungus Gaumanomyces graminis var. tritici (94, 99, 113, 118). These three
strains were obtained from take all-suppressive soils, which exhibit natural processes
of biological control due to the presence of indigenous microflora antagonistic to G.
graminis var. tritici. P. chlororaphis subsp. aureofaciens O6 induces host resistance of
tobacco and cucumber to several diseases (45, 66). P. protegens Pf-5 (previously
called P. fluorescens Pf-5), P. fluorescens SBW25, and Pseudomonas sp. SS101
suppress damping-off diseases caused by Oomycete pathogens (22, 52-53, 70, 83, 105,
114). Pseudomonas sp. BG33R can suppress ring and root knot nematodes (69). Eight
of the strains in this study were isolated from bulk or rhizosphere soil, but P.
fluorescens strains SBW25 and A506 were isolated from leaf surfaces. Strain A506,
which was isolated from a pear tree in California (77), is a registered biopesticide
5
named Blight Ban A506, which is used to suppress frost damage of several crops and
fire blight, caused by the bacterium Erwinia amylovora, of pear and apple (61-62, 110,
128).
Toxicity of bacteria to insects
In addition to suppressing plant pathogens, many strains of Pseudomonas spp.
have been described recently as causing both oral and injectable insect lethality (4, 20,
75-76, 79, 88, 116, 120-121). For the vast majority of these Pseudomonas spp., the
mechanisms of insect lethality are still unknown. At least two processes are important
in insect lethality by bacteria: evasion of the insect immune response and production
of toxins. Bacteria can evade the insect immune response by degrading or avoiding
insect-produced antimicrobial peptides and hemocytes. After the insect immune
system is negated, some bacteria can produce a variety of toxins that will kill their
host. Insect toxins have been described most thoroughly in entomopathogenic bacteria
such as Bacillus thuringiensis, Serratia entomophila and Yersinia pestis and in
bacterial endosymbionts of entomopathogenic nematodes such as Photorhabdus spp.
and Xenorhabdus spp. that produce potent insecticidal toxins (9, 30-31, 33, 37, 57,
103, 112).
Counteracting insect immune response
Bacterial defense against insect antimicrobials often involves the production of
extracellular proteases. In Photorhabdus spp., the serralysin-type metalloprotease PrtA
is generated early during infection and can degrade specific recognition proteins in the
tobacco hornworm, M. sexta, a member of the Lepidoptera order (8, 10, 27-28, 47,
81). In Xenorhabdus spp., two distinct proteases have been identified, Protease I and
II. Purified Protease II was shown to degrade antibacterial peptides, such as cecropin
A, and suppress the antibacterial activity of insect hemolymph in the larva of the wax
moth, Galleria mellonella (Lepidoptera) and the armyworm, Pseudaletia unipuncta
6
(Lepidoptera) (13). This protease, later characterized as XrtA, shares 60% amino acid
identity with the alkaline metalloprotease, PrtA, of P. luminescens (91). Members of
the genus Serratia also produce proteases that degrade proteins involved in host
protective functions, such as antimicrobial peptides (AMPs), in a wide variety of
insects such as Drosophila melanogaster (Diptera) and G. mellonella (36, 65). Liehl et
al. (75) demonstrated that AprA, an abundant secreted metalloprotease, is an important
virulence factor that protects Pseudomonas entomophila from antibacterial peptides in
D. melanogaster. In Pseudomonas spp., the production of AprA is controlled by the
GacS/GacA regulatory system (71), and a gacA mutant of P. entomophila lacks
virulence in D. melanogaster (121).
In response to infection, insect larvae will also produce hemocytes as a cellular
immune response that will aggregate and phagocytose the foreign entity. There are
various bacterial mechanisms that suppress phagocytosis, but the Type III secretion
system is best characterized. In Photorhabdus spp., many Type III effectors have been
shown to overcome phagocytosis. A Type III effector termed Cif (cell inhibiting
factor) contributes to the control of host cell proliferation and apoptotic death during
infection of Locusta migratoria (Orthoptera) by P. luminescens (16). Cif was first
characterized in Escherichia coli as a cyclomodulin that inhibits the G/M transition of
the cell cycle (102, 130). Since then, Cif homologs have been found in P. luminescens,
Photorhabdus asymbiotica, Yersinia pseudotuberculosis and Burkholderia
pseudomallei. These Cif homologs induce cytopathic effects identical to those
observed in response to Cif from pathogenic E. coli (63). Another Photorhabdus spp.
effector, LopT, has also been shown to contribute to evasion of the immune response.
This effector is activated only at sites of cellular defense during infection of the
cutworm Spodoptera littorali (Lepidoptera) and the locust L. migratoria, which
indicates that specific effectors have a role in suppression of the insect immune
response (11-12). LopT is similar to YopT, an effector that causes cytoskeletal
disruption and contributes to the antiphagocytic effect of Yersinia spp. (58, 104).
7
Pseudomonas aeruginosa also produces Type III effectors that suppress insect cellular
immunity. Four effectors have been identified in P. aeruginosa that are involved in
cytotoxicity: ExoS, T, U and Y (35, 64). The Type III secretion system of P.
aeruginosa is necessary for the rapid killing of D. melanogaster, specifically in the
first hours of infection, when cellular immunity is engaged (4, 26). The
aforementioned effectors overcome insect immune defenses, but are not the sole cause
of insect morbidity.
Insect toxins
Insect toxins with various modes of action have been identified in many
bacteria. The best studied of these are the Cry or Cyt toxins of B. thuringiensis (51,
112), Tc-toxins of Photorhabdus spp. and Xenorhabdus spp. (7, 9, 72-73, 78, 103),
Mcf (makes caterpillars floppy) toxins of Photorhabdus spp. (21, 24, 119, 123, 127),
and PirAB binary toxins of Photorhabdus spp. (1, 25, 126). Many additional insect
toxins have been described more recently: monalysin, a pore-forming toxin of P.
entomophila (88, 116); a rhamnolipid of Pseudomonas sp. EP-3 (38, 67); XaxAB, a
cytotoxin of X. nematophila (117); Ucp, the “you cannot pass” membrane-bound toxin
of Pantoea stewartii (109); Afp, the “antifeeding prophage” toxin of S. entomophila
(55-56) and its homolog, Pvc (or Photorhabdus virulence cassette) of Photorhabdus
spp. (129). Two types of insect toxin genes have been identified to date in strains in
the P. fluorescens group. Genes encoding the FitD (fluorescens insecticidal toxin)
toxin were found in the genome of P. protegens Pf-5 and the same locus was
identified in the closely-related strain CHAO (93). More recently, the fitD locus was
found in genomes of P. chlororaphis O6 and 30-84, which, with strain Pf-5, compose
the Sub-clade 1 strains evaluated in this study (Figure 1.1A). FitD is homologous to
the Mcf toxin of P. luminescens. The Mcf1 and Mcf2 toxins of P. luminescens have
shown injectable lethality towards insects via apoptosis (21, 24, 123). Similarly, when
injected into the hemocoel, even low doses of P. fluorescens CHAO or Pf-5 kill larvae
8
of the tobacco hornworm, M. sexta, and the wax moth, G. mellonella (93). When
heterologously expressed in E. coli, the Fit toxin was lethal to both hosts. Putative
genes for Tc toxins have also been annotated in the genome of P. fluorescens strain
Pf0-1 (32) and, very recently, in the genomes of seven strains within the P. fluorescens
group that are a focus of this study (80).
Toxin complex genes
Toxin complex (Tc) proteins are large, high molecular weight toxins that are
exhibited on the surface of the bacteria. They cause oral insecticidal activity similar to
the Cry toxins of B. thuringiensis (32). The Tc toxins are composed of three
components: A, B, and C. All three protein components are necessary for full toxicity
(30) but there is evidence that the A component is the main source of toxicity. The
gene encoding the A component from P. luminescens strain W14 was shown to confer
insect resistance in transgenic Arabidopsis (78, 124). Nevertheless, the A component
commonly is not enough for full toxicity. Waterfield et al. (116) found that the
expression of the A component in E. coli can result in insect toxicity, but higher levels
of toxicity are exhibited by E. coli co-expressing genes for components A, B, and C
(125). This provided evidence that all toxin complex components are necessary for full
toxicity towards insects. Furthermore, genes encoding the A, B, and C components
must be expressed together in the same organism to confer full toxicity; expressing B
and C individually, then adding them to A, does not result in full activity (125). This
same phenomenon is seen when Tc genes from Xenorhabdus spp. are expressed in E.
coli, providing evidence that A components from different bacteria confer insect
toxicity (103). This also presented support that component A is a toxin whereas B and
C are potentiators (33). The C component is thought to modify B thereby generating a
new B-C conformation, which is the active potentiator (33). By combining a single BC potentiator with A components from several bacteria, Hey et al.. (48) showed that
different combinations vary in efficacy and show distinctive spectra of toxicity against
9
a panel of insects. For example, a Paenibacillus B-C component can increase the
effectiveness of A toxins from both Photorhabdus spp. and Xenorhabdus spp. A
components also can be combined to increase the spectrum of toxicity against a range
of pests (48). There is an incentive to investigate new sources of Tc proteins, which
may be lethal against different insects, thereby expanding the spectrum of insects that
may be managed by biological control with Tc toxin-producing bacteria.
Genes for toxin complexes are found in many bacterial genomes, including
bacteria associated with entomopathogenic nematodes (Photorhabdus spp. and
Xenorhabdus spp.), insects (Serratia spp., Yersinia spp. and Paenibacillus spp.) and
bacteria with no known association with insects (Pseudomonas spp., Erwinia spp. and
Burkholderia spp.). Genes encoding Tc proteins are best characterized in P.
luminescens strain W14, where four types of Tc gene clusters have been identified:
tca, tcb, tcc and tcd (Figure 1.2). In the tca and tcc clusters, two genes (tcaA and tcaB
or tccA and tccB) encode the A component. In the tcb and tcd clusters, one gene (tcbA
or tcdA) encodes a fusion protein for the A component. Two clusters (tca and tcd)
include a gene (tcaC and tcdB) encoding the B component. The C component is
encoded by tccC, copies of which are present in two of the Tc clusters (tcc and tcd) in
the genome of P. luminescens W14. Only the tcd cluster of strain W14 includes genes
encoding each of the three Tc components.
