David I. Friedman

Integration host factor:a protein for all reasons.

The identification and characterization of the Escherichia coli DNA binding protein integration host factor (IHF) is an elegant example of how a well-characterized virus can be employed in the analysis of a host function. In this case, Nash and coworkers, through their landmark in vitro studies of coliphage h site-specific recombination (reiewed in Nash, 1981), have identified a protein that plays roles not only in other recombination reactions, but also in DNA replication and regulation of gene expression. IHF belongs to a class of structurally related “histonelike” proteins that can wrap DNA into higher-order structures (Drlica and Rouviere-Yaniv, 1987). The most abundant of these proteins in E. coli is HU, and others have been found in a number of bacterial genera as well as archaebacteria. In addition to site-specific recombination, other aspects of h development influenced by IHF have been fertile sources of information about this protein. I will initially focus on studies with h that serve to present the basic information about IHF and then examine the various roles for IHF derived from studies of E. coli and some of its other phages and plasmids.

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H‐19B genome located the genes encoding shiga‐like toxin I (Stx‐I) downstream of the gene encoding the analogue of the phage λ Q transcription activator with its site of action, qut at the associated pR′ late promoter, and upstream of the analogues of λ genes encoding lysis functions. Functional studies, including measurement of the effect of H‐19B Q action on levels of Stx expressed from an H‐19B prophage, show that the H‐19B Q acts as a transcription activator with its associated pR′(qut) by promoting readthrough of transcription terminators. Another toxin‐producing phage, 933W, has the identical Q gene and pR′(qut) upstream of the stx‐II genes. The H‐19B Q also activates Stx‐II expression from a 933W prophage. An ORF in H‐19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx‐I expression can be enhanced by transcription from pR′ as well as a model for toxin release through cell lysis mediated by action of phage‐encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H‐19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N‐like system differs significantly from those of other lambdoid phages.

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

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.

Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli

The stx genes of many Shiga toxin‐encoding Escherichia coli (STEC) strains are encoded by prophages of the λ bacteriophage family. In the genome of the Stx1‐encoding phage H‐19B, the stx1AB genes are found ≈ 1 kb downstream of the late phage promoter, pR′, but are known to be regulated by the associated iron‐regulated promoter, pStx1. Growth of H‐19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H‐19B prophage. We found that replication of the phage genome, with the associated increase in stx1AB copy number, is the most quantitatively important mechanism by which H‐19B induction increases Stx1 production. Three promoters are shown to be involved in stx1AB transcription after phage induction, the iron‐regulated pStx1 and the phage‐regulated pR and pR′ promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the pR′ promoter, contrary to previous findings in the related Stx2‐encoding phage φ361, was found to be unnecessary for high‐level Stx1 production after phage induction. Finally, we present evidence that phage‐mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx1AB copy number, regulating stx1AB transcription and allowing for Stx1 release, the biology of the Stx‐encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

Transcription antitermination: the λ paradigm updated

Coliphage λ employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage‐encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination‐resistant form. Expression of λ late genes is regulated by the phage‐encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination‐resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins, (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination‐resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non‐template DNA strand. (iv) A host‐encoded inhibitor of N antitermination appears to act through the NUT site as well as with the α subunit of RNA polymerase, and is antagonized by NusB protein.

Am E. coli gene product required for λ site-specific recombination

Abstract We report characteristics of him A mutations of E. coli, selected for their inability to support the site-specific recombination reaction involved in the formation of lysogens by bacteriophage λ. The him A allele lies at minute 38 on the chromosome. Three noncomplementing and closely linked mutations define the him A locus; one is a nonsense mutation which shows that the gene product is a protein. Him A mutations reduce both λ integrative and excisive site-specific recombination. Since dominance tests demonstrate that him A mutations are recessive, it is probable that the him A protein is either a necessary component for site-specific recombination or, alternatively, regulates the expression of such a function. Him A mutations exhibit pleiotropic effects. They reduce integration of phages that have different attachment specificities from λ and inhibit the growth of phage mu. In addition, him A mutations reduce precise excision of integrated phage mu as well as Tn elements. This pleiotropy suggests that the role of him A protein is nonspecific. Since all of the processes affected by him A mutations ultimately rely on protein-DNA interactions, we suggest that him A protein may act in an auxillary manner to facilitate these interactions.

Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability

tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s).

Genetic characterization of a bacterial locus involved in the activity of the N function of phage λ

Abstract We report the genetic mapping of a locus of the Escherichia coli chromosome involved in the expression of the N gene function of phage λ. This phage specified function regulates the subsequent transcription of most of the λ genome. The bacterial locus involved in N expression, called nus for N u tilization s ubstance, maps between aspB at minute 62 and argG at minute 61 of the E. coli chromosome. Two different bacterial variants in which λ N function is not active have been used in mapping the nus locus, a mutant of E. coli K12, Nus, and a hybrid bacterium formed by genetic transfer between E. coli and S. typhosa . Although these two bacterial variants exhibit slightly different phenotypes, chromosome transfer studies demonstrate that the same genetic region is involved in the observed N-ineffective phenotype. Dominance studies show that in the case of the Nus mutant, the nus + allele is dominant. This suggests that the nus + allele is responsible for the expression of a function necessary for N product activity. In the case of transfer of the nus region from a Nus mutant to an E. coli-S. typhosa hybrid, the resulting hybrid assumes the phenotype of the Nus mutant. Genetic studies using P1 transduction demonstrate that the same genetic region is involved in the N-ineffective phenotype of the two bacterial variants.

Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system

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 PR 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.

Lytic Mode of Lambda Development

Lytic growth of bacteriophage requires the concerted expression of a number of functions, products of both viral and host genes. These functions promote transcription, replication, DNA processing, and packaging. Additionally, temperate phage such as λ , which can grow either lytically or lysogenically, must insure that once the decision to adopt one of the alternative modes of existence has been made, functions that can interfere with that mode are not expressed. In the lytic mode, the phage functions must be sequentially expressed, i.e., replication must precede packaging which, in turn, must precede lysis. Thus, although phage propagation occurs in a relatively short time—some 45 minutes for λ —it follows a strictly coordinated developmental plan. In this paper we review current knowledge of λ lytic development. The lysogenic program is discussed by Wulff and Rosenberg and by Echols and Guarneros (both this volume). Our discussion focuses on (1) gene organization, (2) patterns of mRNA synthesis, and (3) regulatory controls of gene expression. The role of the λ N function in regulating the lytic cycle is stressed. This is, in part, because N function is among the best characterized regulatory proteins and, also, its action typifies a mechanism of regulation based upon transcription termination-antitermination. For further background material and discussions from different perspectives, see Echols (1971), Weisberg et al. (1977), and Herskowitz and Hagen (1980). GENETIC ORGANIZATION The λ genome expresses approximately 50 proteins (Szybalski and Szybalski 1979). In this section we discuss the arrangement of the λ genes with respect to the...