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

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

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

Substrate flexibility of the flavin-dependent dihydropyrrole oxidases PigB and HapB involved in antibiotic prodigiosin biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5a (resulting from transamination of the aldehyde of 3-acetyloctanal) and 6 (resulting from transamination of the ketone), were synthesised. Only 5a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colorimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5a were synthesised, varying in the C2 and C3 side-chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side-chain tested at C3 were accepted but only short side-chains at C2 were accepted. The knowledge that 5a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5a makes a range of prodigiosin analogues readily available by mutasynthesis.We acknowledge the Frances and Augustus Newman foundation, the Cambridge Commonwealth Trust, Emmanuel College, Cambridge, and the B.B.S.R.C. (award codesBB/N008081/1 and BB/K001833/1) for funding this research

A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.The authors would like to acknowledge several funding sources. D. Smith was supported by a PhD studentship from the BBSRC. Work in the MW lab is supported by the BBSRC (grants BB/G015171/1 and BB/M019411/1). K. Roberts was funded by an MRC studentship. R. Monson and the Salmond lab were supported by grants from the BBSRC (Grant No Provisional BB/K001833/1). M.A. Matilla was supported by the EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF), grant number 298003. B. Richardson was supported by a Harry Smith vacation studentship from the SGM, UK. The authors would also like to thank Ray Chai for careful reading and comments on this manuscript. Alison Drew provided technical support. Work with plant pathogens was carried out under DEFRA licence No. 50864/197900/1.This is the final version of the article. It was first available from Frontiers via http://dx.doi.org/10.3389/fmicb.2015.0144

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

Citrobacter rodentium is an unstable pathogen showing evidence of significant genomic flux.

Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen. In this study, through PFGE, sequencing of isolates showing variation, whole genome transcriptome analysis and examination of the mobile genetic elements, we found that, consistent with our previous hypothesis, the genome of C. rodentium is unstable as a result of repeat-mediated, large-scale genome recombination and because of active transposition of mobile genetic elements such as the prophages. We sequenced an additional C. rodentium strain, EX-33, to reveal that the reference strain ICC168 is representative of the species and that most of the inactivating mutations were common to both isolates and likely to have occurred early on in the evolution of this pathogen. We draw parallels with the evolution of other bacterial pathogens and conclude that C. rodentium is a recently evolved pathogen that may have emerged alongside the development of inbred mice as a model for human disease

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