The organization of Tc gene clusters differ among bacterial genomes (Figure
1.2). Within the P. luminescens strain W14 genome, single genes encoding A, B, or C
components are scattered throughout the genome in addition to the tca, tcb, tcc and tcd
clusters described above. The tcd gene cluster appears to be on a pathogenicity island,
as the cluster is flanked by repeats and tRNAs and has a G+C content that differs from
the overall genome (33). Some Tc genes in other bacterial genera are also present in
mobile genetic elements, such as the Tc genes found on the plasmid of S. entomophila
or those found on a genomic island of Burkholderia pseudomallei (49).
10
Research objectives
In this study, I evaluated ten strains of the P. fluorescens group for lethality
against the tobacco hornworm M. sexta and determined if genes commonly required
for insect toxicity in other bacteria are present in the genomes of the ten strains. Genes
encoding putative insect toxins (FitD and Tc) and possible modes of
immunosuppression (the AprA metalloprotease and GacS/GacA regulatory system)
were of primary focus. The functionality of the toxin complex, extracellular protease
and GacS genes of strain A506 were assessed by creating or utilizing existent knockout mutants and testing them for lethality to larvae of M. sexta.
Here, I established that six of ten strains representing diverse lineages of the P.
fluorescens group exhibited injectable lethality to larvae of the tobacco hornworm, M.
sexta, while four of the strains (Q2-87, Q8R1-96, Pf0-1 and SBW25) exhibited no
significant lethality. The presence of Tc gene clusters in seven strains was established
and, on the basis of the organization and genomic context, G+C content, and
phylogeny of the Tc genes, the Tc gene clusters were classified into six distinct types.
One of the types included genes from strain A506, which was selected for further
study because of its injectable lethality towards M. sexta, and its importance as a
model strain for studies of phyllosphere microbial ecology and as a commercial
biological control agent. Of the genes (aprA, gacS and tcaABC-tccC) evaluated for
their roles in insect lethality, the tcaABC-tccC cluster had the greatest effect on insect
lethality by A506. Nevertheless, none of the genes were wholly responsible for the
injectable lethality caused by A506, indicating that loci other than those evaluated here
contribute to insect toxicity by this strain.
11
Table 1.1. Strains of the Pseudomonas fluorescens group evaluated in this study.
a
Strain
Source
Target disease(s) for biological
control
Genome
sequence
P. chlororaphis subsp. aureofaciens:
30-84
Wheat rhizosphere,
Washington, USA
Take-all of wheat (94, 113)
(80)
O6
Soil, Utah, USA (115)
Wildfire of tobacco (45),
Corynespora target spot of
cucumber (66)
(80)
Soil, Texas, USA
Seedling emergence (52-53)
(92)
Wheat rhizosphere,
Washington, USA
Take-all of wheat (99)
(80)
P. protegens:
Pf-5
P. brassicacearum:
Q8r1-96
P. fluorescens:
Pf0-1
Soil, Massachusetts, USA
(106)
Q2-87
Wheat rhizosphere,
Washington, USA
Take-all of wheat (118)
(80)
SBW25
Sugar beet phyllosphere,
Oxfordshire, UK
Seedling emergence
(106)
A506
Pear phyllosphere,
California, USA
Fire blight of pear and apple; frost
injury, fruit russeting (110, 128)
(80)
SS101
Wheat rhizosphere, The
Netherlands
Diseases caused by Pythium spp.
and Phytophthora spp. (22, 70, 83,
114)
(80)
Peach rhizosphere, South
Carolina, USA
The plant-parasitic nematode
Mesocriconema xenoplax (69)
(80)
Pseudomonas sp.:
BG33R
a
In previous publications, strain Pf-5 has been designated as P. fluorescens Pf-5 , strain Q8r1-96 as P.
fluorescens Q8r1-96, and strain BG33R as either P. synxantha or Pseudomonas sp. BG33R.
12
Figure 1.1 Phylogenetic relationships and genomic diversity of ten strains of the
Pseudomonas fluorescens group. A) The phylogenetic tree is based on concatenated
alignments of ten core housekeeping genes: acsA, aroE, dnaE, guaA, gyrB, mutL,
ppsA, pyrC, recA, and rpoB, and was generated using the MrBayes package (101). The
interior node values of the tree are clade credibility values, which represent the
likelihood of the clade existing, based on the posterior probability values produced by
MrBayes. Phylogenetic relationships between these ten strains produce three subclades, which are colored pink (Sub-clade 1), purple (Sub-clade 2), or green (Subclade 3). B) Each strain is represented by an oval that is colored according to subclade, as in A. The number of orthologous coding sequences (CDSs) shared by all
strains (i.e., the core genome) is in the center. Overlapping regions show the number
of CDSs conserved only within the specified genomes. Numbers in non-overlapping
portions of each oval show the number of CDSs unique to each strain. The total
number of protein coding genes within each genome is listed below the strain name.
Reprinted with permission from (80).
Figure 1.2 Toxin complex (Tc) gene clusters in diverse bacterial genera. In P. luminescens W14, Tc genes are
in clusters of four types: tca, tcb, tcc and tcd. Genes are color-coded according to the Tc component encoded: red
(Component A), blue (Component B) and yellow (Component C). Component A can be encoded by two genes or
by a single, large gene.
13
.
14
Chapter 2: METHODS
BIOINFORMATIC ANALYSIS OF TC SEQUENCES IN GENOMES OF THE P.
FLUORESCENS GROUP
Identification of candidate Tc genes
Using well-characterized Tc genes from P. luminescens W14 as queries, a
BLAST search was performed against the ten genomes of P. fluorescens listed in
Table 1.1. A total of 38 putative Tc genes were identified in the ten fully-sequenced
genomes.
Domain analysis
Conserved domains in putative Tc sequences were identified by submitting all
38 candidate sequences to the Pfam domain prediction search program (97). Domains
were assigned using the Pfam protein family search query using default settings with
an E-value cutoff of 1.0.
Organization and genomic context of Tc gene clusters
The organization and genomic context of Tc gene clusters was determined
from the genome viewer function of the Manatee program (J. Craig Venter Institute,
Gaithersburg, Maryland). Tc genes present in genomes of different strains within the
P. fluorescens group were comparing using a match table constructed from a ten-way
best-match BLASTp, and putative orthologs were identified with an E-value cutoff of
10-5. Using the match table, genes in each genome were listed in ascending order, and
homologous genes from the other nine genomes were aligned horizontally with each
gene in the first genome. This allowed for cross species comparison regarding
genomic positions of the Tc genes, and conservation and gene context was noted for
each of the genes.
15
G+C content of Tc genes
The percent of G+C in each Tc gene was compared to that of the overall
genome and analyzed statistically to detect significant differences between them. The
G+C content was determined using the MBCF Oligo Calculator from the Dana-Farber
Cancer Institute, Molecular Biology Core Facilities
(http://mbcf.dfci.harvard.edu/docs/oligocalc.html) and normalized to gene size. The
percentage G+C for each gene encoding a putative Tc component was compiled and
those differing significantly from the genomic average were identified by chi square
analysis. Probabilities were derived from chi square test giving a two-tailed p-value.
Alignment and Phylogenetic analysis of Tc sequences
Amino acid sequences of all putative Tc proteins were submitted to the
National Center for Biotechnology Information (NCBI) database of non-redundant
protein sequences to identify the five best hits for each using the BLASTp algorithm
(2). The amino acid sequences of all Tc proteins in addition to the five best BLASTp
hits were utilized for phylogenetic analysis (2). Multiple sequence alignments of the
Tc proteins were executed using the CLUSTALW option in BioEdit (44). The BioEdit
alignment program was used to create PHYLIP and NEXUS formats for maximum
likelihood phylogenetic analysis of each of the individual gene types. Three
phylogenetic trees were created using each putatively annotated amino acid sequence
for each toxin complex component: A (TcaA, TcaB, and TcdA), B (TcaC and TcdB)
and C (TccC). Each tree was created using the top five BLASTp hits resulting from
the search of non-redundant protein sequences at NCBI plus one outgroup, which was
the last BLASTp hit on the list of sequences resulting from that search. The three trees
have different numbers of taxa represented because the query sequences from the P.
fluorescens genomes varied in the number of overlapping top BLASTp hits. Using the
CIPRES portal, maximum likelihood analysis with RAxML was used and 100
16
bootstrap values were assigned (107). For each tree created, joint branch length
optimization was used. The Gamma+P-Invar model of rate heterogeneity was used
and the substitution matrix BLOSUM62 was implemented for each tree.
BIOASSAYS ASSESSING LETHALITY OF THE TOBACCO HORNWORM M. SEXTA
Strains and conditions of bacterial growth
The ten strains evaluated in this study are listed in Table 1.1. Pseudomonas
spp. were grown on King’s medium B (KMB) (68) at 27oC. E. coli was grown in
lysogeny broth (LB) (6) or on LB agar at 37oC. Antibiotics were used for selection at
the following concentrations (µg/ml): gentamicin (Gm) 40 (P. fluorescens) and 12.5
(E. coli), kanamycin (Km) 50, Rif (Rif) 100, tetracycline (Tet) 200, cyclohexamide
(Cyc) 50.
