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David I Friedman

Publications and source records attributed to David I Friedman.

12 recordsLinked to original sources
PubMed2006

Evidence that the promoter can influence assembly of antitermination complexes at downstream RNA sites.

The N protein of phage lambda 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 lambda 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.

Artificial Gene Fusion↗
PubMed2005

Phage regulatory circuits and virulence gene expression.

In many pathogenic bacteria, genes that encode virulence factors are located in the genomes of prophages. Clearly bacteriophages are important vectors for disseminating virulence genes, but, in addition, do phage regulatory circuits contribute to expression of these genes? Phages of the lambda family that have genes encoding Shiga toxin are found in certain pathogenic Escherichia coli (known as Shiga toxin producing E. coli) and the filamentous phage CTXphi, that carries genes encoding cholera toxin (CTX), is found in Vibrio cholerae. Both the lambda and CTXphi phages have repressor systems that maintain their respective prophages in a quiescent state, and in both types of prophages this repressed state is abolished when the host cell SOS response is activated. In the lambda type of prophages, only binding of the phage-encoded repressor is involved in repression and this repressor ultimately controls Shiga toxin production and/or release. In the CTXphi prophage, binding of LexA, the bacterial regulator of SOS, in addition to binding of the repressor is involved in repression; the repressor has only limited control over CTX production and has no influence on its release.

Bacteriophages↗
PubMed2005

The utility of bedside ultrasound and patient perception in detecting soft tissue foreign bodies in children.

OBJECTIVE: The purpose of the study was to determine if bedside ultrasound (US) and perception of wound foreign bodies (FBs) are useful screening tools for detecting wound FBs in children. METHODS: Prospective consecutive sample of children aged 18 years or younger presenting to a pediatric emergency department with wounds considered by the pediatric emergency department attending physician to be at risk for FBs was enrolled. Patients were asked if they had FB sensation in their wound(s). A bedside US of each wound was performed by the pediatric emergency department attending physician. A radiograph of each wound was obtained and interpreted by a radiologist blinded to US results and patient perception. Wound FBs were defined by the removal of a FB. The utilities of US and US with FB perception were compared with radiography for screening for wound FBs. Differences in performance characteristics among the 3 modalities were assessed using Fisher exact test. RESULTS: One hundred thirty-one wounds were studied in 105 patients. FBs were identified in 12 wounds (9.2%). A subanalysis was performed on patients able to answer questions regarding their perception of wound FBs. There were no significant differences in the test performance characteristics of bedside US alone compared with radiography for detecting wound FBs. Except for specificity, there were no significant differences in the test performance characteristics of bedside US combined with perception compared with radiography for detecting wound FBs. CONCLUSIONS: Bedside US is comparable to the performance of radiography interpreted by an attending pediatric radiologist. Bedside US alone or combined with patient perception may be an adequate initial screening tool for detecting wound FBs.

Child↗
PubMed2004

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 lambda 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.

Animals↗
PubMed2004

Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W.

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 cI 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.

Amino Acid Sequence↗
PubMed2004

The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression.

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 lambda, 933W has three operator repeats in the right operator region (OR), but unlike lambda and all other studied lambdoid phages, which have three operator repeats in the left operator region (OL), 933W only has two operator repeats in OL. As was observed with lambda, the 933W OR and OL regions regulate transcription from the early PR and PL 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.

Bacteriophage lambda↗
PubMed2003

A salvage pathway for protein structures: tmRNA and trans-translation.

Transfer-messenger RNA (tmRNA, or SsrA), found in all eubacteria, has both transfer and messenger RNA activity. Relieving ribosome stalling by a process called trans-translation, tmRNAala enters the ribosome and adds its aminoacylated alanine to the nascent polypeptide. The original mRNA is released and tmRNA becomes the template for translation of a 10-amino-acid tag that signals for proteolytic degradation. Although essential in a few bacterial species, tmRNA is nonessential in Escherichia coli and many other bacteria. Proteins known to be associated with tmRNA include SmpB, ribosomal protein S1, RNase R, and phosphoribosyl pyrophosphate. SmpB, having no other known function, is essential for tmRNA activity. trans-translation operates within ribosomes stalled both at the end of truncated mRNAs and at rare codons and some natural termination sites. Both the release of stalled ribosomes and the subsequent degradation of tagged proteins are important consequences of trans-translation.

Codon↗
PubMed2002

Evidence that the KH RNA-binding domains influence the action of the E. coli NusA protein.

The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. An essential function in E. coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of lambda. Tandem KH domains have been identified downstream of the S1 domain. We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains. The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action. The changes in both KH domains interfere with NusA binding to RNA. A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action. However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein. These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the lambda N protein.

Amino Acid Substitution↗
PubMed2002

The biological roles of trans-translation.

The unique transfer-messenger RNA (tmRNA) molecule has been identified in all bacterial species examined, suggesting that its action confers an important survival advantage to bacteria. Acting both as a tRNA and an mRNA, in a process known as trans-translation, tmRNA adds a short peptide tag to undesirable proteins. Trans-translation plays at least two physiological roles: removing ribosomes stalled upon mRNA, and targeting the resulting truncated proteins for degradation by proteases. The first of these roles is required for all known activities of tmRNA, whereas the second may be dispensed with in most cases with little biological effect. However, tmRNA-targeted proteolysis may be important for fine-tuning expression of certain genes by altering the concentration of regulatory proteins. Here, we review recent literature that addresses the biological functions of tmRNA.

Bacteria↗
PubMed2002

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 lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). 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 stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, 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 stx(1)AB copy number, regulating stx(1)AB 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.

Bacteriophage lambda↗