20 research outputs found
A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.
Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.This work was supported by a Commonwealth Scholarship from the Commonwealth Scholarships Commission (UK) and Sir Henry Wellcome Postdoctoral fellowship to FLS, and the Biotechnology and Biological Sciences Research Council (UK).
.This is the final version of the article. It first appeared from Taylor & Francis via http://dx.doi.org/10.1080/15476286.2015.107343
Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria.
Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, Serratia sp. ATCC 39006 (S39006). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006, identifying how this differs from the archaeon Halobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in Escherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E. coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain.The authors would like to thank Professor Tony Walsby (Emeritus, Bristol university) for advice, technical help and donation of the pressure nephelometry and volumetric calculation apparatus. We would also like to thank Alison Drew for technical support and Chin Mei Lee and Andrew Day for critical reading. REM and GPCS were supported through the BBSRC (Grant ID BB/K001833/1). YT was supported by a Scientific Research Fellowship from the Japan Society for the Promotion of Sciences (JSPS) and JPR was supported by a Herschel Smith Post Doctoral Fellowship while at Cambridge University.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1111/1462-2920.1320
Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.
Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.GPCS and REM were supported by BBSRC grant (Grant ID BB/K001833/1). YT was supported by a KAKENHI (15H01315) from the Japan Society for the Promotion of Sciences (JSPS). The authors declare that they have no conflicts of interest.This is the author accepted manuscript. The final version is available from the Microbiology Society at http://dx.doi.org/10.1099/mic.0.000347
Recommended from our members
Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.
Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Cambridge Commonwealth TrustA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate
The rsmS (ybaM) mutation causes bypass suppression of the RsmAB post-transcriptional virulence regulation system in enterobacterial phytopathogens.
Plant cell wall degrading enzymes (PCWDEs) are the primary virulence determinants of soft rotting bacteria such as the potato pathogen, Pectobacterium atrosepticum. The regulation of secondary metabolite (Rsm) system controls production of PCWDEs in response to changing nutrient conditions. This work identified a new suppressor of an rsmB mutation - ECA1172 or rsmS (rsmB suppressor). Mutants defective in rsmB (encoding a small regulatory RNA), show reduced elaboration of the quorum sensing molecule (N-3-oxohexanoyl-homoserine lactone; OHHL) and PCWDEs. However, OHHL and PCWDE production were partially restored in an rsmB, rsmS double mutant. Single rsmS mutants, overproduced PCWDEs and OHHL relative to wild type P. atrosepticum and exhibited hypervirulence in potato. RsmS overproduction also resulted in increased PCWDEs and OHHL. Homology searches revealed rsmS conservation across pathogens such as Escherichia coli (ybaM), Dickeya solani, Klebsiella pneumoniae and Shigella flexneri. An rsmS mutant of Pectobacterium carotovorum ATCC39048 showed bypass of rsmB-dependent repression of PCWDEs and OHHL production. P. carotovorum ATCC39048 produces the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a carbapenem). Production of the antibiotic was repressed in an rsmB mutant but partially restored in an rsmB, rsmS double mutant. This work highlights the importance of RsmS, as a conserved pleiotropic regulator of virulence and antibiotic biosynthesis.James Hutton Institut
The LacI-Family Transcription Factor, RbsR, is a Pleiotropic Regulator of Motility, Virulence, Siderophore and Antibiotic Production, Gas Vesicle Morphogenesis and Flotation in Serratia
Gas vesicles (GVs) are proteinaceous, gas-filled organelles used by some bacteria to enable upward movement into favorable air/liquid interfaces in aquatic environments.