Constructing mutants of P. fluorescens strain A506
Mutants of A506 and plasmids used for their construction are listed in Table
2.1. The mutant A506Δtc, which has a 14.5-kb deletion encompassing the entire Tc
cluster of A506, was constructed using an overlap extension PCR method modified
from Choi and Schweizer (17). All PCR was performed using KOD Hot Start DNA
polymerase (Novagen, Madison, WI, USA). In the first round of PCR, 5′ and 3′ gene
fragments were amplified using the primers listed in Table 2.2. Amplification of the
Gm-resistance gene cassette from pPS856 (50) was performed with the Gm-F and
Gm-R primers essentially as described (17). In the second round PCR, 50 ng each of
the Up and Dn gene-specific PCR products and the FRT-Gm-FRT cassette PCR
product were combined and two cycles of amplification and extension were done
without added primers. The corresponding UpF and DnR primers were added halfway
through the third cycle extension. Amplification of the full-length product was
completed with an additional 25 cycles of PCR. The second-round PCR product was
17
digested with HindIII and cloned into the HindIII site of pEX18Tc (50).The resulting
plasmid was transformed into One Shot TOP10 Chemically Competent E. coli
(Invitrogen, Carlsbad, CA, USA) and the identity of the cloned PCR product was
confirmed by sequencing. The plasmid was introduced into A506 via triparental
conjugation using E. coli DH5α (pRK2013) (59) as a helper. A506 transconjugants
were selected on KMB containing Rif and Gm. Resulting colonies were grown for 3 h
without selection in LB and then plated on LB agar amended with Gm and 5% sucrose
to favor growth of resolved merodiploids. Sucrose-resistant colonies were patched
onto KMB containing Rif to confirm resolution of merodiploids. Tet-sensitive, Gmresistant clones were considered to be putative mutants. To remove the Gm resistance
gene, the plasmid pFLP2Km expressing Flp recombinase was introduced into the
mutant strains by conjugation (46). Transconjugants were selected on KMB with Rif
and Km. Colonies were replica-plated onto KMB and KMB containing Gm to screen
for loss of the Gm-resistance gene. Gm-sensitive colonies were transferred onto LB
agar supplemented with 5% sucrose to promote loss of the pFLP2Km plasmid.
Sucrose-resistant colonies were replica plated onto KMB and KMB containing Km to
confirm loss of the plasmid. The resulting mutants, containing only an FRT scar at the
site of the deletion, were confirmed as such by performing PCR across the deletion
site (including all flanking sequence introduced by recombination) and sequencing the
resultant product.
Rearing M. sexta
Eggs of M. sexta (Carolina Biological Supply, Charlotte, NC, USA) were
hatched in mass on meridic diet
(http://www.entm.purdue.edu/Entomology/outreach/recipe/ artificialdiet.pdf,
Department of Entomology, Purdue University) at a 16:8 h light to darkness (L:D)
regime and 27°C. Five days after hatch, larvae were transferred into larger containers
and additional meridic diet was added as needed. Larvae were maintained at 16:8 h
18
(L:D) and 27°C for approximately 2-3 weeks until they reached the fifth/ultimate
instar.
Insect toxicity assays
For insect toxicity assays, bacteria were grown in 5 ml of KMB broth for 24h
at 27ºC. Cells were collected by centrifugation, washed, resuspended in sterile water at
OD600= 0.01 (~1 x 107 CFU/ml), and diluted to obtain cell densities specified in
Results. Dilutions of the cell suspensions were spread on KMB to determine titers of
the inoculum injected into larvae of M. sexta.
Prior to injection, each larva was rinsed with tap water and dried with a tissue
to remove any diet or frass adhering to the surface of the insect. Larvae were injected
between the second and third abdominal segments with 10 µl of water or bacterial
suspension using a Dutky-Fest microinjector mounted with a 1 ml syringe with a 27gauge needle (Becton Dickinson & CO., Model# 309623) (93). For toxicity assays,
larvae were placed back into their individual containers, returned to the incubator at
16:8 h (L:D) and 27°C, and assessed for mortality over time. This was repeated for all
bacterial strains at least twice using ten larvae per replicate. Those ultimate instars that
were more developed, or later in their fifth instar, were monitored for the onset of
pupal ecdysis, as this could affect responses to treatment. This was seen in experiment
three in strain lethality tests and in experiments two and three when observing
mortality curves for A506 and derivatives.
Bacterial colonization of larvae
To monitor bacterial colonization of M. sexta, larvae were inoculated by
injection, as described above, with ca. 105 bacterial cells per larva and placed in an
incubator held at 27°C and 16:8 h (L:D). RifR derivatives described in Table 2.1 were
used as inoculum in these experiments. Larvae with a healthy appearance were
19
sampled immediately after inoculation and at 6, 12, 18, 24, and 30 h after injection.
Each larva was surface-disinfested for 30 s in 70% ethanol, rinsed with sterile water,
and then homogenized for 30 s in 10 ml of sterile distilled H2O using a Tekmar SDT
Tissumizer (Cincinnati, OH, USA). Serial dilutions were prepared from the resulting
homogenate and plated onto KMB containing Rif and Cyc to select for the injected P.
fluorescens strains. As a control, extracts of larvae injected with sterile H2O and were
homogenized and plated on KMB with and without Rif to estimate background
populations of culturable bacteria in the larvae. Plates were incubated for 24 h at 27°C
prior to enumeration of bacterial colonies. This was repeated twice for strains from
Sub-clade 3 and A506 derivatives.
Statistical analysis
A chi-square analysis was performed for lethality tests evaluating strains in the
P. fluorescens group. The negative control (sterile water) was compared to each
treatment individually. A chi-square analysis was also used to compare the G+C
content of each Tc gene to the average G+C content of the corresponding genome. For
colonization experiments, bacterial population sizes were log transformed and the
means and standard errors were calculated for each strain at each time point. To
compare differences in hornworm lethality caused by A506 versus derivative strains,
the numbers of dead and living larvae were counted over time following inoculation
with inoculum doses varying from 103 to 105 CFU/larva. The counts were analyzed
under a Fisher’s protected least significant difference test at P = 0.05 using the
analysis of variance (ANOVA) procedure of Statistical Analysis Systems (SAS
Institute, Cary, NC). The relative area under the mortality curve (RAUMC) was
calculated for each dose in each trial. The area under the mortality curve (AUMC) was
calculated for each replication, as described by Hagen et al. (43). Because the sample
period time points varied among trials, the AUPC was converted to the RAUMC by
dividing the AUMC by the total duration of each experiment, or 48h. Mean RAUMC
20
values obtained for each bacterial strain at each dose were separated with Fisher’s
protected least significant difference test at P = 0.05 using the ANOVA procedure of
SAS.
21
Table 2.1 Bacterial strains and plasmids used in this study
Strain or Plasmid
Reference or Source
Description
P. sp.
Rhizosphere isolate, RifR (JL4870)
D. Kluepfel (80)
Pf0-1
Rhizosphere isolate (JL4803)
M. Silby (106)
Pf0-1 gacA+
Pf0-1 containing a plasmid-borne gacA+ from P.
protegens Pf-5 (LK079)
(80)
SBW25
RifR derivative of SBW25 (JL4802)
J. Raaijmakers
A506
Isolated from the phyllosphere of pear in
California, RifR (LK001)
S. E. Lindow (77)
A506∆tc
∆tcaABC/ tccC derivative of A506, KmR, RifR
(LK196)
This study
A506∆aprA
aprA::miniTn5 derivative of A506, (aprA
previously called aprX), KmR, RifR
(JL4983)
(3)
A506∆gacS
gacS::miniTn5 derivative of A506, KmR , RifR
(JL4640)
(3)
SS101
Rhizosphere isolate, RifR (LK002)
J. Raaijmakers (80)
hsdR, mcrA, lacZΔM15,endA1, recA1
Invitrogen
pEX18Tc
Tetr, oriT, sacB, pUC MCS
(50)
pRK2013
TraRK21, ColE1 replicon, KmR,
(34)
pFLP2Km
KmR, oriI600 oriV, sacB, flp, cI857 derivative of
pFLP2
(50)
BG33R
P. fluorescens
Escherichia coli
Top10
Plasmids
Abbreviations: KmR, kanamycin resistant; RifR, rifampicin-resistant.
22
Table 2.2 Primers used in the construction of A506Δtc, a mutant of P. fluorescens
A506 lacking the entire tc cluster.
Primer
Sequence 5’
Toxin Complex:
1195 UpF
GACCAAGACTAAAGCTTTCAAGACCCTGCTGACCCAGT
1195 UpR
TCAGAGCGCTTTTGAAGCTAATTCGGTGACCTTGGGCTGTTTGCCT
1198 DnF
AGGAACTTCAAGATCCCCAATTCGAGCGTGGGCGTGTTGTGATG
1198 DnR
GTAGTTGTAGAAGCTTTTACCGTCGCCCTGGTGTTCCT
Gentamycin-resistance cassette:
Gm-F
CGAATTAGCTTCAAAAGCGCTCTGA
Gm-R
CGAATTGGGGATCTTGAAGTTCCT
23
Chapter 3: RESULTS
CHARACTERIZATION OF TC GENES IN P. FLUORESCENS
Identification of Tc genes in genomes of the P. fluorescens group
A total of 38 candidate Tc genes were identified from a BLASTp search of the
predicted proteomes of ten strains within the P. fluorescens group using characterized
Tc genes of P. luminescens W14 as queries (Table 3.1). Candidate Tc genes were
identified in seven of the ten genomes, but were not found in P. fluorescens SBW25,
P. protegens Pf-5 or P. chlororaphis O6. To identify additional Tc genes that may
have been missed in this initial search, representative Tc genes from the seven
genomes were used as queries in a second BLASTp search against each of the ten
genomes. No additional putative Tc genes were detected. Homologs of P. luminescens
W14 genes for each of the characterized four Tc components were identified in the
seven genomes of the P. fluorescens group (Table 3.1). Based on the size of putative
Tc genes and the results of BLASTp analysis, I tentatively identified seven tcaA,
seven tcaB, one tcdA, one tcdB, eight tcaC and fifteen tccC genes in the seven
genomes (Table 3.1).
