Library-on-a-slide identification of novel enediyne encoding genes
Nick Allenby, JEM. Stach, Lucy Bock, and Alan Ward
University of Newcastle upon Tyne, Institute for Research on Environment and Sustainability,
School of Biology, Newcastle upon Tyne. NE1 7RU.
email: N.Allenby@surrey.ac.uk Alan.Ward@ncl.ac.uk
Analysis of DNA extracted from environmental samples has shown that molecular genetic
diversity is much greater in natural habitats than was previously recognised (1). Can we use
this diversity to help alleviate the need for new anti-infective compounds? How do we know
where to look? Using standard Microarray spotting techniques to print the genomic DNA
(rather than PCR/oligo DNA) of multiple species onto a glass microarray slide we have
created a “Library-on-a-slide”. These slides are then probed for the presence of specific genes,
allowing the determination of genetic content.
As a family the enediyne antibiotics are the most potent cytotoxic antitumoral agents
ever to be discovered. They have three distinct structural elements: a DNA-recognition unit;
an activation component; and the enediyne “warhead” which can be either a 9- or 10membered ring, this warhead cycloaromatises to a diradical species and in the presence of
DNA results in oxidative strand scission (2).
Recent studies have shown that the genes encoding enediynes are present in the
genomes of actinomycetes at a higher rate than predicted by fermentation studies (3). We
wanted to determine if library-on-a-slide can be used to identify novel enediyne gene clusters
from diverse actinomycetes.
Fluorescently labelled probes created from enediyne genes were hybridised to the
“Library-on-a-slide” and species with good signals were investigated for the presence of
enedyine loci by degenerate PCR (4). Six of the eleven candidates identified from the
hybridisations were confirmed to harbor enediyne biosynthetic loci by PCR and sequence
analysis a similar hit rate to Zazopoulos and co-workers (3).
The application of “Library-on-a-slide” methodology to the study of phylogeny and
diversity of species and genes could be useful in predicting which species should be targeted
for antibiotic discovery programs.
REFERENCES
1. Bull, A. T., A. C. Ward, and M. Goodfellow. 2000. Search and discovery strategies for biotechnology: the paradigm shift. Microbiol Mol
Biol Rev 64:573-606.
2. Galm, U., M. H. Hager, S. G. Van Lanen, J. Ju, J. S. Thorson, and B. Shen. 2005. Antitumor antibiotics: bleomycin, enediynes, and
mitomycin. Chem Rev 105:739-58.
3. Zazopoulos, E., K. Huang, A. Staffa, W. Liu, B. O. Bachmann, K. Nonaka, J. Ahlert, J. S. Thorson, B. Shen, and C. M. Farnet. 2003. A
genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nat Biotechnol 21:187-90.
4. Liu, W., J. Ahlert, Q. Gao, E. Wendt-Pienkowski, B. Shen, and J. S. Thorson. 2003. Rapid PCR amplification of minimal enediyne
polyketide synthase cassettes leads to a predictive familial classification model. Proc Natl Acad Sci U S A 100:11959-63.
Robert Bell poster abstract
Lantibiotics are ribosomally synthesised, post-translationally modified antimicrobial peptides.
They are produced by Gram-positive bacteria and are effective against a wide variety of
bacterial pathogens. Lantibiotics contain the dehydrated amino acids 2,3-didehydroalanine
(Dha) and (Z)-2,3-didehydrobutyrine (Dhb) formed from serine and threonine residues,
respectively. These modified amino acids are then able to form lanthionine rings which are
characteristic of lantibiotics via cyclisation reactions carried out by lantibiotic specific
enzymes. The characteristic lanthionine (Lan) residue consists of a Dha residue cross-linked
via a thioether bridge to a cysteine, while Dhb bound to cysteine forms a methyl-lanthionine
(MeLan) bridge. Lantibiotic biosynthetic genes are typically organised into clusters encoding
all the necessary components for production, modification, export of and immunity towards
the peptide. The lanA gene encodes the lantibiotic precursor, LanA. The LanA peptide
contains a C-terminal region that undergoes post translational modification by either LanBC
or LanM modification enzymes. The LanA pre-lantibiotic also contains a relatively long (23
to 59 amino acids) N-terminal leader sequence which is not modified. The final step of
lantibiotic maturation is carried out by two enzymes, the LanP protease, and the LanT
transporter. Removal of the leader sequence is carried out by the serine-type protease LanP, or
by the protease domain of the ATP-binding cassette LanT. Once exported from the cell and
cleaved from the leader sequence, the cell must then protect itself from the mature lantibiotic.
Two systems have been identified that confer host cell immunity. These are the lanFEG and
lanI systems. lanFEG encodes an ABC transporter which is able to remove lantibiotics such as
nisin from the cytoplasmic membrane and export it out into the extracellular space. Both
lantibiotic systems also contain a LanI lipoprotein, which binds lantibiotics on the outside of
the cytoplasmic membrane preventing them from affecting the host cell. The majority of
lantibiotics are regulated by two component regulatory systems. These consist of a LanK
sensor kinase which is able to bind to its lantibiotic and transmit a signal to the response
regulator LanR, which in turn binds to its cognate promoters to activate transcription of the
biosynthetic gene cluster.
IRON AND COPPER COOPERATE TO PAVE THE ROAD TO
DEVELOPMENT IN STREPTOMYCES LIVIDANS
S.M. Bialek1, B.J.F. Keijser1, R. van der Heijden2, G.W. Canters1, E. Vijgenboom1
1
2
Leiden Institute of Chemistry, Einsteinweg 55, Leiden, The Netherlands
Leiden/Amsterdam Center for Drug Research, Einsteinweg 55, Leiden, The Netherlands
In Streptomyces an aerial mycelium that produces spores is the end product of
the development. Although a lot of data are available on the intracellular action that is
needed for the development, little is known about the extracellular activity and
changes during development.
Copper ions have been shown to be essential for morphological development
in Streptomyces lividans [1, 2]. If all Cu+1 is chelated by the BCDA (bathocuproine
disulphonic acid) development is arrested in vegetative mycelium phase. Studies of
homeostasis of other heavy metals in S. lividans, showed a strong influence of iron on the
development. In the presence of a Fe+2 chelator (bathophenantroline disulphonic acid), no
aerial hyphae formation was observed, as was also seen with Cu chelation.
Analysis of the copper and iron effect on S. lividans development is studied on the
extracellular proteome level. Comparison of the 2-dimensional profiles shows, that
expression levels of several proteins are induced by both Cu and Fe starvation,
among which Sco1 and proteins of the iron-siderophor uptake system. The copperchaperone Sco1 is required for the maturation of the respiratory terminal oxidase
cytochrome c oxidase, COX. Higher Sco1 levels could signal that the terminal
oxidase lacks its co-factor, Cu. A S. lividans knock-out mutant of sco1 is devoid of
COX activity and has a bald phenotype in contrast to the wild type strain that has
high COX activity and full development.
