Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces.

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.

Fecal prevalence of Escherichia coli O157, Salmonella, Listeria, and Bacteriophage Infecting E. coli O157:H7 in feedlot cattle in the Southern Plains region of the United States.

Escherichia coli O157:H7, Salmonella, and Listeria are foodborne pathogens of critical importance that often colonize cattle. E. coli O157:H7 can be specifically killed by lytic bacteriophage, and lytic bacteriophage treatment has been suggested as a pre-harvest intervention strategy to reduce foodborne pathogens in cattle. To date, no systematic approach to determine the incidence of E. coli O157:H7-infecting lytic bacteriophage has been published. Therefore, the current study was designed to determine (1) the incidence of E. coli O157, Salmonella spp., and Listeria and (2) the incidence of E. coli O157:H7-infecting bacteriophage in the feces of feedlot steers in commercial feedlots in the United States. Fecal samples (n=60) were collected from four feedlots in two Southern Great Plains states (total (n=240 fecal samples). Salmonella and E. coli O157:H7 were found in 3.8% and 11.7% of the fecal samples, respectively. Bacteriophage targeting E. coli O157:H7 were found in all four feedlots, in 15% of the individual fecal samples, and in 55% of the cattle pens. Our results indicate that such bacteriophage are widespread in feedlot cattle, suggesting that further research into the ecological role of bacteriophage in the gastrointestinal tract is needed.

Identification of bacteriophage T4 prereplicative proteins on two-dimensional polyacrylamide gels.

Bacteriophage T4 makes a large number of prereplicative proteins, which are involved in directing the transition from host to phage functions, in producing the new T4 DNA, and in regulating transcriptional shifts. We have used two-dimensional gel electrophoresis (nonequilibrium pH gradient electrophoresis gels in the first dimension and sodium dodecyl sulfate-polyacrylamide gradient slab gels in the second) to identify a number of new prereplicative proteins. The products of many known genes are identified because they are missing in mutants with amber mutations of those genes, as analyzed by us and/or by previous workers. Some have also been identified by running purified proteins as markers on gels with labeled extracts from infected cells. Other proteins that are otherwise unknown are characterized as missing in infections with phage carrying certain large deletions and, in some cases, are correlated with sequence data.

Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4.

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with [gamma-32P]ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template.

Bacteriophage T4 alc gene product: general inhibitor of transcription from cytosine-containing DNA.

The alc gene of bacteriophage T4 was originally defined on the basis of mutations which allow late protein synthesis directed by T4 DNA containing cytosine rather than hydroxymethylcytosine. The question remained whether the normal alc gene product (gpalc) also blocks the transcription of early genes from cytosine-containing DNA. Complementation experiments were performed between hydroxymethylcytosine-containing phage which direct gpalc synthesis but carry mutations in a given gene(s) and cytosine-containing phage carrying that gene(s). The required protein would then have to be directed by the cytosine-containing DNA: it is looked for directly on polyacrylamide gels or through its physiological effects or both. For all early proteins examined in this way, no synthesis was observed when 95 to 100% of the hydroxymethylcytosine was substituted by cytosine in the infecting DNA, whereas there was significant synthesis with 75% substitution or less. The results indicate that gpalc is carried in with the infecting DNA or is made very early to block transcription of all cytosine-containing DNA.

Synthesis of T4 DNA and bacteriophage in the absence of dCMP hydroxymethylase.

Several lines of research have suggested that the dCMP hydroxymethylase (HMase) coded by bacteriophage T4 is an essential protein in a DNA replication complex, as well as a supplier of hydroxymethyl dCMP for phage DNA synthesis. We show that a mutant [HMase, dCTPase, endonuclease II, endonuclease IV] which lacked this enzyme made cytosine-containing DNA at about two-thirds of the normal rate. When coupled with an alc mutation which permitted synthesis of late proteins, a small burst of phage was produced whose DNA contained no hydroxymethylcytosine. This pentuple mutant made both early and late proteins with abnormal kinetics, whereas the HMase+ parent showed normal kinetics. However, intracellular phage DNA showed no gross abnormalities in alkaline sucrose gradients. We conclude that HMase is not required for DNA synthesis when hydroxymethyl dCMP is not needed as a substrate; however, its absence still impairs both replication and transcription.

Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4.

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.