Detection of conserved domains of Tc genes
Amino acid sequences of candidate Tc genes were searched for the presence of
domains conserved among Tc proteins of entomopathogenic bacteria. Conserved
domains were found in the TcaA/TcdA and TcaC/TcdB components of the toxin
complexes. Amino acid sequences of candidate tcaA genes from five genomes and the
much larger tcdA of strain 30-84 have a conserved domain called VRP1 (Table 3.2).
The VRP1 domain corresponds to SpvA, the product of a plasmid-borne gene
associated with virulence of Salmonella spp. (40). The VRP1 domain is also present in
known TccA, TcaA, TcbA TcdA proteins of entomopathogenic bacteria such as P.
24
luminescens strain W14 (122). Likewise, amino acid sequences of candidate tcaC/tcdB
genes from seven genomes have three conserved domains called SpvB, MidN and
MidC (Table 3.3). These domains are also found in TcaC/TcdB sequences in insectassociated bacteria (122). The presence of these conserved domains in candidate genes
provides further evidence for their identity as tcaA/tcdA and tcaC/tcdB genes.
Organization of Tc gene clusters
Most of the Tc genes are clustered with one another in the genomes of strains
in the P. fluorescens group. The Tc gene clusters vary among the genomes, falling into
six types (I-VI) distinguished by gene content and organization (Figure 3.1). Type I is
a gene cluster containing tcdA1 and tcdB1 in P. chlororaphis 30-84. These Tc genes
are approximately twice as large as any other Tc gene found in the P. fluorescens
group (Table 3.1), which is typical for tcdA and tcdB in entomopathogenic bacteria
(33). Type II is present in strain Pf0-1, which lacks tcaA but has a single tcaC and five
tccC genes in two clusters distal to one another in the genome. Type III, which is
present in strains Q8r1-96 and Q2-87, is a cluster composed of four genes (tcaABC
and tccC) located at the same site in both genomes. Type IV, which is present in strain
Q8r1-96 only, is composed of all three Tc components located at three sites distributed
over 169 kb of the genome. One cluster contains tcaB2 and tcaA2; a second cluster
contains tcaC2 and tccC2; and a single tccC3 gene occupies a third site on the
genome. Type V, which is present in strains A506, SS101 and BG33R, is composed of
four contiguous genes (tcaABC and tccC) located at the same site in all three genomes.
Type VI is a single cluster in BG33R containing contiguous genes: tcaA2, tcaB2,
tcaC2 and three copies of tccC. Four of the cluster types (I, II, IV, and VI) are present
in only one strain, but two types of clusters (III and V) are in more than one strain.
Strains having type III or V clusters that fall within a single sub-clade defined by
Bayesian phylogeny of the P. fluorescens group (Figure 1.1) (80).
25
The relationships of the Tc genes in the P. fluorescens group to those in other
organisms were explored through BLASTp analysis of the NCBI database. The top
BLASTp hits for all tcaA, tcaB, tcaC and tccC genes were peptide sequences from
other Pseudomonas spp. (Table 3.1), including several pathovars of P. syringae, P.
brassicacearum, and P. entomophila. In contrast, the top BLASTp hits for TcdA and
TcdB were in distantly-related bacterial species such as Mesorhizobium alhagi and a
Wolbachia endosymbiont of the almond moth, Cadra cautella (Lepidoptera) (Table
3.1). Highly dissimilar to the other Tc genes, which are widely distributed in genomes
of Pseudomonas sp., TcdA and TcdB appear to be relatively rare in this genus.
Phylogenetic analysis
Phylogenetic analyses were performed using maximum likelihood methods for
amino acid sequences of the Tc components present in the seven genomes of the P.
fluorescens group and the top five BLASTp hits for each.
The A-component tree contains seven TcaA sequences, seven TcaB sequences,
one TcdA sequence from the P. fluorescens group, all of the characterized A-like toxin
complex peptide sequences from P. luminescens W14, and a sequence from
Catenulispora acidiphila that serves as an outgroup (Figure 3.2). In this tree, the Type
V and VI TcaB sequences cluster together in a well-supported clade, whereas Type III
TcaB sequences cluster with one another between the Type V and VI TcaB sequences
and TcaB sequences from P. syringae. In contrast, the Type IV TcaB (PflQ8_4570) is
a member of a well-supported clade containing sequences from P. luminescens and
Salmonella. enterica. As for the TcaA sequences, the Type V and VI TcaA sequences
cluster together in a well-supported clade. The Type III TcaA sequences also cluster
with one another and between Type V and VI sequences and TcaA sequences from P.
syringae. In contrast, the Type IV TcaA (PflQ8_4571) is a member of a wellsupported clade containing the TcaA sequence of P. entomophila L48. The type I
26
TcdA of P. chlororaphis 30-84 (Pchl3084_2947) is most closely related to the
products of uncharacterized genes in the soil bacterium Mesorhizobium alhagi and a
bacterial endosymbiont of the deep sea mussel Bathymodiolus sp.
The TcaC/TcdC tree contains nine sequences from the P. fluorescens group as
well as sequences from 21 other taxa, including P. luminescens W14 and Burkholderia
thailandensis as an outgroup (Figure 3.3). Again, sequences from Type V and VI
cluster together in a well-supported clade. These sequences are also members of a
larger well-supported clade containing Type III, II, and IV sequences and TcaC
sequences of P. syringae. In contrast, the Type I TcdC sequence fell outside of this
clade of sequences from Pseudomonas spp.
Lastly, the TccC tree contains 15 sequences from the P. fluorescens group as
well as sequences from 21 other taxa, including P. luminescens W14 and Erwinia
pyrifoliace as an outgroup (Figure 3.4). All of the five top BLASTp hits to TccCs in
the seven P. fluorescens genomes were sequences in other strains of Pseudomonas
spp. (Table 3.1). The TccC sequences from the P. fluorescens group fell into three
distinct clades. The first clade contains one Type IV TccC and two Type II sequences
as well as uncharacterized genes from P. brassicacearum NFM421 and P. fluorescens
F113. The second clade is composed of three Type II, the one Type IV, and two Type
III TccC sequences. Type V and VI TccC sequences formed the third distinct clade,
which contains no other taxa.
Inference of Tc gene inheritance
To further explore the inheritance of the Tc genes in the P. fluorescens strains,
I evaluated the genomic locations of Tc clusters. Five of the six types of Tc clusters
are in conserved regions of the genome shared by all ten strains of the P. fluorescens
group, with Tc genes inserted at these sites in only a sub-set of the strains. Only the
tcdA-tcdC genes of P. chlororaphis 30-84 (Type I) are in a non-conserved region of
27
genome. These genes are in a genomic region, unique to P. chlororaphis 30-84, that
has characteristics of a phage, including the presence of genes encoding a phage
integrase, a transposase and cointegrate resolution proteins. The location of the tcdAtcdB genes in a phage-like region suggests that the genes could have been acquired by
strain 30-84 through horizontal gene transfer.
To further explore the possibility that horizontal gene transfer played a role in
inheritance of certain Tc genes by strains of the P. fluorescens group, I calculated
G+C content for each Tc gene and compared it to average G+C content of the entire
genome for all seven strains having Tc genes. Probabilities were derived from a chi
square test giving a two-tailed p-value. Nine genes had G+C contents that differed
significantly (p<0.001) from the average of their respective genomes (Table 3.4;
Figure 3.1). Both toxin complex genes found in P. chlororaphis 30-84 were
significantly different from the overall G+C content of the genome.
Another interesting feature relating to inheritance of the Tc genes is that their
locations within the genome correspond, in some cases, to their phylogeny. This is
particularly apparent in the Sub-clade 2 strains (P. brassicaearum Q8R1-96 and P.
fluorescens Q2-87 and Pf0-1), which have the Type II, III, and IV Tc clusters (Figure
3.5). For example, tcaC1 Pf0-1 (Type II) and the tcaC2 from Q8R1-96 (Type IV)
share genomic locations and are also closely related phylogenetically. Tc sequences
for tccC3, tccC4, and tccC5, which are adjacent to one another in the genome of Pf0-1
(Type II), are at the same location as the Type IV tccC2 and Type III tccC1 in their
respective genomes. All of these TccCs are also closely related phylogenetically. In
the same manner, TccC1 and TccC2 of Pf0-1 are similar phylogenetically to TccC3 of
Q8R1-96; genes encoding these proteins are also in the same locations in their
respective genomes. The close phylogenetic relationships and identical genomic
context of the aforementioned genes suggests that these genes may be ancestral in
Pseudomonas spp. and have undergone divergence and duplication in some lineages.
28
On the other hand, Type IV TcaA and TcaB are more closely related to proteins
present in strains outside of the P. fluorescens group, and could have been acquired
via horizontal gene acquisition. Based on these observations, I conclude that the Tc
genes have complex mechanisms of inheritance that likely involve both vertical and
horizontal acquisition.
INSECT TOXICITY OF STRAINS IN THE P. FLUORESCENS GROUP
P. fluorescens A506, SS101 and P. sp. BG33R show insect toxicity
After identifying the presence of full sets of toxin complex genes in the P.
fluorescens group, each strain was tested for toxicity against the tobacco hornworm M.
sexta. In the first set of two experiments, each P. fluorescens strain was injected into
the insect hemocoel of ten fifth/ultimate instars at 1 x 105 cells per larva. In both
experiments, six of the ten strains caused lethality of the hornworms within 48h after
injection (Figure 3.6). None of the strains falling in Sub-clade 2 (P. brassicaearum
Q8R1-96 and P. fluorescens Q2-87 and Pf0-1) (Figure 1.1) were lethal to M. sexta in
these experiments. In contrast, all of the strains falling into Sub-clade 1 (P. protegens
Pf-5 and P. chlororaphis 30-84 and O6) and three of four strains falling in Sub-clade 3
(P. fluorescens strains A506, BG33R and SS101) caused lethality. I performed two
additional experiments testing the lethality of all strains in Sub-clades 2 and 3 (Figure
3.6), and again observed no significant effect of injection of Sub-clade 2 strains
(Q8R1-96, Q2-87 or Pf0-1) or the Sub-clade 3 strain SBW25 on survival of M. sexta
larvae. In contrast, injection with strains A506, SS101 and BG33R consistently
resulted in death of the larvae. Among these three strains, A506 and BG33R
consistently caused significant lethality by 48h following injection, whereas SS101
caused significant levels of lethality at 48h only in experiment 2 (Figure 3.6). All three
strains caused significant levels of lethality by 72h following injection (p<0.05, χ2
test).