An increased expression of a siderophore-binding lipoprotein under Fe and Cudepleted conditions was observed. Analysis of the extracellular metabolite- profile of Cu-rich,
Cu-depleted and Fe-depleted growth conditions with MALDI-TOF resulted in the identification
of several different siderophores and the changes in their expression patterns. The results
strongly suggest a tight relation between copper- and iron-homeostasis. This relation will be
discussed in the framework of the proteomics and metabolomics studies.
[1] Keijser, BJF, van Wezel, GP, Canters, GW, Kieser, T and Vijgenboom, E. 2000. The ramdependence of Streptomyces lividans differentiation is bypassed by copper. J Mol Microbiol Biotechnol
2(4):565-74
[2] Keijser, BJF and Vijgenboom, E. 2002. Copper and the morphological development of
Streptomyces. In: Handbook of copper pharmacology and toxicology (eds. E. Massaro), Humana
press, chapter: 31:503-525
Screening of endophytic metagenomic libraries
using Bacillus species as hosts
Ruth Breitschädel1, Sabine Feichtenhofer1, Branislav Nikolic2, Angela Sessitsch2,
Helmut Schwab
1Institute of Molecular Biotechnology TU Graz; Austria
2Department of Bioresources ARC Seibersdorf, Austria
Screening of new enzymes is of increasing interest for industrial, pharmaceutical, agricultural
or biotechnological applications. Therefore it’s very important to use an inexpensive but fast
method to handle large gene libraries in an economic manner. To increase the probability of
finding enzymes with special substrate specificities new biological sources are investigated.
In this study we use the wide diversity of endophytic bacterial small-insert metagenomic
libraries combined with highly efficient transformation procedures.
RELATIONSHIP BETWEEN ACTIVATING AND EDITING FUNCTIONS OF THE
ADENYLATION DOMAIN OF APO-TYROCIDIN SYNTHETASE 1 (APO-TY1)
V. Bucevic-Popovic1, M. Pavela-Vrancic1, R. Dieckmann2, H.Von Döhren2
1
Department of Chemistry, Faculty of Natural Sciences, Mathematics and Kinesiology,
University of Split, N. Tesle 12, 21000 Split, Croatia
2
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität
Berlin, Franklinstrasse 29, 10587 Berlin, Germany
Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine
biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the
mis-activated amino acid provides for fidelity of product formation. We chose to analyse the
consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1,
on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1
depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such
as L-PheSer, implies that editing is not designated to select a specific mis-activated amino
acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that
active site residues which interact with the adenylate are essential for both activation and
editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the
protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant
proteins suggests a role of active site residues in maintaining the structural determinants for
substrate selection. Inadequate conformational changes, induced by non-cognate amino acid
substrates, promote ATP breakdown yielding Pi and ADP. Replacement of residue Lys186 or
Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate
chain for adenylate formation and pyrophosphate cleavage.
Two novel lantibiotics produced by Streptomyces venezuelae
Jan Claesen, Mervyn J. Bibb
Department of Molecular Microbiology, John Innes Centre, Norwich, UK
Lantibiotics are ribosomally synthesised peptide antibiotics containing a number of unusual
amino acids (e.g. lanthionine, methyllanthionine)1,2. These are introduced post-translationally
by dedicated modification enzymes (e.g. LanBC or LanM), encoded by genes which are
clustered alongside genes for transport, immunity and regulation. Because of their
proteinaceous nature, lantibiotics are ideal candidates for structural engineering.
Organisms belonging to the genus Streptomyces are Gram-positive soil bacteria that are
generally known as important antibiotic producers. Among the broad spectrum of antibiotic
compounds, several lantibiotics have been described and recently the gene cluster for
cinnamycin has been characterised3.
While most lanthionine-containing peptides have been identified by their biological activities,
genome sequencing of S. venezuelae has revealed two gene clusters potentially encoding
lanthionine-containing peptides with novel biological functions. This study focuses on the
genetic and biochemical analysis of both clusters.
1) Chatterjee et al. (2005). Biosynthesis and mode of action of lantibiotics. Chem Rev, 105: 633-84.
2) McAuliffe et al. (2001). Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol Rev, 25: 285-308.
3) Widdick et al. (2003). Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus
cinnamoneus DSM 40005. Proc Natl Acad Sci U S A, 100: 4316-21.
Abstract: Emma Doud
Moenomycin A (MmA) is a potent antibiotic that inhibits cell wall biosynthesis by reversibly
binding the transglycosylase portion of penicillin binding protein 1b (PBP1b); however, few
studies have been done to investigate its biosynthetic pathway due to the difficulty of
identifying the relevant genes.
Our lab has recently located the MmA biosynthetic gene cluster in the Streptomyces
ghanaensis genome, and we have begun to annotate this cluster by analyzing MmA
biosynthetic intermediates that accumulate upon deletion of specific Moe genes. The
information gained has given us clues to the order and general mechanism of MmA
production. To further elucidate the mechanism of MmA biosynthesis, we have also begun to
biochemically characterize many of the enzymes involved, specifically the unique synthetase
(MoeO5) that is believed to couple phosphoglycerate to moenocinol, the lipid portion of
MmA. MoeO5 has been cloned into a vector for expression in Streptomyces lividens as
expression of the functional protein in E.coli has been challenging. Following purification of
functional protein, we will develop an assay to biochemically characterize the enzymatic
reaction in vitro. Since the phosphoglyceric acid moiety is unique to the MmA family of
antibiotics, results from these experiments will allow for a better understanding of a new
synthetase with potential use in chemoenzymatic syntheses of novel bioactive molecules as
well as of the overall biosynthesis of MmA.
Isolation and characterisation of actinomycete bacteria
from sediments of the Trondheim fjord, Norway
Kerstin Engelhardt1, Harald Bredholt1#, Espen Fjærvik1, Håvard Sletta2, Geir Klinkenberg2,
Trond E. Ellingsen2, Sergey B. Zotchev1
1 Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
E-mail: kerstin.engelhardt@biotech.ntnu.no
# Present address: Alpharma AS, Harbitzaleen 3, Oslo, Norway
2 Department of Biotechnology, SINTEF Materials and Chemistry, SINTEF, N-7034 Trondheim, Norway
.:: Abstract ::.
The aim of this study was to isolate rare actinomycete bacteria from sediment samples taken
from different depths in the Trondheim fjord, Norway, to find new producers of antibiotics.
Five different selective treatment methods of sediment samples were used to enrich and obtain
particularly rare actinomycete genera. Pure, single isolates were conserved in a strain library.
A selection of strains was chosen for a screening for antibacterial and antifungal activity. In
addition, the selected strains were characterised by molecular taxonomy via determination of
16S rDNA sequences.
