Lon protease- and temperature-dependent activity of a lysis cassette located in the insecticidal island of Yersinia enterocolitica

The Yersinia genus comprises pathogens that are able to adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19 kb pathogenicity island Tc-PAIYe. All tc genes follow a strict temperature regulation in that they are silenced at 37°C, but activated at lower temperatures. Four highly-conserved phage-related genes, located within the Tc-PAIYe, were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C (1). Conditions transcriptionally activating the cassette are yet unknown. In contrast to E. coli, the overproduction of holin and endolysin did not result in cell lysis of strain W22703 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to a complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease negative mutant of W22703 in which overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703, but remained stable in strain W22703 Δlon or in Y. pseudotuberculosis. Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703. IMPORTANCE The investigation of the mechanisms that help pathogens to survive in the environment is a prerequisite to understand their evolution and their virulence capacities. In members of the genus Yersinia, many factors involved in virulence, metabolism, motility or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying activation of determinants at body temperature have been analysed in detail, the molecular basis of low temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica, does not lyse its own host following overexpression at 15°C, and that the Lon protease is involved in this phenotype.

The Complete Genome Sequence of the Staphylococcus Bacteriophage Metroid

Phages infecting bacteria of the genus Staphylococcus play an important role in their host’s ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities – including a conservation of the lysis cassette – with other Staphylococcus cluster C bacteriophages, several of which were previously characterized for therapeutic applications.

The complete genome sequence of the Staphylococcus bacteriophage Metroid

Phages infecting bacteria of the genus Staphylococcus play an important role in their host’s ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities – including a conservation of the lysis cassette – with other Staphylococcus cluster C1 bacteriophages, several of which were previously characterized for therapeutic applications.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Siskin

Bacteriophage Siskin is a member of the χ-like siphovirus phage cluster that infects Salmonella enterica serovar Typhimurium strain LT2. Here, we report the complete 58,476-bp sequence of the Siskin genome, provide confirmation of its genomic termini, and describe a potentially new class of holins and endolysins found in the lysis cassette.

Pectobacterium atrosepticum Phage vB_PatP_CB5: A Member of the Proposed Genus ‘Phimunavirus’

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal–arrest–release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

Activity of a Holin-Endolysin System in the Insecticidal Pathogenicity Island ofYersinia enterocolitica

ABSTRACTYersinia enterocoliticais a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAIYe), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity inEscherichia colicells upon overexpression. When a set ofYersiniastrains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAIYewas demonstrated in this study, its biological role remains to be elucidated.IMPORTANCEThe knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity ofYersiniaspp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAIYe. While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyseYersiniastrains.

Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots

ABSTRACT R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere. IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.