29
During the course of these experiments, a parallel study revealed that the
sequenced strain Pf0-1 has a mutation in gacA (80), which could influence its lethality
to M. sexta. Therefore, I compared the lethality of Pf0-1 to a derivative gacA+ strain,
which contains a plasmid-borne gacA+ from Pf-5 (Table 2.1). Neither strain caused
significant lethality of M. sexta larvae although some lethality occurred in one
experiment following injection of both strains (Figure 3.6B, Experiment 4).
To further explore the toxicity of the ten strains, I evaluated the lethality of
each strain at a high inoculum density. When ultimate instars of M. sexta were injected
with 1 x 106 cells per larva, all P. fluorescens strains caused some lethality by 72h
(Figure 3.7). At these very high inoculum densities, it is possible that any strain within
the P. fluorescens group can kill larvae of M. sexta. In contrast, the lethalities of the
strains could be distinguished when the insects were injected with 1 X 105 cells/larva
(Figure 3.6). Consequently, our subsequent experiments were done with inoculum
densities of 105 cells per larva or below.
Insect colonization by strains of P. fluorescens
After observing differences in lethality between strains in the three sub-clades
of the P. fluorescens group, I selected the strains of Sub-clade 3 for additional study.
Strains in other sub-clades were not investigated further because those in Sub-clade 2
were less toxic to M. sexta and strains in Sub-clade 1 have the fitD gene cluster (80),
which was shown previously to be a primary determinant of lethality against M. sexta
(93).
As noted above, the four strains in Sub-clade 3 differ markedly in lethality,
which could be due to their differential capacities to colonize the larvae of M. sexta.
To evaluate this possibility, I assessed the growth of each strain in larvae of M. sexta
over time. Ultimate instar M. sexta were injected with 1 x 105 CFU per larva and
sampled every six hours until death. Initial populations of the strains, assessed
30
immediately after injection of the larvae, were between 3.9 and 5.7 log (CFU/larva)
(Figure 3.8). The populations of all strains increased over time, and strains A506,
BG33R, and SS101 reached population sizes of 10.0 to 10.3 log (CFU/larva) at 24h
after injection. By 30h, A506 and BG33R populations had reached 10.9 and 10.4 log
(CFU/larvae), respectively. Strain SS101 was not sampled at 30h. In contrast to the
other strains, SBW25 established lower population sizes in the larvae, reaching only
8.1 log (CFU/larva) at 24h, and was not assessed at later time points. All hornworms
inoculated with A506 or BG33R died by 36h. As observed earlier (Figure 3.6),
SBW25 did not kill larvae of M. sexta in this experiment, which suggests a correlation
between the lethality and larval colonization phenotypes of the strains. It seems
evident that a strain that colonizes larvae would be more likely to cause lethality, but it
is also possible that a dying animal provides a habitat more conducive to colonization
by P. fluorescens than a healthy animal. Both of these possibilities may contribute to
correlation between colonization and lethality of P. fluorescens strains observed here.
Identification of genetic determinants of insect lethality in P. fluorescens
I next set out to identify genes contributing to insect lethality by P. fluorescens,
and selected strain A506 for this analysis for the following reasons. Of the strains
evaluated, A506 was one of the most lethal to larvae of M. sexta and had the capacity
to colonize the larvae. A506 is also of interest because it is an important model strain
for studies of phyllosphere microbial ecology (43). Of the seven strains in the P.
fluorescens group that exhibited lethality to M. sexta, A506 is the only strain to be
isolated from leaf surfaces and, as a phyllosphere inhabitant, it is likely to come into
contact with foliar feeding insects such as the tobacco hornworm. Furthermore,
derivatives of A506 with mutations in genes with putative roles in insect lethality were
constructed earlier (3) and were available for this study. These mutants include
A506∆gacS, which lacks the global GacA/S regulatory system that controls the
production of exoprotease and other exproducts, and A506∆aprA, which is deficient in
31
the production of an exoprotease. Because of the potential roles of Tc toxins in insect
lethality, I constructed a derivative of A506 (A506∆tc) in which the entire tcaABCtccC cluster was deleted from the genome.
P. fluorescens strain A506 and the mutants A506∆tc, A506∆aprA and
A506∆gacS were tested for lethality against M. sexta. For each strain, a range of
inoculum densities, from 103 to 105 cells per ultimate instar, were tested. Insect
lethality was monitored every few hours for 48h. As observed in the experiments
described above, injection of wild-type A506 at 1 x 105 consistently resulted in nearly
100% lethality by 36h after inoculation (Figure 3.9 and Figure 3.10). When injected at
1 x 103 to 1 X 104 CFU/larva, A506 killed ca. 80% to 100% of the larvae by 48h
(Figure 3.9 and Figure 3.10). A506 was compared to A506∆gacS at 48h and no
significant difference was seen at any dose or when all doses were combined (Table
3.5). When A506 was compared to A506∆gacS, lethality caused by A506∆gacS
appeared to be slightly delayed relative to lethality caused by A506 (Figure 3.10), but
no significant difference in the relative area under mortality curve (RAUMC) was
observed (Table 3.6). At 48h after injection, both strains killed a similar proportion of
larvae (i.e., 90-100%) (Table 3.5). A506 was compared to A506∆aprA in three
experiments, and again, lethality caused by A506∆aprA appeared to be delayed
relative to lethality caused by A506 (Figure 3.9). When the results of all doses and
experiments were combined, A506∆aprA killed significantly fewer larvae by 48h than
were killed by A506 (Table 3.5). A506 also was compared to A506∆tc in three
experiments and, again, lethality caused by A506∆tc appeared to be delayed relative to
lethality caused by A506 (Figure 3.9). In addition, A506∆tc killed a smaller proportion
of larvae by the end of the experiment than were killed by the parental strain (Table
3.5). When injected at 1 x 103 CFU/larva, for example, A506∆tc killed an average of
43% of the larvae whereas A506 killed 80% of the larvae (Table 3.5). To determine if
differences in lethality of A506 vs. A506∆tc could be explained by differences in
colonization of the injected larvae, I quantified the population sizes of both strains in
32
larvae over time (Figure 3.11). The A506∆tc mutant did not colonize as quickly as the
wild-type strain, which could possibly explain the delay in lethality caused by this
mutant. The final population sizes of both strains were similar, however, suggesting
that the Tc genes were not required by A506 for colonization of the larvae.
Differences in lethality of A506 versus the mutants A506∆tc, A506∆aprA and
A506∆gacS were evaluated further by calculating the relative area under the mortality
curve (RAUMC) for each of the injection experiments (Figures 3.9 and 3.10).
Significant differences in the RAUMC were observed between A506 and A506Δtc
when inoculated at 105 CFU/larva and when all inoculum levels were considered
together, indicating that tcaABC-tccC contributes to lethality (Table 3.6). Similarly,
significant differences in the RAUMC were observed between A506 and A506ΔaprA
when inoculated at 104 CFU/larva and when all inoculum levels were pooled,
indicating that aprA contributes to lethality (Table 3.6). Because the deletion of the
toxin complex or exoprotease genes of A506 did not eliminate lethality, however,
these genes are not wholly responsible for insect mortality.
33
Table 3.1 BLASTp analysis of Tc sequences in strains of the P. fluorescens group.
Top BLAST hit
Gene
#AA
Closest
Homolog
Bacterial
Straina
P. luminescens W14
% ID
Closest
Homolog
#AA
% ID
Component A:
TcaA:
PflQ2_0670
843
ZP_03398423.1
Pst T1
38%
AAL18449
TcaA
1095
24%
PflQ8_0739
844
YP_237102.1
Pss B728a
37%
AAL18449
TcaA
1095
24%
PflQ8_4571
1203
YP_606442.1
Pe L48
26%
AAL18449
TcaA
1095
23%
PflA506_3065
878
YP_237102.1
Pss B728a
30%
AAL18449
TcaA
1095
25%
PflSS101_2971
878
YP_237102.1
Pss B728a
31%
AAL18449
TcaA
1095
25%
PseBG33_3189
869
YP_237102.1
Pss B728a
34%
AAL18449
TcaA
1095
29%
PseBG33_3800
931
YP_237102.1
Pss B728a
36%
AAL18449
TcaA
1095
29%
PflQ2_0669
1253
NP_794097.1
Pst DC3000
45%
AAL18450
TcaB
1189
33%
PflQ8_0738
1533
YP_237103.1
Pss B728a
38%
AAL18450
TcaB
1189
33%
PflQ8_4570
1364
NP_931351.1
Pll TTO1
37%
AAL18450
TcaB
1189
32%
PflA506_3066
1559
ZP_03398424.1
Pst T1
35%
AAL18450
TcaB
1189
31%
PflSS101_2972
1559
ZP_03398424.1
Pst T1
33%
AAL18450
TcaB
1189
31%
PseBG33_3190
1599
ZP_03398424.1
Pst T1
34%
AAL18450
TcaB
1189
30%
PseBG33_3801
1611
ZP_03398424.1
Pst T1
35%
AAL18450
TcaB
1189
32%
2729
ZP_09294416.1
Mal
33%
AAL18486
TcdA
2516
27%
2459
BAH22314.1
Wolbachia sp.