Differential regulation of carbapenem and prodigiosin production in Serratia sp. ATCC
39006 by a TetR family transcriptional regulator, PigZ
T. Gristwood & G.P.C. Salmond
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK
Serratia sp. ATCC 39006 (39006) is a Gram-negative enterobacterium which produces a
carbapenem antibiotic, pectinase, cellulase and an intracellular red pigment, prodigiosin.
Prodigiosin has been reported to have immunosuppressive and anti-cancer properties although
the physiological role in 39006 is not yet known. Exoenzyme and secondary metabolite
production are under the control of a LuxIR-type quorum sensing system. In addition,
numerous factors have been identified that alter exoenzyme and secondary metabolite
production in response to multiple environmental cues, forming a complex hierarchal
regulatory network. Many of these regulators are under quorum sensing control. We have
identified a hyper-pigmented mutant containing a transposon insertion in a gene (pigZ)
encoding a TetR family transcriptional regulator. The pigZ mutant also shows reduced levels
of carbapenem, cellulase and pectinase. We have shown that pigZ regulates both the
prodigiosin and carbapenem biosynthetic operons at the level of transcription but does not
affect transcription of other known secondary metabolite regulators. The PigZ protein
represses transcription from its own promoter and that of a divergently transcribed gene, zrpA,
apparently via direct binding to the 248 base pair intergenic region. The zrpA gene is
predicted to encode a resistance-nodulation-cell division (RND) membrane fusion protein and
forms part of a putative operon encoding a complete RND efflux pump. A mutation in zrpA
restores production of prodigiosin and carbapenem in the pigZ mutant to wild type levels.
Therefore, our data suggests that it is the overexpression of this putative RND pump in the
pigZ mutant which is responsible for the alterations in pigment and carbapenem production.
To date, pigZ is the only regulator identified that differentially regulates prodigiosin and
carbapenem production in this strain. This differential regulation may imply a different
‘fitness role’ for the two secondary metabolites. Therefore, determining the physiological
signals to which PigZ is responding may help to shed light on the biological role of
prodigiosin.
Tenellin Biosynthesis in the Entomopathogenic Fungus Beauveria bassiana
Laura M. Haloa*, Kirstin L. Eley a, Zhongshu Song a, Russell J. Cox a, Andrew M. Bailey
b
, Colin M. Lazarus b and Thomas Simpson a
a School
b School
of Chemistry, University of Bristol
of Biological Sciences, University of Bristol
Tenellin 1 is a 2-pyridone produced by the entomopathogenic fungus B. bassiana. 1 is
derived from a polyketide and tyrosine. The first step in tenellin biosynthesis is catalyzed
by a polyketide synthase and non-ribosomal peptide synthetase hybrid (PKS-NRPS)
encoded by tenS1 and an auxiliary trans-acting enoyl reductase.
HO
OH
O
N
O
OH
1
New tenellin analogues have been produced by expressing tenS in Aspergillus oryzae.
Three main products, prototenellins A- C 2-4 have been isolated. The lack of enoyl
reduction after first extension cycle results to errors in biosynthesis. Prototenellin-A has a
different methylation pattern, prototenellin-B has shorter chain length and prototenellin-C
is less reduced than tenellin. TenS has a putative releasing domain, which is similar to
reduction domain found in bacterial NRPS, but prototenellins are released unreduced.
OH
OH
HN
HO
O
HN
HO
O
O
O
2
3
or isomer
OH
OH
HO
O
HN
O
O
4
B. bassiana is a broad spectrum insect pathogen and can be used as biological pesticide.
The role of tenellin in insect pathogenesis was studied by comparing the ability of B.
bassiana wt and tenS knock-out (non tenellin producer) to kill wax moth larvae. No
significant differences were detected indicating that tenellin production is not essential for
insect pathogenicity.
1
K. L. Eley, L. M. Halo, Z. Song, H. Powles, R. J. Cox, A. M. Bailey, C. M. Lazarus, T. J.
Simpson, ChemBioChem, 2007, 8:289-297.
Abstract: Diane Hatziioanou
Discovery & Analysis of Novel Bacteriocins from Commensal Bacteria
Bacteriocins are antimicrobial proteins or peptides produced by bacteria with the purpose of
killing or inhibiting the growth of other bacteria. They have been grouped into 3 major classes
but each class has a diverse content with regard to bacteriocin structures, genetics, modes of
secretion and action as well as their target organisms. This project uses two approaches to
identify a novel bacteriocin from the gut: the classical biochemical methods for bacteriocin
screening and a genome mining approach which focuses on the class I bacteriocins: the
lantibiotics. Lantibiotics are small lanthionine-containing peptides produced by Gram positive
bacteria and exhibit nanomolar potency against other Gram positive bacteria including human
pathogens.
Spread of Antibiotic Biosynthesis Genes by Mobile Genetic Element?
Hanne Jørgensen1, Espen Fjærvik1, Sigrid Hakvåg1, Per Bruheim1, Harald Bredholt2, Geir
Klinkenberg3, Sergey B. Zotchev1.
1
Department of Biotechnology ,The Norwegian University of Science and Technology, N-7491 Trondheim,
Norway.
2
Alpharma, Harbitzalleen 3, P.O.Box 158 Skoyen, N-0212 Oslo, Norway.
3
SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, N-7034 Trondheim, Norway.
Extracts of bacterial isolates from the Trondheim fjord were screened for the presence of
antifungal antibiotics. Approximately one thousand potential producers of antifungal
antibiotics were identified, and extracts of almost half of them exhibited a UV-spectrum
characteristic of heptaene macrolides. LC-MS analysis of 52 of these extracts revealed that all
contained the same compund: the aromatic polyene antibiotic candicidin D. As the isolates
differed morphologically, the wide occurance of producers of this particular antibiotic
suggested that candicidin biosynthesis genes might be transferred from one strain to another
by a mobile genetic element. To investigate this possibility Southern blot analyses, pulsed
field gel electrophoresis and mating experiments were performed. A giant linear plasmid
harbouring the candicidin biosynthetic gene cluster was identified in one of the Trondheim
fjord streptomycete isolates, but we were not able to establish a procedure for interspecific
transfer. However, the wide distribution of the can gene cluster among the Trondheim fjord
isolates suggests that this GLP might possess the ability for conjugal transfer and integration
into the chromosome in its natural environment.
CHARACTERIZATION
AND
ENGINEERING
OF
THE
CONGOCIDINE BIOSYNTHETIC PATHWAY
M. Juguet, S. Lautru, F-X. Francou, J-L. Pernodet
Institut de Génétique et Microbiologie, Université Paris-Sud 11 - Orsay, France
Streptomyces are bacteria remarkable for the diversity of the secondary
metabolites that they produce. Streptomyces ambofaciens was known to produce
two antibiotics: the macrolide spiramycin and the pyrrole amide congocidine.