Serratia sp. ATCC39006 (S39006) was the first enterobacterium discovered to produce GVs naturally. The regulation of GV assembly in this host is complex and part of a wider regulatory network affecting various phenotypes, including antibiotic biosynthesis. To identify new regulators of GVs, a comprehensive mutant library containing 71,000 insertion mutants was generated by random transposon mutagenesis and 311 putative GV-defectivemutants identified. Three of thesemutants were found to have a transposon inserted in a LacI family transcription regulator gene (rbsR) of the putative ribose operon. Each of these rbsR mutants was GV-defective; no GVs were visible by phase contrast microscopy (PCM) or transmission electron microscopy (TEM). GV deficiency was caused by the reduction of gvpA1 and gvrA transcription (the first genes of the two contiguous operons in the GV gene locus). Our results also showed that a mutation in rbsR was highly pleiotropic; the production of two secondary metabolites (carbapenem and prodigiosin antibiotics) was abolished. Interestingly, the intrinsic resistance to the carbapenem antibiotic was not affected by the rbsR mutation. In addition, the production of a siderophore, cellulase and plant virulence was reduced in the mutant, whereas it exhibited increased swimming and swarming motility. The RbsR protein was predicted to bind to regions upstreamof at least 18 genes in S39006 including rbsD (the first gene of the ribose operon) and gvrA. Electrophoretic mobility shift assays (EMSA) confirmed that RbsR bound to DNA sequences upstream of rbsD, but not gvrA. The results of this study indicate that RbsR is a global regulator that affects the modulation of GV biogenesis, but also with complex pleiotropic physiological impacts in S39006
Recommended from our members
Draft Genome Sequence of Pectobacterium carotovorum subsp. carotovorum ATCC 39048, a Carbapenem-Producing Phytopathogen.
Pectobacterium carotovorum subsp. carotovorum ATCC 39048 was originally isolated in the 1980s and studied because it produced the β-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid. The draft genome for this strain was 4,637,928 bp with a G+C content of 51.98%. The genome contained the carbapenem biosynthetic cluster, genes encoding plant virulence determinants, and a single metallo-β-lactamase.BBSR
Recommended from our members
Environmental potassium regulates bacterial flotation, antibiotic production and turgor pressure in Serratia through the TrkH transporter.
Serratia sp. strain ATCC 39006 (S39006) can float in aqueous environments due to natural production of gas vesicles (GVs). Expression of genes for GV morphogenesis is stimulated in low oxygen conditions, thereby enabling migration to the air-liquid interface. Quorum sensing (via SmaI and SmaR) and transcriptional and post-transcriptional regulators, including RbsR and RsmA, respectively, connect the control of cell buoyancy, motility and secondary metabolism. Here, we define a new pleiotropic regulator found in screens of GV mutants. A mutation in the gene trkH, encoding a potassium transporter, caused upregulation of GV formation, flotation, and the prodigiosin antibiotic, and downregulation of flagellar motility. Pressure nephelometry revealed that the mutation in trkH affected cell turgor pressure. Our results show that osmotic change is an important physiological parameter modulating cell buoyancy and antimicrobial production in S39006, in response to environmental potassium levels.Cambridge Trust International scholarshi
Recommended from our members
Biosynthesis of Antifungal Solanimycin May Involve an Iterative Nonribosomal Peptide Synthetase Module.
Funder: FP7 People: Marie-Curie Actions; doi: https://doi.org/10.13039/100011264; Grant(s): 298003Dickeya solani, a plant-pathogenic bacterium, produces solanimycin, a potent hybrid polyketide/nonribosomal peptide (PKS/NRPS) anti-fungal compound. The biosynthetic gene cluster responsible for synthesis of this compound has been identified. Because of instability, the complete structure of the compound has not yet been elucidated, but LC-MS2 identified that the cluster produces two main compounds, solanimycin A and B, differing by a single hydroxyl group. The fragmentation pattern revealed that the central part of solanimycin A is a hexapeptide, Gly-Dha-Dha-Dha-Dha-Dha (where Dha is dehydroalanine). This is supported by isotopic labeling studies using labeled serine and glycine. The N-terminal group is a polyketide-derived C16 acyl group containing a conjugated hexaene, a hydroxyl, and an amino group. The additional hydroxyl group in solanimycin B is on the α-carbon of the glycine residue. The incorporation of five sequential Dha residues is unprecedented because there is only one NRPS module in the cluster that is predicted to activate and attach serine (which is subsequently dehydrated to Dha), meaning that this NRPS module must act iteratively. While a few other iterative NRPS modules are known, they all involve iteration of two or three modules. We believe that the repetitive use of a single module makes the solanimycin biosynthetic pathway unique among NRPSs so far reported
Biosynthesis of antifungal solanimycin may involve an iterative nonribosomal peptide synthetase (NRPS) module
The work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC;UK) through award BB/N008081/1.Work with plant pathogens was carried out under DEFRA license no. 50864/197900/1.M.A.M.was supported by an EU Marie-Curie Intra-European Fellowshipfor Career Development (FP7-PEOPLE-2011-IEF) grant number 298003. Workin the Matilla laboratory was supported by grant from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00)