33%
AAL18487
TcdB
1476
31%
Pfl01_4453
1488
YP_004355903.1
Pbb NFM421
52%
AAL18451
TcaC1
1485
34%
PflQ2_0668
1495
YP_237104.1
Pss B728a
48%
AAL18451
TcaC1
1485
38%
PflQ8_0737
1493
YP_237104.1
Pss B728a
49%
AAL18451
TcaC1
1485
38%
PflQ8_4580
1447
YP_004351856.1
Pbb NFM421
99%
AAL18451
TcaC1
1485
35%
PflA506_3067
1476
YP_237104.1
Pss B728a
45%
AAL18451
TcaC1
1485
37%
TcaB:
TcdA:
Pchl3084_2947
Component B:
TcdB:
Pchl3084_2950
TcaC:
34
Top BLAST hit
Gene
#AA
Closest
Homolog
Bacterial
Straina
P. luminescens W14
% ID
Closest
Homolog
#AA
% ID
PflSS101_2973
1476
YP_237104.1
Pss B728a
45%
AAL18451
TcaC1
1485
37%
PseBG33_3191
1477
YP_237104.1
Pss B728a
45%
AAL18451
TcaC1
1485
36%
PseBG33_3802
1527
YP_237104.1
Pss B728a
46%
AAL18451
TcaC1
1485
36%
Pfl01_0947
893
YP_004356021.1
Pbb NFM421
52%
AAL18492
TccC2
915
33%
Pfl01_0948
942
YP_004356021.1
Pbb NFM421
56%
AAL18473
TccC1
1043
36%
Pfl01_4454
928
YP_004355904.1
Pbb NFM421
49%
AAL18492
TccC2
915
36%
Pfl01_4455
944
YP_004355904.1
Pbb NFM421
45%
AAL18492
TccC2
915
35%
Pfl01_4456
891
YP_004355904.1
Pbb NFM421
45%
AAL18473
TccC1
1043
34%
PflQ2_0667
929
YP_237106.1
Pss B728a
50%
AAL18473
TccC1
1043
39%
PflQ8_0736
927
YP_237101.1
Pss B728a
49%
AAL18492
TccC2
915
39%
PflQ8_4581
937
YP_004355904.1
Pbb NFM421
99%
AAL18492
TccC2
915
40%
PflQ8_4696
905
YP_004356021.1
Pbb NFM421
100%
AAL18492
TccC2
915
36%
PseBG33_3799
968
ZP_03395262.1
Pst T1
43%
AAL18473
TccC1
1043
37%
PseBG33_3803
995
ZP_03395262.1
Pst T1
45%
AAL18473
TccC1
1043
36%
PseBG33_3804
916
ZP_03395262.1
Pst T1
42%
AAL18473
TccC1
1043
35%
PseBG33_3192
966
YP_237106.1
Pss B728a
47%
AAL18473
TccC1
1043
39%
PflA506_3068
964
ZP_03395262.1
Pst T1
46%
AAL18473
TccC1
1043
40%
PflSS101_2974
967
AAL18473
TccC1
1043
Component C:
TccC:
a
ZP_03395262.1
Pst T1
47%
40%
Strains: Pst T1, Pseudomonas syringae pv. tomato T1; Pss B728a, Pseudomonas
syringae pv. syringae B728a; Pe L48, Pseudomonas entomophila strain L48; Pst
DC3000, Pseudomonas syringae pv. tomato DC3000; Pll TT01, Photorhabdus
luminescens subsp. laumondii strain TT01; Mal, Mesorhizobium alhagi CCNWXJ12-2;
Wolbachia sp., Wolbachia endosymbiont of Cadra cautella; Pbb NFM421,
Pseudomonas brassicacearum subsp. brassicacearum NFM421.
35
Table 3.2 All tcaA/tcdA-like gene products possess
the VRP1 (PF03538) domain
a
b
Locus tag
AA
Alignment
Pchl3084_2947
2729
57-292
9.70E-12
PflQ8_4571
1203
34-311
8.70E-27
PflQ8_0739
844
29-318
2.20E-45
PflQ2_0670
843
30-319
8.20E-48
PseBG33_3800
931
5-282
7.30E-45
PseBG33_3189
869
5-219
1.70E-33
PflA506_3065
878
5-229
3.10E-35
PflSS101_2971
878
6-229
1.60E-35
a/b
E-value
alignment and e-values taken from pfam.sanger.ac.uk
Table 3.3 All tcaC/tcdB-like gene products possess SpvB (PF03534) and
MidN/MidC (PF12255) domains
SpvB
a
Locus tag
AA
Alignment E-value
Pchl3084_2950
2459
42-316
Pfl01_4453
1488
PflQ8_0737
MidN
b
MidC
Alignment E-value
Alignment E-value
9.90E-78
647-824
6.90E-41
369-879
5.90E-32
27-310
1.80E-102
628-806
6.80E-56
855-997
2.00E-41
1493
28-302
7.70E-101
627-806
5.80E-50
854-1000
1.00E-41
PflQ8_4580
1447
27-303
1.30E-91
627-809
2.60E-49
857-1000
1.60E-30
PflQ2_0668
1495
28-303
1.50E-99
627-805
1.60E-50
853-1002
1.50E-40
PseBG33_3802
1527
29-316
1.90E-103
642-821
2.80E-48
876-1025
5.90E-36
PseBG33_3191
1477
30-314
2.50E-103
644-814
1.40E-44
861-1009
3.40E-39
PflA506_3067
1476
30-314
8.60E-99
643-815
4.20E-45
862-1010
4.70E-39
PflSS101_2973
1476
30-314
5.09E-100
643-815
3.10E-44
862-1010
1.40E-39
a/b
alignment and e-values taken from pfam.sanger.ac.uk
36
Table 3.4 Comparison of G+C content of individual genes to the genomic
average.
Strain
P. chlororaphis 30-84
Locus tag
Pchl3084_2947
Pchl3084_2950
P. fluorescens Pf0-1
Pfl01_0947
Pfl01_0948
Pfl01_4456
Pfl01_4455
Pfl01_4454
Pfl01_4453
P. brassicacearum Q8R1-96
PflQ8_4696
PflQ8_0739
PflQ8_0738
PflQ8_0737
PflQ8_0736
PflQ8_4581
PflQ8_4580
PflQ8_4571
PflQ8_4570
P. fluorescens Q2-87
PflQ2_0670
PflQ2_0669
PflQ2_0668
PflQ2_0667
P. sp. BG33R
PseBG33_3804
PseBG33_3803
PseBG33_3802
PseBG33_3801
PseBG33_3800
PseBG33_3799
PseBG33_3192
PseBG33_3191
PseBG33_3190
PseBG33_3189
P. fluorescens A506
PflA506_3065
PflA506_3066
PflA506_3067
PflA506_3068
P. fluorescens SS101
PflSS101_2971
PflSS101_2972
PflSS101_2973
PflSS101_2974
G+C content (%)
62.9
55.4***
56.3***
60.5
58.5
59.1
59.6
59.5
60.2
59.2
61
59.3
58.9
56.4***
60.2
59.0
60.6
60.4
59.8
60.7
60.7
59.5
58.2***
60.9
60.6
59.6
59.8
62.1
60.1
54.1***
54.9***
61.4
60.8
62.8***
58.5
59.5
60
60.9
59.7
64.1***
61.9
60
61.9
59
64.0***
61.5
*Genes having G+C contents that differ significantly from the average
G+C content of the entire genome for that strain ( p<0.001).
37
Table 3.5 Lethality of strain A506 and derivatives against larvae of M. sexta. In
each experiment, the numbers of dead larvae at 48hrs were compared between A506
and derivative treatments. An ANOVA was performed for each dose for each strain
and also when pooling all doses (Mean). Significant differences (P<0.05) were seen
between A506 and A506∆tc at 105 and 103 CFU/larva, A506 and A506∆aprA at 104
CFU/larva and when pooling all doses for these two comparisons. Values are means of
three experiments and no significant interactions between experiment, treatment, or
dose were observed.
Cell
Density
103
104
105
Mean
A506
8.0
9.7
10.0
9.2
A506
Δtc
4.3
6.7
6.7
5.9
# Dead Larvae at 48 h following inoculation
P>F A506 A506
P>F A506
A506
ΔaprA
ΔgacA
0.008
8.3
6.7 0.130
7.0
6.5
0.272
10.0
7.3 0.015
8.5
6.0
0.063
10.0
8.0 0.074
9.5
8.0
0.001
9.4
7.3 0.003
8.3
6.8
P>F
0.500
0.344
0.205
0.161
Table 3.6 Relative area under the mortality curve (RAUMC) of M. sexta
inoculated with strain A506 and derivatives. In each experiment the RAUMC was
compared between A506 and derivative treatments. An ANOVA was performed for
each dose for each strain and also when pooling all doses (Mean). Significant
differences (P<0.05) were seen between A506 and A506∆tc at 105 CFU/larva, A506
and A506∆aprA at 104 CFU/larva and when pooling all doses for these two
comparisons. Values are means of three experiments and no significant interactions
between experiment, treatment, or dose were observed.
Cell
Density
103
104
105
Mean
A506
22.7
34.1
40.9
32.6
A506
Δtc
5.3
23.5
23.9
19.8
P>F
A506
0.119
0.330
0.025
0.010
26.5
38.4
39.5
34.8
RAUMC
A506 P>F
ΔaprA
21.2 0.161
25.7 0.019
29.9 0.093
25.6 0.008
A506
27.5
37.0
41.9
35.5
A506
ΔgacA
25.2
26.2
33.5
28.3
P>F
0.500
0.335
0.217
0.125
38
Figure 3.1 Types of toxin complex (Tc) gene clusters in Pseudomonas spp. Types
I-VI are separated by gene organization and genome location. Genes are color-coded
according to the Tc component encoded: red (Component A), blue (Component B) and
yellow (Component C). Green circles denote genes with amino acid sequences most
closely related to proteins in taxa outside of the P. fluorescens group. Black circles
denote genes whose G+C content differs significantly from the genomic average
(p<0.0001). Locus tags are shown next to each strain.