The spiramycin biosynthetic gene cluster has been characterized but nothing was
known on congocidine biosynthesis although the molecule has been isolated in
1951 and characterized in 1967. Congocidine belongs to the pyrrole amide
family and is interesting for its ability to bind DNA with some sequence
specificity.
The sequencing of the terminal parts of the S. ambofaciens chromosome (about
1,3Mb on each side) was undertaken in order to discover gene clusters possibly
involved in secondary metabolite biosynthesis. This sequencing has revealed
several such gene clusters. In each of these clusters, one gene was inactivated
and the biological activity of the resulting mutant strain was studied.
This allowed the identification, on the right arm of S. ambofaciens of a gene
(cgc18) involved in the biosynthesis of congocidine and belonging to a gene
cluster containing 22 genes (cgc3* to cgc19). Two genes (cgc1*, cgc2*),
encoding an ABC transporter, confer congocidine resistance to the producer
organism. One gene (cgc1) might be involved in the regulation of congocidine
production. Several genes encode proteins related to NRPS domains which
could be involved in the assembly of congocidine. The function of the other
genes cannot be predicted by sequence analysis and comparison. To characterize
the involvement of each gene in congocidine biosynthesis, a functional analysis
of the cluster, overexpressing or inactivating several genes and analyzing the
molecules accumulated by the mutant strains is in progress.
When this cluster was expressed in an heterologous host, Streptomyces lividans,
it conferred to this host the ability to synthesize congocidine in amount
comparable to the one obtained in the original host.
A cryptic type I PKS gene cluster in Streptomyces ambofaciens ATCC23877:
towards the discovery of a new antibiotic.
Luisa Laureti
Laboratory of Genetics and Microbiology, University Henri Poincaré, BP 239, 54506,
Vandoeuvre-lès- Nancy, France.
E-mail: Luisa.Laureti@scbiol.uhp-nancy.fr
Streptomycetes and related actinomycetes are Gram-positive bacteria well known for being a
great source of secondary metabolites; in particular they produce about 60-70% of all the
natural discovered antibiotics. The most important chemical class of antibiotics is the
polyketides. Modular polyketide synthases (PKSs) are multifunctional enzyme assemblies
that catalyze the stepwise condensation of small carboxylic acids into structurally diverse
polyketides. For their ability to yield a multitude of different and complex compounds
together with the constant need of new pharmaceuticals, in the last few years these enzymes
came to the fore as ideal biotech tools for the production of novel hybrid compounds.
During the sequence project of Streptomyces ambofaciens ATCC23877 chromosome, several
clusters encoding secondary metabolites were found, some of them probably involved in
antibiotic production, as already demonstrated for alpomycin cluster1. One of these clusters
corresponds to a cryptic type I PKS gene cluster which contains a large number of PKS genes
(9) and of modules responsible for the polyketide chain elongation (25). In silico analysis of
all the modules showed that they should be functional; hence the idea that a new interesting
compound, probably a macrolide, can be produced.
Generally the expression of secondary metabolite clusters is under the control of different
regulatory genes. We identified five regulatory genes in the cluster: a two component system
and three other genes belonging to deoR, gntR and luxR transcriptional regulator families,
respectively. In order to trigger the expression of this cryptic cluster and possibly isolate its
compound, our main strategy rests upon deregulating the regulatory network. Firstly we
constructed mutants in which the genes are overexpressed under the control of a strong
constitutive or inducible promoter. Secondly we are making deletion mutants. In both cases
our purpose is to screen for a particular phenotype that differs from the wild type. Therefore
HPLC profiling will be a proper way to identify the possible new compound produced by this
cluster. We are also interested in possible cross effects on the other antibiotics, since this was
already observed for the spiramycin cluster2.
REFERENCES:
1
Pang X., Aigle B, Girardet J-M., Mangenot S., Pernodet J-L., Decaris B. and Leblond P. (2004)
Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces
ambofaciens linear chromosome. Antimicrob. Agents Chemother. 48:575-588.
2
Aigle B., Schneider D., Morilhat C., Vandewiele D., Dary A., Holl A.C., Simonet J.M. and Decaris B.
(1996) An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene
homologous to polyketide synthase genes. Microbiol. 142:2815-24.
2-PHENYLETHANOL SYNTHESES BY YEAST SACCHAROMYCES
CEREVISIAE
Mameeva Olga, PhD
Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of
Ukraine, D03680 Kiev, Zabolotnogo str. 154, Ukraine, mameeva@ukr.net
Aromas and flavors from natural sources, received by biotechnological processes
have high quality. One of compound advantages of such origin is absence of impurity,
which is formed at chemical synthesis. The high aromatic alcohol of the 2-phenylethanol (2PE) has a strongly pronounced smell of roses. It is widely applied in various industry areas.
Natural 2-PE is mainly extracted from huge amounts of rose, hyacinths, jasmine, narcissi and
lilies petals. Therefore the biotechnological production can be a cost-efficient alternative.
Various yeasts have been reported to be capable of producing 2-PE. Saccharomyces
cerevisiae are able to convert the amino acid L-phenylalanine directly to 2-PE via the Ehrlich
pathway.
Production of 2-PE strain specific, thus 16 yeast strains of S. cerevisiae were screened
from different sources like as winemaking and beer making for the production of this aroma.
Used for screening was Etschmann medium containing glucose and L-phenylalanine
(Etschmann et all., 2002). Yeast was cultivated in Erlenmeyer flasks 48 hours at 28-300C, on
rotary shaker 240 r/m. 2-PE in the aqueous phase was analyzed after centrifugation (6000 g,
10 min.) by HPLC (RP-C18 column, methanol/water 60/40 v/v as eluant at 2 ml/min and
detection at 216 nm) according to control sample of 2-PE strong solution (Merck, Germany).
The effect of carbon sources (glucose, sucrose, maltose, trehalose, raffinose), cultivating
temperatures (12-140C, 20-220C, 28-300C, and 42-480C), different sucrose concentrations 214 %, on the biomass production and a level of synthesis 2-PE and ethanol were investigated.
Ethanol in the aqueous phase was analysed by GC and detection by FID. Sucrose was
determined by the dinitrosalicylic acid method. Cell dry weight was determined by drying
centrifuged and washed 10 ml samples at 1050C for 48 h and weighing. All data are
standardized and carried out in triple frequency.
It was determined, that in the beginning of stationary growth phase (18-24 h, strain
specific), presence 2-PE in the medium reaches the maximal values at primary screening. S.
cerevisiae UCM Y-514 and UCM Y-524 rely on sucrose, and to a lesser degree, glucose as a
carbon source. The amount of 2-PE peaked at 23-42 % in presence of sucrose. Growth of S.
cerevisiae UCM Y-514 and UCM Y-524 were completely inhibited at 1.5 g 2-PE/l,
respectively. These yeasts are Crabtree positive, meaning they can produce ethanol under
aerobic conditions. Ethanol in combination with 2-PE was more toxic for S. cerevisiae than
predicted by the summation of the individual effects. At different sucrose concentrations from
10 to 80 g/l were extended ethanol concentrations from 0.08 % to 2.16 % in cultivating
medium. S. cerevisiae UCM Y-514 and UCM Y-524 has resistance both to high
concentrations of 2-PE, and to high concentrations of ethanol. Thus the total effect of alcohols
on yeast cells not show up to a full degree, as during researches we looked after constantly the
high increase of biomass.
