Lysogenic Conversion Research Articles

Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3.

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM.

Exfoliative toxin A (ETA) causes staphylococcal scalded-skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA-producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated phi-ZM-1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage phi-ZM-1 carries the structural gene for ETA.

Lysogenic conversion of environmental Vibrio mimicus strains by CTXPhi.

The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.

Bacteriophages and virulence evolution

A major virulence factor for Salmonella spp., as well as for a variety of other animal and plant bacterial pathogens, is the type III secretion system (TTSS). The TTSS allows the transfer of bacterial effector proteins into eukaryotic cells and can thus modulate host-cell signal transduction. The majority of Salmonella typhimurium TTSS effector proteins are encoded by genes located on pathogenicity island 1 (SPI-1). Now, Mirold et al.1xIsolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain. Mirold, S. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9845–9850Crossref | PubMed | Scopus (161)See all References1 have reported that the sopE gene, encoding one of the effectors of the SPI-1 TTSS, is located outside this pathogenicity island on the temperate bacteriophage SopEΦ. sopE is present only in a small number of S. typhimurium isolates, but these isolates all carry SopEΦ sequences. By lysogenic conversion, SopEΦ can horizontally transfer genes between different S. typhimurium strains and could, therefore, cause the emergence of new epidemic strains. Indeed, most of the isolates harboring SopEΦ belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large proportion of human and animal salmonellosis and have persisted for a long period of time. These data suggest that bacteriophages might provide an efficient mechanism for fine-tuning or acquiring new methods of interaction between bacterial pathogens and their hosts.

Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage phi3538 isolated from Escherichia coli O157:H7.

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage phi3538(Deltastx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi3538(Deltastx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi3538(Deltastx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi3538(Deltastx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that phi3538(Deltastx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain.

Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPhi) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPhi belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts.

The clinically isolated FIZ15 bacteriophage causes lysogenic conversion in Pseudomonas aeruginosa PAO1.

FIZ15 bacteriophage, from a human clinical isolate of Pseudomonas aeruginosa, causes lysogenic conversion in the P. aeruginosa strain PAO1. The prophage-conferred phenotypes are: (1) increased resistance to phagocytosis by mouse peritoneal macrophages; (2) increased resistance to killing by normal human serum, and (3) increased adhesion to human buccal epithelial cells. These phenotypes are related to the prophage-induced change at the level of its own bacterial receptor, which appears to be the O-antigen.

Analysis of Clinical and Environmental Strains of Nontoxigenic Vibrio cholerae for Susceptibility to CTXΦ: Molecular Basis for Origination of New Strains with Epidemic Potential

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.

Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae.

Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients. Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water. Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXPhi. Although the mechanism by which CT causes diarrhea is known, it is not clear why V. cholerae should infect and elaborate the lethal toxin in the host. Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V. cholerae strains and a continual emergence of new epidemic clones. In view of lysogenic conversion by CTXPhi as a possible mechanism of origination of new toxigenic clones of V. cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V. cholerae strains and genetic elements that mediate the transfer of virulence genes. The ecosystem comprising V. cholerae, CTXPhi, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.

Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer.

A combined effort integrating studies of gonococcal Tfp biogenesis, the data made available from the gonococcal genome sequence project and applied molecular genetics have been used to identify the fibrillar filaments themselves, the PilT protein and the ComP protein as essential components for the DNA uptake phase of competence for natural transformation. Our ongoing studies are focused on identifying and understanding the complex interactions which exist between these essential constituents. These studies may be relevant not only to the early steps of genetic transformation but also to the two other venues for horizontal gene transfer based on recent findings. First, the thin pili of IncI1 conjugal plasmids required for liquid mating belong to the type IV family of pili (Yoshida et al., 1998). Secondly, type IV pili are required for lysogenic conversion of Vibrio cholerae by a filamentous phage encoding cholera toxin (Waldor and Mekalanos, 1996). How these highly conserved surface organelles contribute to such diverse forms of DNA translocation across membranes remains to be seen.