Figure 3.2 Maximum likelihood tree of TcaAB/TcdA amino acid sequences. Roman numerals indicate the
Tc type of each TcaA, TcaB or TcdA sequence from the P. fluorescens group. Bootstrap values equal to or
greater than 50% are shown. GenBank accession numbers are shown next to each taxon.
39
Figure 3.3 Maximum likelihood tree of TcaC/TcdB amino acid sequences. Roman numerals indicate the Tc
type of each TcaC or TcdB sequence from the P. fluorescens group. Bootstrap values equal to or greater than
50% are shown. GenBank accession numbers are shown next to each taxon.
40
Figure 3.4 Maximum likelihood tree of TccC amino acid sequences. Roman numerals indicate the Tc type of
each TccC sequence from the P. fluorescens group. Bootstrap values equal to or greater than 50% are shown.
GenBank accession numbers are shown next to each taxon.
41
42
Figure 3.5 Homologous genes in Type II, III and IV Type Tc clusters.
Homologous genes, defined by genomic location and phylogenetic relationships, are
connected with shading of the same color. Green circles denote genes with amino acid
sequences most closely related to proteins in taxa outside of the P. fluorescens group.
Black circles denote genes whose G+C content differs significantly from the genomic
average (p<0.0001).
43
Figure 3.6A Lethality of P. fluorescens strains to fifth instars of the tobacco
hornworm, M. sexta. Mortality was assessed at 24h, 48h, or 72h following injection
with 1 x 105 CFU per larva or water, as a control. Two experiments, each evaluating
ten replicate larvae per treatment, are presented. Chi square analysis showed that
mortality greater than 40% was significant (P<0.05, d.f.= 1, χ2 test).
44
Figure 3.6B Lethality of P. fluorescens strains to fifth instars of the tobacco
hornworm, M. sexta. Mortality was assessed at 24h, 48h, or 72h following injection
with 1 x 105 CFU per larva or water, as a control. Two experiments, each evaluating
ten replicate larvae per treatment, are presented. Chi square analysis showed that
mortality greater than 40% was significant (P <0.05, d.f.= 1, χ2 test). Seven larva in
experiment three were beginning pupal ecdysis.
45
Figure 3.7 Lethality of high cell densities of P. fluorescens strains to fifth instars
of the tobacco hornworm, M. sexta. Mortality was assessed at 24h, 48h, or 72h
following injection with 1 x 106 CFU per larva or water, as a control. Two
experiments, each evaluating ten replicate larvae per treatment, are presented. Chi
square analysis showed that mortality greater than 40% was significant (P <0.05, d.f.=
1, χ2 test).
46
Figure 3.8 Colonization of larvae of M. sexta by strains in the P. fluorescens
group. Strains were: SBW25 (orange line, ▬), BG33R (red line, ▬), SS101 (green
line, ▬) and A506 (blue line, ▬). A RifR derivative of each strain was injected into
5
fifth instar at 1 x 10 CFU per larva, and culturable population sizes of each strain
were estimated from ten replicate larvae over time. No RifR pseudomonads were
reisolated from control larvae injected with sterile water. Background populations of
culturable bacteria in the control were between 1.1 x 102 and 7.8 x 105 CFU per larva.
47
Figure 3.9A Progress curves depicting mortality of larvae of M. sexta following
inoculation with P. fluorescens A506 or derivative strains. Larvae were injected
with A506 (blue line, ▬), A506∆tc (red line, ▬), A506∆aprA (green line, ▬) or
sterile water (purple line, ▬) as a negative control. Ten larvae of M. sexta were
5
4
3
inoculated with three inoculum levels (1 x 10 , 1 x 10 and 1 x 10 CFU per larva) of
each strain. In experiment two, 19 of 40 larvae began pupal ecdysis by 48 hrs.
48
Figure 3.9B Progress curves depicting mortality of larvae of M. sexta following
inoculation with P. fluorescens A506 or derivative strains. Larvae were injected
with A506 (blue line, ▬), A506∆tc (red line, ▬), A506∆aprA (green line, ▬) or
sterile water (purple line, ▬) as a negative control. Ten larvae of M. sexta were
5
4
3
inoculated with three inoculum levels (1 x 10 , 1 x 10 and 1 x 10 CFU per larva) of
each strain. For experiment three, nine of 40 larvae began pupal ecdysis by 48 hrs.
49
Figure 3.10 Progress curves depicting mortality of larvae of M. sexta following
inoculation with P. fluorescens A506 or derivative strains. Larvae were injected
with A506 (blue line, ▬), A506∆gacS (orange line, ▬), or sterile water (purple line,
▬ ) as a negative control. Ten larvae of M. sexta were inoculated with three inoculum
5
4
3
levels (1 x 10 , 1 x 10 and 1 x 10 CFU per larva) of each strain.
50
Figure 3.11 Colonization of larvae of M. sexta by A506 (blue line, ▬) and A506∆tc
(purple line, ▬). A RifR derivative of each strain was injected into fifth instar at 1 x
5
10 CFU per larva, and the population size of each strain was estimated from ten
replicate larvae over time. No RifR pseudomonads were reisolated from control larvae
injected with sterile water. Background populations of culturable bacteria in the
control were between 1.1 x 102 and 7.8 x 105 CFU per larva.
51
Chapter 4: DISCUSSION
In this study, I established that six of ten strains representing diverse lineages
of the P. fluorescens group exhibited injectable lethality to larvae of the tobacco
hornworm, M. sexta. The ten strains evaluated in this study are known for their
biological control of plant pathogens via secondary metabolite and antibiotic
production, and only two strains (Pf-5 and SBW25) of the ten evaluated here were
known to be lethal to any insect previously (87, 93). Until recently, P. fluorescens was
not known to be lethal to insects but a variety of insect orders now are known to be
susceptible to infection by P. fluorescens strains. Commare et al. observed that a
mixture of P. fluorescens strains PF1 and FP7 were able to increase larval mortality of
the leaf-folder larvae Cnaphalocrocis medinalis (Lepidoptera) and increase the
numbers of their natural enemies in the field (18). P. protegens strain Pf-5 and P.
fluorescens SBW25, which were also evaluated in the present study, have insecticidal
activity when fed to Drosophila melanogaster (Diptera) (87), and strains Pf-5 and
CHAO have injectable lethality towards G. mellonella and M. sexta (Lepidoptera)
(93). P. protegens strain CHAO strain also kills termites, Odontotermes obesus
(Isoptera) via cyanide poisoning (23). P. fluorescens strain KPM-018P has been used
to control larvae of phytophagous ladybird beetles, Epilachna vigintioctopunctata
(Coleoptera) (90) while strains F26-4C and 88-335 can elevate supercooling points
and kill overwintering Colorado potato beetles, Leptinotarsa decemlineata
(Coleoptera) (14). P. fluorescens also has been reported to decrease numbers of wood
ants, Formica paralugubris (Hymenoptera) (15) and mosquitoes, Culex
quinquefasciatus (Diptera) (96). These entomopathogenic capabilities of P.
fluorescens have been observed, yet for most of these strains, mechanisms of action
for insect lethality have yet to be uncovered.
Of the ten strains of P. fluorescens evaluated in this study, six killed the insect
host M. sexta when injected at ca. 105 CFU into fifth instar, and four strains (Q2-87,
52
Q8R1-96, Pf0-1 and SBW25) exhibited no significant lethality. These results confirm
the negligible insect toxicity of Pf0-1, which exhibited virtually no oral toxicity to D.
melanogaster in an earlier study (87). Because Pf0-1 is now known to have a mutation
in gacA (80), I also tested a gacA+ derivative of Pf0-1, which did not cause significant
lethality when injected into larvae of M. sexta. Therefore, the mutation in the central
regulator gacA did not account for the benign phenotype of Pf0-1. It is interesting that
strain SBW25, which exhibits oral toxicity to D. melanogaster (87), did not kill larvae
of M. sexta in this study. Insect mortality resulting from a strain of P. fluorescens has
often been observed in one order of insects but not another, implying that distinct
bacterial traits are responsible for lethality in different insect hosts. In this study, the
greatest levels of hornworm lethality were caused by strains in Sub-clade 1 and three
of the four strains in Sub-clade 3 of the P. fluorescens group (Figure 1.1). All three
strains (Pf-5, O6 and 30-84) in Sub-clade 1 have the fitD gene, which encodes the FitD
toxin. Because FitD is known to be a lethal to M. sexta and a primary determinant of
the injectable insect lethality phenotype of Pf-5 (93), I did not explore genetic
mechanisms of insect lethality in the fitD-containing strains. Instead, I focused on the
strains A506, SS101 and BG33R, which also caused high levels of lethality to M.
sexta. Following a search of these genomes for genes with potential roles in insect
lethality, I selected three types for consideration: those encoding toxin complexes,
extracellular proteases and the GacS/GacA regulatory system.
I began my analysis of toxin complexes with a bioinformatic survey of the
genomes, and I identified candidate Tc genes in eight of the ten genomes. Among
those were genes encoding putative A components (tcaA/tcdA) and B components
(tcaC/tcdC) of Tc toxins. I then found that TcaA/TcdA and TcaC/TcdC sequences
have conserved domains characteristic of the extensively-studied insect Tc genes from
entompathogenic bacteria. For example, the SpvA (also called VRP1) domain found in
TcaA/TcdA and the SpvB domain, found in TcaC/TcdB, correspond to products of
spvA and spvB, which are present on a 65-kb virulence plasmid and necessary for full
53
virulence of S. enterica. Although SpvA and SpvB domains are present in insect
toxins of many bacteria that infect insects, they do not appear to have an essential role
in toxicity in all bacteria (74). The SpvB domain is within a protein family that resides
in the bacterial cytoplasm and is expressed most during early stationary phase, but
lessens as the latent phase approaches, suggesting a role in initiating virulence (41).