At maintenance of the above-stated cultivating conditions the concentration of 2-PE in the
medium increased for yeast S. cerevisiae UCM Y-514 21.5 % and for S. cerevisiae UCM Y524 24 %. The given results testify perspective biotechnological ability of the data strains to
substantial growth with synthesis of 2-PE.
The author gratefully acknowledge support from Acad. Podgorsky V.S. and Dr.
Nagornaya S.S. and Federation of European Microbiological Societies for a FEMS Meeting
Attendance Grant.
Poster abstract, Christian Mandt
Generation of new aminoglycosides using pathway-specific glycosyltransferases
Aminoglycoside antibiotics (AGAs) are potent broad host antibiotics which act as
translational inhibitors, which are produced by actinobacteria and Bacilli. They fall into
different families according to their chemical structures (1). The most important family
contains the aminocyclitol 2-deoxystreptamine (2-DOS) as a common precursor. During the
biosynthesis different sugars become attached to the aminocyclitol and after further
modifications the bioactive endproducts are formed. These can be pseudo-di-, tri- or tetrasaccharides (Fig. 3). The different sugars are transferred by various glycosyltransferases
(GTs), which differ in their substrate and acceptor specifities (Figs. 1, 2 and 3). However, it
has been shown that many GTs have broad substrate recognition abilities (3). Therefore the
GTs are a promising tool for the generation of activated sugars or new aminoglycoside
derivatives which are naturally not produced (cf. Figs 4 and 5). Recently, we have cloned and
sequenced 13 AGA biosynthetic gene clusters (1, 2). We have cloned and expressed the genes
encoding the various GTs involved in the biosynthesis of the aminoglycosides of the
Kanamycin-, Neomycin-, and Hygromycin-family which will be used for biocombinatoric
approaches for the development of new AGA-derivatives (Fig. 5).
References:
(1) Piepersberg, W., Aboshanab, K., Schmidt-Beißner, H. and U. F. Wehmeier (2007). THE
BIOCHEMISTRY AND GENETICS OF AMINOGLYCOSIDE PRODUCERS. In press for: D. P.
Arya, B. Wang (editors): Aminoglycoside Antiobiotics: From Chemical Biology to Drug Discovery.
Wiley and Sons, New Jersey, USA (ISBN-13: 978-0471743026 )
(2) Piepersberg, W., Aboshanab, K., Schmidt-Beissner, H., Wehmeier, U., Welzel, K., Vente, A.
(2005). Patent PCT WO2005/095591 A2
(3) Zhang C., Griffith B.R., Fu Q., Albermann C., Fu X., Lee I.K., Li L., Thorson J.S. Science. 2006
Sep 1;313(5791):1291-4
Modelling and mutagenesis of aminoglycoside resistance methyltransferase
Sgm
Gordana Maravic(1), Sonja Cubrilo(1), Karolina Tkaczuk(2,3) and Janusz M. Bujnicki (2,4)
(1) Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and
Biochemistry, University of Zagreb, Ante Kovacica 1, 10000 Zagreb, Croatia ; (2) Laboratory
of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell
Biology, Trojdena 4, 02-109 Warsaw, Poland ; (3) Institute of Technical Biochemistry,
Technical University of Lodz, B. Stefanowskiego 4/10, 90-924 Lodz, Poland ; (4) Institute of
Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, 61-614
Poznan, Poland
Methyltransferases that carry out posttranscriptional N7-methylation of G1405 in 16S
rRNA confer bacterial resistance to aminoglycoside antibiotics, including kanamycin and
gentamicin. Genes encoding enzyme from the Arm (aminoglycoside resistance
methyltransferases) family have been recently found to spread by horizontal gene transfer
between various human pathogens. The knowledge of the Arm protein structure would lay the
groundwork for the development of potential resistance inhibitors, which could be used to
restore the potential of aminoglycosides to act against the resistant pathogens. We analyzed
the sequence-function relationships of Sgm MTase, a member of the Arm family, by limited
proteolysis and site-directed and random mutagenesis. We have also modeled the structure of
Sgm using bioinformatics techniques and used the model to provide a structural context for
experimental results. We found that Sgm comprises two domains and we characterized a
number of functionally compromised point mutants with substitutions of invariant or
conserved residues. Our study provides a low-resolution (residue-level) model of sequencestructure-function relationships in the Arm family of enzymes and reveals the cofactorbinding and substrate-binding sites. These functional regions will be prime targets for further
experimental and theoretical studies aiming at defining the reaction mechanism of m7G1405
methylation, increasing the resolution of the model and developing Arm-specific inhibitors.
Protoplast formation and transformation to manipulate Nonomuraea ATCC 39727,
the producer of A40926 glycopeptide
Giorgia Letizia Marcone1, Flavia Marinelli1, Fabrizio Beltrametti2.
1
Department of Biotechnology and Molecular Sciences, University of Insubria, Via J. H.
Dunant 3, 21100 Varese Italy
2
Actygea S.r.l, Via R. Lepetit 34, 21040 Gerenzano (VA) Italy
Glycopeptides are antibiotic molecules produced by actinomycetes and active against grampositives. They inhibit the late stages of cell wall assembly by forming complexes with the Dalanyl-D-alanine (D-ala-D-ala) C termini of the peptidoglycan precursors. Glycopeptide
producing actinomycetes develop self-resistance mechanisms in order to avoid suicide during
antibiotic production. Nonomuraea sp. ATCC 39727 is a rare actinomycete that produces the
glycopeptide antibiotic A40926, precursor of the semi-synthetic derivative dalbavancin
currently under clinical development. Our aim is to analyze the genetic bases of Nonomuraea
resistance to glycopeptides. The ancillary resistance gene vanY has been recently identified in
the Nonomuraea gene cluster for A40926 production (dvb cluster), but its effective role in the
resistance phenotype should be verified by knocking it out. Unlikely, the complex life cycle
and the lack of genetic manipulation tools in this microorganism make this investigation quite
challenging. Hereby, we present a method to efficiently produce Nonomuraea protoplasts and
to transform them with the integrative plasmid pSET152. Protoplast yield is conditioned by
the age of the culture and by the medium components. Transformation efficiency appears to
be affected by the use of different agents such as PEG1000 or lipofectamine. In the optimized
conditions, 1x108 protoplasts/ml are achieved with a transformation frequency of 3x103 per
μg of DNA (with lipofectamine), while with PEG 1000 the transformation frequency per μg
of DNA is only of 1x102. This protocol is now being experimented to inactivate vanY gene,
but it will be of a more general usefulness for the A40926 cluster manipulation.
