Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field <Emphasis Type="Italic">E. coli</Emphasis> O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.     

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.     

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment

Background

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Methodology/Principal Findings

The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg²⁺. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction.

Conclusions/Significance

Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.     

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx₂ gene in coliform bacteria in sewage samples of different origins. The presence of the stx₂ gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10² genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx₂ gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx₂ gene in the wastewater studied.     

Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx_1a subtype, while human strains carried mainly stx_1a or stx_2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.     

Shiga Toxin and Shiga Toxin-Encoding Phage Do Not Facilitate Escherichia coli O157:H7 Colonization in Sheep

Isogenic strains of Escherichia coli O157:H7, missing either stx₂ or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of E. coli O157:H7.     

Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

Aim: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. Methods and Results: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Φ3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx₂. A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Φ3538 (Δstx₂::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. Conclusions: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host’s DNA. Significance and Impact of the Study: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.     

Heterogeneity in Induction Level,Infection Ability,and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.     

Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx₂ loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

Spread of a Distinct Stx2-Encoding Phage Prototype among Escherichia coli O104:H4 Strains from Outbreaks in Germany, Norway, and Georgia

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.     

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli

Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.     

Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 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 stx₂ gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage 3538(Δstx₂::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 3538(Δstx₂::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 3538(Δstx₂::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 3538(Δstx₂::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that 3538(Δstx₂::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.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Differences in Virulence among Escherichia coli O157:H7 Strains Isolated from Humans during Disease Outbreaks and from Healthy Cattle

Escherichia coli O157:H7 causes life-threatening outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans and significant economic loss in agriculture and could be a potential agent of bioterrorism. Although the prevalence of E. coli O157:H7 in cattle and other species with which humans have frequent contact is high, human infections are relatively uncommon, despite a low infectious dose. A plausible explanation for the low disease incidence is the possibility that not all strains are virulent in humans. If there are substantial differences in virulence among strains in nature, then human disease may select for high virulence. We used a gnotobiotic piglet model to investigate the virulence of isolates from healthy cattle and from humans in disease outbreaks and to determine the correlation between production of Shiga toxin 1 (Stx1) and Stx2 and virulence. Overall, E. coli O157:H7 strains isolated from healthy cattle were less virulent in gnotobiotic piglets than strains isolated from humans during disease outbreaks. The amount of Stx2 produced by E. coli O157:H7 strains correlated with strain virulence as measured by a reduction in piglet survival and signs of central nervous system disease due to brain infarction. The amount of Stx1 produced in culture was not correlated with the length of time of piglet survival or with signs of central nervous system disease. We suggest that disease outbreaks select for producers of high levels of Stx2 among E. coli O157:H7 strains shed by animals and further suggest that Stx1 expression is unlikely to be significant in human outbreaks.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin‐producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin‐producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx‐related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx‐related events and enhance Stx absorption during STEC infection.     

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by 相似文献   

X-ray sensitivity of Escherichia coli lysogenic for bacteriophage P2.

Summary Strains of Escherichia coli C or K lysogenic for the non-inducible phage P2 show a lower survival following X-ray irradiation as compared to nonlysogenic strains. This difference in X-ray sensitivity is not accompanied by a significant difference in X-ray induced mutability. The capacity of X-irradiated P2 lysogens to multiply any of a number of unirradiated infecting phages is severely impaired. These effects of X-ray treatment can be most simply explained as a consequence of the fact that protein and RNA syntheses are strongly inhibited in P2 lysogens after X-irradiation. All the above events specifically occurring in X-rayed P2 lysogens are dependent on the P2 gene old.