Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome.

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli.

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.

A Novel Inducible Prophage from Burkholderia Vietnamiensis G4 is Widely Distributed across the Species and Has Lytic Activity against Pathogenic Burkholderia.

Burkholderia species have environmental, industrial and medical significance, and are important opportunistic pathogens in individuals with cystic fibrosis (CF). Using a combination of existing and newly determined genome sequences, this study investigated prophage carriage across the species B. vietnamiensis, and also isolated spontaneously inducible prophages from a reference strain, G4. Eighty-one B. vietnamiensis genomes were bioinformatically screened for prophages using PHASTER (Phage Search Tool Enhanced Release) and prophage regions were found to comprise up to 3.4% of total genetic material. Overall, 115 intact prophages were identified and there was evidence of polylysogeny in 32 strains. A novel, inducible Mu-like phage (vB_BvM-G4P1) was isolated from B. vietnamiensis G4 that had lytic activity against strains of five Burkholderia species prevalent in CF infections, including the Boston epidemic B. dolosa strain SLC6. The cognate prophage to vB_BvM-G4P1 was identified in the lysogen genome and was almost identical (>93.5% tblastx identity) to prophages found in 13 other B. vietnamiensis strains (17% of the strain collection). Phylogenomic analysis determined that the G4P1-like prophages were widely distributed across the population structure of B. vietnamiensis. This study highlights how genomic characterization of Burkholderia prophages can lead to the discovery of novel bacteriophages with potential therapeutic or biotechnological applications.

The phage gene wmk is a candidate for male killing by a bacterial endosymbiont.

Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

Cryptic prophages as targets for drug development.

Bacterial chromosomes may contain up to 20% phage DNA that encodes diverse proteins ranging from those for photosynthesis to those for autoimmunity; hence, phages contribute greatly to the metabolic potential of pathogens. Active prophages carrying genes encoding virulence factors and antibiotic resistance can be excised from the host chromosome to form active phages and are transmissible among different bacterial hosts upon SOS responses. Cryptic prophages are artifacts of mutagenesis in which lysogenic phage are captured in the bacterial chromosome: they may excise but they do not form active phage particles or lyse their captors. Hence, cryptic prophages are relatively permanent reservoirs of genes, many of which benefit pathogens, in ways we are just beginning to discern. Here we explore the role of active prophage- and cryptic prophage-derived proteins in terms of (i) virulence, (ii) antibiotic resistance, and (iii) antibiotic tolerance; antibiotic tolerance occurs as a result of the non-heritable phenotype of dormancy which is a result of activation of toxins of toxin/antitoxin loci that are frequently encoded in cryptic prophages. Therefore, cryptic prophages are promising targets for drug development.

The role of prophage in plant-pathogenic bacteria.

A diverse set of phage lineages is associated with the bacterial plant-pathogen genomes sequenced to date. Analysis of 37 genomes revealed 5,169 potential genes (approximately 4.3 Mbp) of phage origin, and at least 50% had no function assigned or are nonessential to phage biology. Some phytopathogens have transcriptionally active prophage genes under conditions that mimic plant infection, suggesting an association between plant disease and prophage transcriptional modulation. The role of prophages within genomes for cell biology varies. For pathogens such as Pectobacterium, Pseudomonas, Ralstonia, and Streptomyces, involvement of prophage in disease symptoms has been demonstrated. In Xylella and Xanthomonas, prophage activity is associated with genome rearrangements and strain differentiation. For other pathogens, prophage roles are yet to be established. This review integrates available information in a unique interface ( http://propnav.esalq.usp.br ) that may be assessed to improve research in prophage biology and its association with genome evolution and pathogenicity.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Lysogenic Streptococcus suis isolate SS2-4 containing prophage SMP showed increased mortality in zebra fish compared to the wild-type isolate.

Streptococcus suis (S. suis) infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2) being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.

Characteristics of defective phage particles of Pectobacterium carotovorum ZM1.

It is shown for the first time that the expression products of defective prophages are typical of defective lysogenic systems of phytopathogenic Pectobacterium carotovorum. It is established that virus-like particles (LP) such as phage capsids are packing bacterial DNA which size is determined by pulse field gel electrophoresis separation. Based on data about capsid structures which are formed by the virulent mutant ZF40/421, there is made a suggestion about the forming mechanism of defective virions of P carotovorum.

The essential function of B. subtilis RNase III is to silence foreign toxin genes.

RNase III-related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi)-RNAs and small interfering (si)-RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPß, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPß that are sensitive to RNase III. We further explore the mechanism of RNase III-mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.

Inhibitory effect of prophage SPß fragments on phage SP10 ribonucleotide reductase function and its multiplication in Bacillus subtilis.

Bacteria have evolved various kinds of defense mechanisms against phage infection and multiplication. Analysis of these mechanisms is important for medical and industrial application of phages as well as for their scientific study. Strains of Bacillus subtilis Marburg strain carrying both nonA and nonB mutations are susceptible to the Bacillus phage SP10. The nonB mutation has been shown to have a compromised intrinsic restriction system. The nonA mutation represents the cured state of prophage SPß whose genome is 135 kb in length and contains 187 ORFs. For this study we investigated the molecular mechanism behind the inhibitory activity of the wild type nonA function against phage SP10 development. The progression of phage-developmental stages was examined in cells harboring wild type nonA, i.e. prophage SPß. After phage adsorption and DNA injection into host cells, the synthesis of phage specific mRNA proceeded normally. However, phage DNA synthesis was severely inhibited by some effect of wild type nonA. We thus systematically deleted portions of the prophage SPß region from the B. subtilis genome and the resultant mutant strains were examined as to whether they still retained sufficient wild type nonA functionality to inhibit SP10 phage development. The SPß region encompassing the bnrdEF gene, which codes for a putative ribonucleotide reductase (RRase), turned out to be responsible for the wild type nonA function. The phage SP10 possesses its own xnrdE gene coding for a putative RRase that complements the temperature-sensitive mutation of the host RRase gene nrdE. This complementation was blocked by an artificially induced transcription from a non-coding strand of the bnrdEF region. It is thus likely that the transcript from the bnrdEF region of SPß inhibits ribonucleotide reductase function of SP10, resulting in arrest of DNA synthesis during phage SP10 development.