Investigations on the pristinamycin biosynthesis in Streptomyces pristinaespiralis
Yvonne Mast, Eva Schinko & Wolfgang Wohlleben.
University of Tübingen, Institute of Microbiology, Dpt. Microbiology / Biotechnology,
Tübingen, Germany
Streptomyces pristinaespiralis produces the streptogramin antibiotic pristinamycin, which is a
mixture of two types of chemically unrelated compounds: pristinamycin PI and PII.
Pristinamycin PI is a cyclic hexadepsipeptide, while pristinamycin PII has the structure of a
polyunsaturated macrolactone. Both components inhibit the elongation of the protein
biosynthesis by affecting the peptidyltransferase in the 50S subunit of the ribosome. Each
compound alone exhibits only a weak bacteriostatic activity whereas the combination of both
substances leads to a synergistic effect that results in the bactericidal activity of pristinamycin.
The genes for pristinamycin biosynthesis, regulation and resistance are organized as a single
large gene region, in which the genes encoding PI and PII are spread over at least 260 kb. So
far the function of most of the pristinamycin biosynthetic genes has been revealed, except the
genes that are responsible for the biosynthesis of the aproteinogenic amino acid phenylglycine
which is part of PI. Recent sequence analysis led to the identification of putative
phenylglycine biosynthetic genes. To analyse their function plasmids for deleting and
overexpressing the genes were constructed. Furthermore six regulatory genes were identified
within the 260 kb region: spbR, papR1, papR2, papR3, papR4 and papR5. SpbR (S.
pristinaespiralis butyrolactone-responsive transcriptional repressor) is a specific receptor
protein for γ-butyrolactones and the global regulator of the papR (regulator of pristinamycin
antibiotic production) genes. PapR1, papR2 and papR4 encode for proteins that are
homologous to SARPs (Streptomyces antibiotic regulatory protein), which are pathwayspecific transcriptional activator proteins, whereas papR3 and papR5 encode both for proteins
that belong to the family of TetR repressors. To investigate the function of the regulatory
genes, deletion and overexpression strains were constructed. Altogether the analysis of the
mutants, RT-PCR- and bandshift-experiments should lead to a better understanding of the
complex mechanisms of pristinamycin biosynthesis and regulation.
SECONDARY METABOLISM AND ITS REGULATION IN STREPTOMYCES
AMBOFACIENS
Šárka Nezbedová
Institute of Microbiology CAS, Prague; Institut de Génétique et Microbiologie,
Univ. Paris-Sud 11, Orsay;
S. ambofaciens has been known to produce the macrolide spiramycin (polyketide), used in
medicine, and the pyrrolamide congocidine. Recently, after the analysis of the sequence of the
terminal parts of S. ambofaciens chromosome, several other PKS and/or NRPS clusters were
found indicating that some other secondary metabolites might be produced by this strain.
This hypothesis was confirmed recently when one of the S. ambofaciens cryptic PKS clusters
was shown to be responsible for the production of an antibacterial compound, named
alpomycin, and an orange pigment (Pang et al. 2004; Antimicrob Agents Chemother. 48:575588, Aigle et al. 2005 J Bacteriol. 187:2491-2500).
In order to study the products of the other putative secondary metabolite biosynthetic clusters
(the PKS and/or NRPS clusters), as well as to examine the possible cross-regulation between
clusters, we undertook the construction of S. ambofaciens mutants no longer producing the
antibiotics alpomycin, congocidine and spiramycin. Genes from each of the clusters were
inactivated by antibiotic excisable cassette which was afterwards excised in order to obtain a
strain without antibiotic resistance marker.
During the search for the products of cryptic clusters we also studied one of the putative
global regulators - the rep-like gene. The rep gene, previously isolated from gene
environmental library, caused enhanced antibiotic production in S. lividans. We have
sequenced the S. ambofaciens rep homolog. Inactivation of the S. ambofaciens rep-like gene
resulted in ceasing of spiramycin and congocidine biosynthesis. In future, a mutant overexpressing the rep-like gene will be prepared and the levels of produced antibiotics will be
measured.
Investigating the biosynthetic pathway of anthrax stealth siderophore
D. Oves-Costales, N. Kadi, M. J. Fogg, L. Song, K. S. Wilson and G. L. Challis
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK and Structural
Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10
5YW, UK
Petrobactin is an iron-chelating siderophore originally isolated from Marinobacter
hydrocarbonoclasticus that has been shown to play an important role in growth under irondeficient conditions and virulence of the deadly bioterrorism agent Bacillus anthracis. It has
recently been shown not to bind to siderocalin, leading it to be designated as a "stealth
siderophore" that can avoid the mammalian immune system. A unique combination of
nonribosomal peptide synthetase (NRPS) and NRPS-independent siderophore (NIS)
synthetase enzymes is known to be required for petrobactin biosynthesis in B. anthracis. Here
it is shown that AsbA from B. anthracis, the first type A NIS synthetase to be biochemically
characterized, catalyzes ATP-dependent regioselective condensation of citric acid with N8 of
spermidine, but not with N1-(3,4-dihydroxybenzoyl)-spermidine. These results rule out a
recently proposed pathway for petrobactin biosynthesis involving AsbA-catalyzed
condensation of N1-(3,4-dihydroxybenzoyl)-spermidine with citric acid and show that
acylation of N1 of spermidine with the 3,4-dihydroxybenzoyl group must occur after acylation
of N8 of spermidine with citrate. They also provide the fundamental knowledge needed to
establish a high throughput screen for inhibitors of AsbA that may provide the basis for
development of new antibiotics for the treatment of deadly anthrax infections.
Elizabeth A. Shank
Kolter Lab
Harvard Medical School
bethshank@gmail.com OR elizabeth_shank@hms.harvard.edu
Using Soil Organisms to Induce Secondary Metabolite Production
In the last number of years, whole genome sequences have been determined for hundreds of
bacterial species. These sequencing efforts have revealed that many bacteria contain orphan
biosynthetic gene clusters not known to produce a metabolite, or whose metabolite has not yet
been identified. In many cases, these biosynthetic gene clusters constitute a significant
portion of the genome, suggesting that they are retained because their products play an
important role in the fitness or survival of the bacteria under natural environmental
conditions.
The sequences of many of these biosynthetic gene clusters suggest that they encode proteins
with novel functions, and therefore may produce new natural products1. Determining
methods to induce the production of these metabolites and subsequently identify them will
expand the repertoire of known natural products, some of which may have therapeutic
function. In addition, recent work has indicated that some of these predicted secondary
metabolites may function as chemical signals in cell-to-cell interactions2. Therefore, by
studying these bacterially produced small molecules, we may also learn about how chemical
communication functions in bacterial interactions.
