Bacteriophage ϕMAM1, a viunalikevirus, is a broad-host-range, high-efficiency generalized transducer that infects environmental and clinical isolates of the enterobacterial genera Serratia and Kluyvera.

Members of the enterobacterial genus Serratia are ecologically widespread, and some strains are opportunistic human pathogens. Bacteriophage ϕMAM1 was isolated on Serratia plymuthica A153, a biocontrol rhizosphere strain that produces the potently bioactive antifungal and anticancer haterumalide oocydin A. The ϕMAM1 phage is a generalized transducing phage that infects multiple environmental and clinical isolates of Serratia spp. and a rhizosphere strain of Kluyvera cryocrescens. Electron microscopy allowed classification of ϕMAM1 in the family Myoviridae. Bacteriophage ϕMAM1 is virulent, uses capsular polysaccharides as a receptor, and can transduce chromosomal markers at frequencies of up to 7 × 10(-6) transductants per PFU. We also demonstrated transduction of the complete 77-kb oocydin A gene cluster and heterogeneric transduction of a plasmid carrying a type III toxin-antitoxin system. These results support the notion of the potential ecological importance of transducing phages in the acquisition of genes by horizontal gene transfer. Phylogenetic analyses grouped ϕMAM1 within the ViI-like bacteriophages, and genomic analyses revealed that the major differences between ϕMAM1 and other ViI-like phages arise in a region encoding the host recognition determinants. Our results predict that the wider genus of ViI-like phages could be efficient transducing phages, and this possibility has obvious implications for the ecology of horizontal gene transfer, bacterial functional genomics, and synthetic biology.This research was supported by the EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF) grant number 298003. The Salmond lab is supported by funding through the Biotechnology and Biological Sciences Research Council (BBSRC, UK). We thank Hazel Aucken and Kornelia Smalla for kindly supplying the environmental and clinical isolates. We would also like to thank Jeremy N. Skepper (Department of Anatomy, University of Cambridge) for assistance in transmission electron microscopy and Alison Rawlinson for technical support.This is the accepted manuscript. The final version is available from the American Society for Microbiology at http://aem.asm.org/content/80/20/6446.lon

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

The bacterial Type III toxin-antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity.

Bacteria have evolved numerous defense systems to protect themselves from viral (bacteriophage) infection. The ToxIN system of Pectobacterium atrosepticum is a Type III toxin-antitoxin complex and "altruistic suicide" anti-phage system, which kills phage-infected cells through the release of a ribonuclease toxin, ToxN. ToxN is counteracted by a co-transcribed antitoxic RNA pseudoknot, ToxI, which self-assembles with ToxN into an inactive 3 ToxI:3 ToxN complex in vitro. However it is not known whether this complex is predominant in vivo, or how the complex is disassembled following infection to trigger a lethal, "altruistic" response. In this study, we characterise ToxI turnover and folding, and explore the link between complex stability and anti-phage activity, with a view to understanding events that lead to ToxN-mediated suicide following phage infection. We present evidence that ToxN constantly cleaves fresh ToxI in vivo rather than staying associated with pre-processed antitoxin, and that the ToxI antitoxin can partially fold spontaneously using conserved nucleotides. We also show that reducing the stability of the ToxIN complex can increase the strength of the antiviral response in a phage-dependent manner. Based on this information, we propose a revised model for ToxN inhibition, complex assembly and activation by infecting bacteriophage

Structure, Evolution, and Functions of Bacterial Type III Toxin-Antitoxin Systems.

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed.Work in the Salmond lab is supported by the BBSRC, UK; N.G. was supported by the Fonds National de la Recherche Luxembourg (9118191); B.C. was supported by a Cambridge International Scholarship from the Cambridge Commonwealth, European & International Trust; and A.D. was supported by a BBSRC -DTP studentship.This is the final version of the article. It first appeared from Molecular Diversity Preservation International via https://doi.org/10.3390/toxins810028

Genome Sequence of Serratia plymuthica A153, a Model Rhizobacterium for the Investigation of the Synthesis and Regulation of Haterumalides, Zeamine, and Andrimid.

The rhizobacterium Serratia plymuthica A153 is a Gram-negative bacterium belonging to the family Enterobacteriaceae Here, we present the genome sequence of this strain, which produces multiple bioactive secondary metabolites, including the halogenated macrolide oocydin A, the polyamino antibiotic zeamine, and the bacterial acetyl-CoA carboxylase inhibitor andrimid.Work in the Salmond laboratory was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, United Kingdom). Miguel A. Matilla was supported by the EU Marie-Curie intra-European Fellowship For Career Development (FP7-PEOPLE-2011-IEF) grant 298003 and the Spanish Ministry of Economy and Competitiveness Postdoctoral Research Program, Juan de la Cierva (JCI-2012-11815). The Tino Krell laboratory is supported by FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grants BIO2013- 42297 and RTC-2014-1777-3).This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/genomeA.00373-1

Biosynthesis of the acetyl-CoA carboxylase-inhibiting antibiotic, andrimid in Serratia is regulated by Hfq and the LysR-type transcriptional regulator, AdmX.

Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia plymuthica A153 produces several bioactive secondary metabolites, including the anti-oomycete and antifungal haterumalide, oocydin A and the broad spectrum polyamine antibiotic, zeamine. In this study, we show that A153 produces a second broad spectrum antibiotic, andrimid. Using genome sequencing, comparative genomics and mutagenesis, we defined new genes involved in andrimid (adm) biosynthesis. Both the expression of the adm gene cluster and regulation of andrimid synthesis were investigated. The biosynthetic cluster is operonic and its expression is modulated by various environmental cues, including temperature and carbon source. Analysis of the genome context of the adm operon revealed a gene encoding a predicted LysR-type regulator, AdmX, apparently unique to Serratia strains. Mutagenesis and gene expression assays demonstrated that AdmX is a transcriptional activator of the adm gene cluster. At the post-transcriptional level, the expression of the adm cluster is positively regulated by the RNA chaperone, Hfq, in an RpoS-independent manner. Our results highlight the complexity of andrimid biosynthesis - an antibiotic with potential clinical and agricultural utility.We thank Kornelia Smalla and Ian Toth for the generous donation of bacterial strains. Work in the Salmond laboratory is supported by funding through the Biotechnology and Biological Sciences Research Council (UK). M.A.M. was supported by the EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF) Grant No. 298003 and the Spanish Ministry of Economy and Competitiveness Postdoctoral Research Program, Juan de la Cierva (BVA-2009-0200). The Krell laboratory is supported by FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy 1and Competitiveness (grants BIO2013-42297 and RTC-2014-1777-3)

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

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