David I. Friedman

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production. Infection with Enterohemorrhagic E. coli (EHEC), and more recently with the Enteroaggregative E. coli strain O104:H4, is a significant health risk, causing bloody diarrhea, kidney failure, and even death. The virulence factor in these bacteria responsible for the severe outcomes is Shiga toxin (Stx). Genes encoding Stx are in the genome of bacterial viruses (prophages) on the pathogenic E. coli chromosomes. The prophage remains quiescent until damage to the bacterial chromosome occurs causing prophage gene expression (called induction), which leads to production of bacteriophages that are released into the environment. Because stx expression is controlled by the phage regulatory system, prophage induction leads additionally to production and release of Stx. This study provides conclusive evidence that in a mouse model of EHEC infection, induction of the prophage carrying the stx genes is specifically required for EHEC to cause disease and that the intestinal environment adds to the induction and therefore to the production of Stx. Similar events likely regulate Stx production and release by the Stx encoding phage in the O104:H4 strain. Controlling prophage induction offers a means to control EHEC infection.

Summary: Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho‐dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus ‐modified transcription complex in rrn operons is as an RNA chaperone insuring co‐ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold‐sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5′ RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.

Fang et al. generate the first genome-wide, strand-specific, single base–pair resolution map of m6A modifications in Escherichia coli. Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

Summary: Bacteria in their struggle for survival have evolved or acquired defences against attacking phage. However, phage often contribute to this defence through mechanisms in which a prophage protects the bacterial population from attack by another, often unrelated, phage. The 933W prophage, which carries Shiga toxin genes that enhance pathogenicity of enterohaemorrhagic Escherichia coli strain O157:H7, also carries the stk gene encoding a eukaryotic‐like tyrosine kinase that excludes (aborts) infection by phage HK97. This exclusion requires the kinase activity of Stk. Little, if any, protein tyrosine phosphorylation can be detected in a 933W lysogen prior to infection with HK97, while extensive Stk‐mediated tyrosine phosphorylation is evident following infection. This includes autophosphorylation that stabilizes Stk protein from degradation. Although increased levels of Stk are found following HK97 infection, these higher levels are not necessary or sufficient for exclusion or protein phosphorylation. An HK97 open reading frame, orf41, is necessary for exclusion and Stk kinase activity. We hypothesize that interaction with gp41 stimulates Stk kinase activity. Exclusion of HK97 appears to be specific since other phages tested, λ, φ80, H‐19B, λ‐P22dis and T4rII, were not excluded. Infection of the 933W lysogen with a non‐excluded phage fails to induce Stk‐determined phosphorylation.

The N protein of phage λ acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the λ early P _R promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P _R , at positions −14 and −45 (yielding P _R-GA ), reduces effectiveness of N modification, while an additional mutation at position −30 (yielding P _R-GCA ) suppresses this effect. In vivo, the level of P _R-GA -directed transcription was twice as great as the wild-type level, while transcription directed by P _R-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P _R-GA in vitro was roughly one-third the rate for wild-type P _R . We ascribe this apparent discrepancy to an effect of the mutations in P _R-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

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 λ, 933W has three operator repeats in the right operator region (O_R), but unlike λ and all other studied lambdoid phages, which have three operator repeats in the left operator region (O_L), 933W only has two operator repeats in O_L. As was observed with λ, the 933W O_R and O_L regions regulate transcription from the early P_R and P_L 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.

The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk , with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with c I from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.

Summary: Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the λ family, such as H‐19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H‐19B prophage encoding the TnpR resolvase gene downstream of the phage P_R promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H‐19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non‐Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H‐19B, usually both prophages in the same cell are induced.