Previous experiments have established that some orphan biosynthetic gene clusters are
activated when the bacteria is exposed to other organisms or their metabolic products. For
instance, experiments performed by Ueda et al.3 examined the interactions between
Streptomyces species. Secreted molecules produced by one strain induced another strain to
generate a new antibiotic activity (one not observed when that strain was grown in isolation).
In addition, preliminary experiments in the Kolter and Walsh labs have demonstrated that
applying sub-inhibitory concentrations of an antibiotic to Streptomyces avermitilis activates a
previously silent biosynthetic gene cluster and a new small molecule is produced. These
examples suggest the plausibility of using co-culture to induce otherwise silent biosynthetic
gene clusters into producing their metabolite.
I am performing two screens based on this concept. In the first I am co-culturing putative
producers (sequenced bacteria containing orphan biosynthetic gene clusters) with soil bacteria
and searching for new antibiotic activity. In the second I am using a similar co-culture to
induce the expression of a fluorescent reporter construct in the producer strain that is driven
by the promoter of an orphan biosynthetic gene cluster. My aim is to not only identify novel
small molecules, but also potentially reveal the role that these compounds may play in
bacterial interspecies communication in more natural contexts.
1.
2.
3.
Van Lanen SG, Shen B (2006). Current Opinion in Microbiology 9:252.
Davies J (2006). Journal of Industrial Microbiology and Biotechnology 33:496.
Ueda K, Kawai S, Ogawa H, Kiyama A, Kubota T, Kawanobe H, Beppu T (2000). Journal of
Antibiotics 53:979.
High throughput cultivation Platform for Streptomyces coeliolor
Sujata V. Sohoni, Prashant M. Bapat, Jens Nielsen, Anna Eliasson Lantz
Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark,
2800 Lyngby, Denmark
Streptomycetes are most intensely studied organisms due to their potential to
produce secondary metabolites which are used in therapeutics. Due to ever increasing demand
for new antibiotics, screening applications such as media optimizations, search for new
bioactive molecules, and lastly characterization of mutagenized strains requires examination
of large number of cultures.
Conventional approaches use individual handling of strains; hence they are
laborious and time consuming. We have established a High throughput Microbioreactor
platform for cultivation of Streptomyces coelicolor. We cultivated Streptomyces coelicolor in
24-square-deep well Microtiter plates with 3 ml culture volume. The growth evaluated with
respect to biomass and secondary metabolite formation was very similar to bioreactors and
shake flask cultivations. We found better shaking in square plates, and we did not observe any
clumping of mycelia. This diminishes need for glass beads to the Microtiter plates.
Process optimization was done at three stages,
 Optimization of Buffer: MOPS buffer was used at 100 mM concentration
 Inoculum percentage: 0.65% inoculum was used in the form of Frozen mycelia.
 Working volume: 3 ml volume was used for cultivation
Since the working volume used is 3 ml one study Fermentation physiology with
respect to Biomass, Carbon and Nitrogen substrate consumption, and Antibiotic concentration
and so on.
We use frozen mycelia as an inoculum for fermentor, instead of Spore
suspension. It decreases one step in fermentation, where spores are inoculated in seed medium
for germination, before inoculating in fermentor.
For further information on Microtiter cultivation and frozen mycelia preparation, you can email me at, sohoni_sujata@yahoo.com or svs@Biocentrum.dtu.dk
Studies on Proteases and the Protease Inhibitor Regulating Differentiation
of Streptomyces coelicolor
Kil Suhmoon1, Dae Wi Kim1 and Kye Joon Lee1
1
School of biological science, Seoul National University, Seoul 151-742, Korea
The aim of this study was to identify protease and protease inhibitor involved in differentiation of
Streptomyces coelicolor. It was studied that production of Streptomyces trypsin inhbitor (STI) is regulated by the
bldA-AdpA dependant mode in proteomics approach of S. coeilcolor. Role of STI was assumed as an inhibitor
of specific proteases during the early growth phase to regulate proper differentiation. In this study, extracellular
serine protease (ScoP1) was identified in relation with STI. From disruption and kinetics studies, it was
considered that the ScoP1 might be responsible to the inactivation of STI. Furthermore, another serine protease
(ScoP2) interacting with STI was identified by yeast two hybrid system. The site of protein-protein interaction
was analyzed.
STI might be inactivated by ScoP1 for the initiation of morphological differentiation after growth phase, and
then ScoP2 could be released from STI inhibition, for translational modification of essential proteins involved in
differentiation of S. coelicolor. It is proposed that proteases and protease inhibitor cascade (ScoP1-STI-ScoP2)
lead to morphological differentiation and antibiotics production of S. coelicolor in programmed manner.
Searching for subunits of lincosamide synthetases – key enzyme complexes in
biosynthesis of lincomycin and celesticetin
Dana Ulanova, Marketa Koberska, Jan Kopecky, Marketa Jelinkova, Jana Olsovska,
Stanislav Kadlcik, Jiri Janata
Institute of Microbiology, Academy of Science of the Czech Republic, Videnska 1083, 14220
Prague 4, Czech Republic, e-mail: ulanova@biomed.cas.cz
Lincosamides and their derivatives are antibiotics with significant antibacterial and
antimalarial effects. Biosynthetic pathways of both naturally occurred lincosamides,
lincomycin and celesticetin, are not completely known. These pathways have one branch in
common, which indicates the presence of homologue genes in their clusters. At first, two
precursors of lincomycin are synthesized separately, the aglycone propyl-L-proline (PPL)
from tyrosine and the methylthiolincosamide (MTL) from D-glucose. These two parts are
condensed to N-demethyllincomycin, which is subsequently methylated to lincomycin. The
antibiotic celesticetin is composed of three precursors: proline, salicylate and
methylthiolincosamide derivative.
Our research is focused on elucidation of function of N-demethyllincomycin synthetase
subunits, which condense two lincomycin precursors, and their analogues in the celesticetin
biosynthetic pathway. Amino acid-activating function of LmbC and CcbC subunits was
proved recently. Also both clusters contain DNA sequence coding for a putative acyl carrier
protein (ACP) important for condensation reaction, but as an integral part of a gene unrelated
to the synthetase activity. Interestingly, the putative ACP coding sequence is located in related
clusters as a part of the lmbN gene (in the lincomycin cluster), while in celesticetin cluster as a
part of the ccbZ gene, which neighbours the ccbN gene – the homolog of lmbN.
In order to describe and explain the function of ACP in lincosamide biosynthesis, disruption
of the lmbN gene and its part coding for the putative ACP or a part coding for the MTL
biosynthetic protein with the PCR targeting system (Gust B. et al 2003) was done. Production
of lincomycin in disruptant Streptomyces lincolnensis strains was measured by bioassay and
ultra performance liquid chromatography (UPLC).