The MidN and MidC domains typically are found with the SpvB domain in toxin
proteins, and these domains are near the N-terminus and C-terminus of the bacterial
insecticidal toxin TcdC (95). These domains are also found in Yersinia
pseudotuberculosis and P. luminescens Tc proteins (95). The detection of these
domains in putative Tc sequences gave rise to the idea that Tc toxins may contribute to
the insect lethality of the P. fluorescens strains, as has been demonstrated in P.
luminescens (31). Conversely, the presence of Tc genes in genomes of strains
exhibiting negligible lethality as well as those exhibiting high levels of lethality
argued against this possibility. To detect any differences that may exist between the Tc
genes of benign and lethal strains of P. fluorescens, I did a detailed bioinformatic and
phylogenetic analysis of the Tc gene clusters in all eight strains.
On the basis of Tc gene cluster organization and genomic location, G+C
content, and phylogeny, the gene clusters were classified into six distinct types. The
genomic context, or relative placement of these genes, revealed clues to inheritance.
The type I Tc gene cluster, which is in P. chlororaphis 30-84, is unlike any of the
others. P. chlororaphis 30-84 is the only strain to have tcdA/tcdB-type genes, which
are in a unique, non-conserved region of the genome also containing genes encoding
phage integrase, transposase and cointegrate resolution proteins. This is similar to the
tcd genes of P. luminescens W14, which are on a pathogenicity island that may be
highly mobile (33). The G+C content of these genes is also significantly lower than
the genome average; only in strain 30-84 was the G+C content of all Tc genes
significantly different than its genomic average (Table 3.3). Phylogenetic analysis
revealed that these genes are more closely related to homologs in other bacterial
54
genera than to Pseudomonas spp. (Figure 3.1 and 3.3). Their phylogenetic
relationships, atypical G+C content, and genomic context give strong evidence that the
toxin complex genes of P. chlororaphis 30-84 were acquired through horizontal
transfer. Interestingly, the Tc cluster of 30-84 does not include a gene encoding a C
component, which is considered a ‘potentiator’ and necessary for full toxicity of the
Tc complex of P. luminescens (33). It is quite possible that the cluster does not confer
insect toxicity, but its functionality was not assessed further in this study.
The Tc gene clusters in seven strains fell into six types that corresponded to
strain phylogeny: Type II, III, and IV clusters are in Sub-clade 2 strains, and Type V
and VI clusters are in Sub-clade 3 strains. This points to similar patterns of inheritance
within each sub-clade, but divergence between Sub-clades 2 and 3, which could
account for the vastly different insect lethality phenotypes exhibited by strains in the
two sub-clades. For example, Pf0-1 does not have a full complement of Tc genes, as it
is missing the A-like component, which is critical for toxicity in P. luminescens (33).
Although a full complement of Tc genes is present in Q2-87 and Q8R1-96, there are
significant differences between the Type III and IV clusters, which are present in these
benign strains, and Type V and VI clusters, which are present in the Sub-clade 3
strains that cause insect lethality. Therefore, I included Tc genes among the loci to be
evaluated for roles in insect lethality.
An important contribution of this study was the evaluation of specific genes for
their roles in insect lethality caused by P. fluorescens. I selected strain A506 for these
studies because it was among the strains showing the highest level of toxicity to M.
sexta, is a widely-studied model strain in phyllosphere microbiology (43), and is a
registered biological control agent that is used commercially for management of fire
blight in the US (110). Three loci with demonstrated or postulated roles in insect
toxicity were evaluated: tcaABC-tccC, gacS, and aprA. Two of the loci (tcaABC-tccC
and aprA) had minor roles in insect toxicity of A506, which was most evident in
55
differences in the RAUMC calculated for A506 versus derivatives with mutations in
these genes. Nevertheless, none of the mutations eliminated lethality caused by A506,
indicating that loci other than those evaluated here contribute to insect toxicity by this
strain. The finding that the Tc genes were not required for the insect lethality
phenotype of A506 is not completely surprising, as the literature includes varied roles
for these genes in insect lethality of other Pseudomonas spp. Although Tc genes have
been found in P. syringae strain B728A, they have not been shown to confer insect
lethality and consequently remain putative toxins. The P. syringae B728A tcdA1 and
tcaA genes were disrupted by single-crossover homologous recombination and each
mutant retained full virulence against the pea aphid, Acyrthosiphon pisum (Hemiptera)
in feeding assays (108). In contrast, when tccC from P. taiwanensis was cloned and
expressed in E. coli, the recombinant E. coli exhibited oral toxicity to D. melanogaster
(79). This seems peculiar, as the tccC gene was initially characterized as a toxin
potentiator and not a toxin in itself (33). Here, the entire set of insecticidal genes was
knocked out in strain A506 and, while the Tc genes were not responsible for virulence
in the tobacco hornworm, they played a significant role in lethality. A506∆tc was still
able to colonize the hornworm, albeit at somewhat reduced levels at early time points
(Figure 3.11), indicating that the mutant retained partial fitness in the insect host.
Therefore, the lack of full toxicity could not be attributed to lack of colonization of the
host by the mutant A506∆tc. This study provides the first evidence for a significant
effect of a Tc cluster on insect lethality by a Pseudomonas species. This could be
attributed to the type of Tc cluster found in A506 or the choice of insect host
evaluated.
The two-component regulators GacS and GacA are key members of a global
regulatory network that control the expression of ca. 10% of the genes in the P.
fluorescens genome (46). In P. protegens Pf-5 (87) and P. entomophila (120), the
GacA/S two component system is required for oral toxicity to D. melanogaster, and
gacA mutants are virtually benign. Furthermore, GacS/GacA are required for the
56
production of many products with possible roles in insect toxicity such as monalysin
(88), biosurfactants (67, 82), type III secretion systems, hemolysins, lipases and other
virulence factors (71, 120). Therefore, it was surprising that the ΔgacS mutation did
not have a significant effect on insect lethality of A506 in this study. As mentioned
above, however, there have been relatively few studies evaluating the mechanisms of
insect toxicity in Pseudomonas spp. to date and it is already clear that distinct
mechanisms are operating in different bacterial strain-insect host combinations. The
GacA/GacS regulatory system has an important role in toxicity of some Pseudomonas
spp. against certain insect hosts, but played a very minor role in injectable toxicity of
A506 against M. sexta.
Among the many phenotypes controlled by GacS/GacA in Pseudomonas spp.
is the extracellular protease (71), and the gacS mutant of A506 is known to be
deficient in extracellular protease production (3). Given that, it is surprising that the
A506ΔaprA differed from A506 whereasA506ΔgacS did not differ markedly from
A506 in lethality towards M. sexta. Because GacA/GacS can have both positive and
negative effects on the expression of many phenotypes of Pseudomonas spp. (46), it is
possible that the gacS mutation increased the expression of another phenotype(s) that
contributes to insect lethality. The role of AprA in insect lethality of A506 observed
here is consistent with results from other studies, which have shown that extracellular
protease production enables pseudomonads to persist more effectively in an insect host
(75, 108). In P. entomophila, AprA is an important virulence factor, enabling the
bacterium to resist the D. melanogaster immune system (75). The aprA gene of A506
is 74.9% identical to that of P. entomophila, suggesting that these similar proteins may
have similar roles in insect lethality of both Pseudomonas species. Nevertheless, a
larger effect of AprA on insect lethality was observed previously in P. entomophila
(75) than I observed here in A506. Again, the strain-host specificity may be the
distinction between results found here versus those found for other bacteria-insect
models. It is possible that A506 has numerous mechanisms to protect itself against the
57
insect immune system, such that loss of one mechanism, such as the AprA protease,
has a negligible effect on its virulence. For example, the genome of A506 has two
genes, in addition to aprA, with conserved domains characteristic of extracellular
proteases (Figure 4.1). These genes are also present strains BG33R and SS101, which
are toxic to M. sexta, but are not present in the related strain SBW25, which is benign
to the hornworm larvae. In contrast, aprA is present in all ten genomes of the P.
fluorescens strains, and therefore is not correlated with insect lethality. The additional
protease genes in the A506 genome would be interesting candidates for further studies
evaluating mechanisms of insect lethality in P. fluorescens.
Insect lethality was an unexpected characteristic of the P. fluorescens group as
there is no known insect association in the life cycles of these bacteria. Here, I have
shown that six strains within this group can be lethal to M. sexta, but the importance of
this lethality in the natural environment is unknown. I also identified two bacterial
genes with potential roles in insect lethality, but none of the three genes that were
tested were completely responsible for killing hornworms. Future efforts to identify
the primary factor(s) responsible for insect lethality could be informed by comparing
the genomes of strains that are lethal versus benign to M. sexta. Some candidate genes
include the exoproteases described above and the distinctive collection of type III
secretion system effectors in these strains (Figure 4.1). Additional candidates may be
found among the genes that are present in the genomes of A506, BG33R and SS101,
but not in the strains that exhibited no lethality to M. sexta in this study. By comparing
the predicted proteomes of these strains (80), we assembled a list of 183 candidate
genes meeting these criteria. By knocking out candidate genes on this list and
evaluating the lethality of the knock-out mutants, as was done in this study, one could
determine if these genes are involved in entomopathogenesis. Future directions
involving the search for novel toxins or other factors in this well-known biocontrol
group have tremendous promise for gaining insight into new strategies for managing
insect pests.
58
Figure 4.1 Exoenzymes and secretion systems found in the sequenced strains of
the P. fluorescens group. Colored boxes represent the presence of a gene or gene
cluster within a genome, while absence of a cluster is represented by a grey circle;
numbers within a box represent the number of copies of a gene or cluster within a
genome. Putative T3SS effectors were not examined for SBW25, therefore no box or
circle is present in that column for SBW25. Genes within regions of atypical
trinucleotide content have half of their boxes blackened. Revised from Loper et al.
2012 (80).
59
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