Assessment of genotoxicity and cytotoxicity of "lixeira" (Curatella americanaL. ) using the prophage λ induction test (SOS inductest) / Avaliação da genotoxicidade e citotoxicidade de "lixeira" (Curatella americana L.) utilizando o teste de indução profago λ (teste de indução SOS)

Curatella americana L., commonly known as "lixeira" in Brazil, has been used in folk medicine to treat ulcers and inflammations. The purpose of the present work was to evaluate the cytotoxic and genotoxic potential of the ethanolic extract of C. americana stem bark using the prophage λ induction test (SOS inductest). To evaluate the cytotoxicity of this plant, after treatment with different concentrations of the extract, Escherichia coli WP2s(λ) cultures were diluted in M9 buffer, inoculated into LB plates, and incubated for 24 h at 37 ºC. To assess genotoxicity, the lysogenic strain E. coli WP2s(λ) was treated with different concentrations of the extract. Then, the lysogenic strain was added to the indicator strain (RJF013), LB(1/2)(malt/amp), seeded into plates with the matches, and incubated for 24 h at 37 ºC. After this period, the total number of colonies and the number of plaques were counted to evaluate C. americana cytotoxicity and genotoxicity, respectively. Our results showed that although the extract of "lixeira" did not modify the survival of bacteria (p > 0.05), it caused a significant increase in prophage λ induction, especially at the higher concentrations (p<0.05). Therefore, we conclude that the ethanolic extract of C. americana stem bark did not present cytotoxic effect, but some genotoxic potential was observed.

Curatella americana L., comumente conhecida como "lixeira" no Brasil, é utilizada em medicina popular para tratamento de úlceras e inflamações. O presente trabalho teve como objetivo avaliar o potencial citotóxico e genotóxico do extrato etanólico das cascas de C. americana utilizando o Induteste SOS. Para avaliar a citotoxicidade da planta, depois de tratadas com diferentes concentrações do extrato, culturas de E. coli WP2s(λ) foram diluνdas em tampão M9 e semeadas em placas LB. Para avaliar a genotoxicidade da planta, a cepa lisogênica WP2s(λ) de E. coli foi tratada com diferentes concentrações do extrato. Em seguida, esta foi adicionada à cepa indicadora (RJF013) e ambas foram semeadas em placas em meio LB(1/2)(malt)(amp). Todas as culturas foram incubadas por 24 h a 37 ºC. Posteriormente, o número total de colônias e o número de centros infecciosos foram computados para a avaliação da citotoxidade e da genotoxicidade desta planta, respectivamente. Os resultados mostraram que embora o extrato de C. americana não tenha modificado a sobrevivência bacteriana (p > 0,05), provocou aumento significativo (p < 0,05) na indução do profago λ, especialmente nas concentrações mais altas. Assim, concluiu-se que o extrato etanólico das cascas de C. americana não apresentou atividade citotóxica, mas foi observada ação genotóxica direta.

The defective prophage pool of Escherichia coli O157: prophage-prophage interactions potentiate horizontal transfer of virulence determinants.

Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1-Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1-Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities.

Host recognition and integration of filamentous phage phiRSM in the phytopathogen, Ralstonia solanacearum.

Two prophages, called varphiRSM3 and varphiRSM4, that are closely related to, but differ from, filamentous phage varphiRSM1, have been detected in strains of the Ralstonia solanacearum species complex. The prophage varphiRSM3, found in host strain MAFF730139, could be converted to infectious phage by means of PCR and transfection. The nucleotide sequence of varphiRSM3 is highly conserved relative to varphiRSM1 except for open reading frame 2 (ORF2), encoding an unknown protein, and ORF9 encoding the presumed adsorption protein that determines host range. The two host ranges differ dramatically and correlate closely with different gel electrophoresis banding patterns for cell surface fimbriae. Infections by varphiRSM1 and varphiRSM3 enhance bacterial cell aggregation and reduce the bacterial host virulence in tomato plants. Database searches in the R. solanacearum strains of known genomic sequence revealed two inovirus prophages, one designated varphiRSM4 that is homologous to varphiRSM1 and varphiRSM3, and one homologues to RSS1, in the genome of strain UW551.

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette.

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies. The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

Genetic and molecular analysis of GogB, a phage-encoded type III-secreted substrate in Salmonella enterica serovar typhimurium with autonomous expression from its associated phage.

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process.

The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression.

The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages. Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium. Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx. We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W. Like lambda, 933W has three operator repeats in the right operator region (OR), but unlike lambda and all other studied lambdoid phages, which have three operator repeats in the left operator region (OL), 933W only has two operator repeats in OL. As was observed with lambda, the 933W OR and OL regions regulate transcription from the early PR and PL promoters, respectively. A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor. This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.

Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1.

The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1vir). In several E. coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF+ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF+ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.