Disruption of the whole lmbN gene, as well as its both parts separately, resulted in
abolishment of lincomycin production. Production of lincomycin by disruptant of
S.lincolnensis with remaining part of the lmbN gene coding for putative ACP was restored
after adding MTL into a fermentation broth.
References:
Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater,K.F. (2003) Gene replacement by
PCR targeting in Streptomyces and its use to identify a protein domain involved in the
biosynthesis of the sesquiterpene odour geosmin.Proc Natl Acad Sci USA 100: 1541–1546.
Molecular Characterization of the DNA-binding activity of the FtsK-like TraB
protein essential for Conjugal Plasmid Transfer in Streptomyces
Jutta Vogelmann, Cordula Gekeler, Wolfgang Wohlleben and Günther Muth
Eberhard-Karls-Universität Tübingen, Fakultät für Biologie, Mikrobiologisches Institut,
Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany
A single plasmid encoded protein TraB is sufficient to promote conjugal plasmid
transfer in Streptomyces. TraB is a multimeric, membrane associated protein that
belongs to the FtsK/SpoIIIE family of septal DNA translocator proteins involved in
segregation of double stranded chromosomal DNA during cell division (FtsK) and
sporulation (SpoIIIE). Whereas SpoIIIE and FtsK seem to interact with chromosomal
DNA quite non-specifically, TraB recognizes a specific non-coding plasmid sequence
(clt) of about 50 bp in length containing several direct repeats and one indirect
repetitive sequence (1). By heterologous gene expression, protein purification and
gel retardation experiments the recognition sequences of TraBpSVH1 and TraBpSG5
were identified.
To characterize the TraB-region involved in the specific interaction with the clt-locus
several chimeric traB genes containing different parts of traBpSVHI, traBpSG5 and
traBpIJ101 were constructed and expressed in E. coli and S. lividans. The ability of
the purified proteins to interact with the clt-loci of the respective plasmids was
analysed in gel retardation. A 772 aa chimeric protein containing the N-terminal half
(1-419 aa) of TraBpSVH1 and the C-terminus (385-738 aa) of TraBpSG5 interacted
specifically with clt of plasmid pSG5. Also, fusion of the last 353 aa of TraBpIJ101
that contained the Walker A and B motifs to the N-terminal half of TraBpSVH1
generated a protein that was no longer capable of recognizing clt of plasmid pSVH1
but bound the clt of plasmid pIJ101. These experiments indicated that the region
around the ATPase domain of TraB is involved in clt recognition.
1. Reuther et al., 2006, Mol Microbiol. 61:436-446
DNA S-modification in S. lividans 66: functional analysis of the determinants for
modification specificity
Zhijun Wang, Jingdan Liang, Xiufen Zhou, Zixin Deng
Shanghai Jiaotong University, China
email: zhijunw@gmail.com jdliang@sjtu.edu. zxdeng@sjtu.edu.cn
The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in
Streptomyces lividans 1326 [1], was strongly aggravated when one (dndB) of the five genes
(dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid
(pHZ209), reflecting DNA degradation, displayed a clear change from a preferential
modification site in strain 1326 to more random modifications in the mutant. Fourteen
randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be
modified only once. Residues in a region (50-c–cGGCCgccg-30) including a highly
conserved 4-bp central core (50-GGCC-30) in a well-documented preferential modification
site were assessed for their necessity by site-directed mutagenesis. While the central core
(GGCC) was found to be stringently required in 1326 and in the mutant, ‘gccg’ flanking its
right could either abolish or reduce the modification frequency only in the mutant, and two
separate nucleotides to the left had no dramatic effect [2].
The lack of essentiality of DndB for S-modification and the result of site-directed
mutagenesis analysis suggests a model for the function of DndB in the modification process:
that by binding to the three direct repeats, it might only be required for enhancing or
stabilizing the activity of a protein complex at the required preferential modification site, or
resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle
for efficient modification.
REFERENCES
1.
2.
Zhou X.F., He X.Y., Liang J.D., Li A.Y., Xu T.G., Kieser T., Helmann J.D. and Deng,
Z.X. (2005) A novel DNA modification by sulphur. Molecular Microbiology, 57,
1428-1438.
Liang J.D. , Wang Z.J., He X.Y., Li J.L., Zhou X.F., and Deng Z.X. (2007) DNA
modification by sulphur: analysis of the sequence recognition specificity surrounding
the modification sites. Nucleic Acids Research, 35(9): 2944-54.
Secondary metabolite engineering in Fusarium moniliforme
Abstract for poster: Katherine Williams
Fusarium moniliforme is a filamentous fungus that is a pathogen of corn, causing
kernel, stalk and ear rot. F. moniliforme produces secondary metabolite mycotoxins, such as
Fusarin C, which consists of a polyketide chain linked to an amino acid. The gene fusA is
involved in the synthesis of Fusarin C and is an unusual PKS linked to an NRPS. A gene
cluster has been identified which includes fusA, and is thought to encode all the enzymes that
synthesise Fusarin C. Gene inactivation and complementation experiments are being
performed to deduce the function of each gene in the cluster. Module swaps are also being
carried out on fusA, substituting the NRPS module which is hypothesised to add a homoserine
to the polyketide chain, for NRPS modules which have a different or unknown hypothesised
substrate. These constructs will then be expressed in the heterologous host, Aspergillus
oryzae, and concurrently in the original organism, F. moniliforme. Subsequently chemical
analysis will be performed to deduce if and what novel compounds have been formed. This
research may lead to the ability to precisely synthesise specific pharmacologically active
compounds.
Alice Morningstar Yaxley, Stephen D. Bentley, Gregory L. Challis and Rosemary Loria.
The single linear chromosome of the potato pathogen Streptomyces scabies 87.22 is the
largest streptomycete genome so far determined (10.1Mbp) and contains short (18.4Kbp)
terminal inverted repeats. Known pathogenicity features from a pathogenicity island (PAI) in
a related strain (Kers et al. 2005 Mol. Microbiol.) are found in at least two loci.
Recombination between copies of an insertion sequence is likely to have caused this
fragmentation of the PAI. Comparisons with S. coelicolor A3(2) and S. avermitilis MA-4680
indicate a high level of rearrangement including at least two inversions of the central region of
the chromosome since divergence of the lineages. Under half of the predicted coding
sequences in S. scabies (n=4180, from total 8806) are common to all three completely
sequenced strains at a suitable level to propose orthology using the Orthomcl clustering
algorithm (Li et al. 2003 Genome Res.). There are many complex natural products in the S.
scabies genome, including phytotoxins, siderophores, and pigments. Production gene clusters
for NIS desferrioxamines, spore pigment, hopanoids, geosmin, and carotenoid pigment, are
found in all three genomes. One unexpected product from S. scabies is the nonribosomal
peptide siderophore pyochelin (Cox et al. 1981 PNAS), previously found only in gram
negative organisms.