Bacteriophage biology and bacterial virulence

Temperate bacteriophages often encode properties that alter the host bacterium following the establishment of lysogeny. This process, referred to as `lysogenic conversion', has played a critical role in the evolution of many Gram-positive and Gram-negative pathogens. The structural genes encoding exotoxins, such as diphtheria toxin, botulinum toxin types C1 and D, streptococcal erythrogenic toxin, staphylococcal enterotoxin A, Shiga toxins 1 and 2 (Stx1 and Stx2), Pseudomonas cytotoxin, and cholera toxin (CT), are located in the genomes of temperate bacteriophages that confer toxinogenicity upon their respective hosts (reviewed in Ref. [1]). Besides toxins, other potential virulence factors encoded by bacteriophages include extracellular enzymes (such as streptococcal hyaluronidase[2]and staphylokinase[3]), outer membrane proteins [such as Lom and Bor of phage λ (Ref. [4])], a G-protein-like open reading frame in Vibrio cholerae phage K139 (Ref. [5]), and enzymes that alter the antigenic properties of lipopolysaccharide (LPS) in Salmonella and Shigella[6]. The diverse bacteriophages that encode these virulence factors have been thought of primarily as means for the horizontal transmission of virulence genes within bacterial populations[7]. Recent studies of the lambdoid phage H-19B, which encodes Stx1 (Ref. [8]), and the filamentous phage CTXφ, which encodes CT (Ref. [9]), raise the possibility that, in addition to serving as vectors for the dissemination of the genes encoding these toxins, the life cycles of these phages may play a role in the regulation of the production and export of these potent toxins.

The Late-Expressed Region of the Temperate Coliphage 186 Genome

The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and φCTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.

Lysogenic conversion as a factor influencing the vancomycin tolerance phenomenon in Staphylococcus aureus

Lysogenic conversion as a factor influencing the vancomycin tolerance phenomenon in Staphylococcus aureus

Lysogenic Conversion by a Filamentous Phage Encoding Cholera Toxin

Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXphi), which is related to coliphage M13. The CTXphi genome chromosomally integrated or replicated as a plasmid. CTXphi used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.

Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor.

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner. DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as 'kappa'. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce beta-lactamase fusions to phage-encoded, exported proteins. All Tn10d-bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein-like ORF) because its amino acid sequence shows similarity to eukaryotic Gs(alpha) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins. LD50 assays with isogenic Glo+ and Glo- K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae.

The return of Corynebacterium diphtheriae: the rise of non-toxigenic strains

With the decline in incidence of diphtheria in Europe and the USA, many laboratories no longer routinely culture throat swabs for Corynebacterium diphtheriae. However, there is an outbreak of infection with toxigenic strains in Russia and most adults do not have protective levels of antibody. Non-toxigenic strains are known to cause local disease and lysogenic conversion probably occurs in vivo as well as in vitro, Non-toxigenic C. diphtheriae var. gravis, formerly quite rare, has been isolated with increasing frequency in the UK over the last five years. During prospective screening at one Sexually Transmitted Disease Clinic, six (1%) of 578 homosexual men were found to harbour the organism in the throat, four of them with clinical pharyngitis. Only one of 1696 heterosexual men and women were found to be carriers. Seven cases of endocarditis due to this organism were reported in a single year in Sydney, Australia and non-toxigenic C. diphtheriae var. mitis has caused four cases of endocarditis in Switzerland. Non-toxigenic strains are responsible for pharyngitis and occasional invasive disease and should be treated. Routine screening of throat swabs should not be abandoned.

Lytic conversion of Escherichia coli by bacteriophage T5: blocking of the FhuA receptor protein by a lipoprotein expressed early during infection.

The nucleotide sequence of the region between the oad gene, encoding the host specificity protein, and the right-terminal repetition of bacteriophage T5 DNA was determined. Five small open reading frames, the first of which was called llp, were detected, which apparently formed an operon transcribed from a promoter that overlapped the oad promoter. Both promoters were confirmed by primer extension assays. Using mRNA isolated at different times after T5 infection, the llp and oad promoters were identified as early and late promoters, respectively. The N-terminus of the llp gene product possess a signal sequence and a processing site characteristic of lipoproteins. After subcloning and expression of llp, its product Llp was identified as a 7.8 kDa polypeptide. Acylation of Llp was confirmed by addition of globomycin, which resulted in the accumulation of the unprocessed precursor form. FhuA+ cells synthesizing Llp were resistant to phage T5. Resistance was caused by inhibition of adsorption of T5 to its FhuA receptor protein. Resistance could be overcome by derepression of fhuA transcription, suggesting a blocking of FhuA by direct interaction with Llp. Since Llp-mediated T5 resistance has several aspects in common with the phenomenon of lysogenic conversion, we suggest that it should be called lytic conversion.

Application of DNA probes to analysis of bacteriophage distribution patterns in the environment.

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.

A case of lysogenic conversion: modification of cell phenotype by constitutive expression of the Mu gem operon

A case of lysogenic conversion: modification of cell phenotype by constitutive expression of the Mu gem operon