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.

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The C1/RIPE3b1 (؊118/؊107 bp) binding factor regulates pancreatic-␤-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse ␤TC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N-and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (؊340/؊91 bp) of the endogenous insulin gene in ␤TC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the ␤-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet ␤ cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet ␤-cell function.Insulin is an essential regulator of metabolism. This hormone, which is synthesized by the ␤ cells of the islets of Langerhans, increases the storage of glucose, fatty acids, and amino acids through its actions in liver, adipose tissue, and muscle. Experiments performed in vivo with transgenic animals have established that the cis-acting elements controlling ␤-cell-selective expression are located within the insulin enhancer region, which is found between nucleotides Ϫ340 and Ϫ91 relative to the transcription start site. Several key control elements within the enhancer have been identified, including C2 (Ϫ317/ Ϫ311 bp), A3 (Ϫ201/Ϫ196 bp), C1 (Ϫ118/Ϫ107 bp), and E1 (Ϫ100/Ϫ91 bp) (37,60,67). Mutations that decrease the binding affinity of the A3, C1, and E1 activators also reduce glucose-regulated transcription (37,60,67).The activator of insulin C2-element stimulated transcription is Pax6 (61). Proteins in the Pax family all contain a paired box bipartite DNA-binding domain, although Pax6 also has a homeodomain. The Pdx-1 homeodomain protein (formerly known as IPF-1, STF-1, and IDX-1) is the regulator of A3 elementactivated expression (46,48,49,50), whereas the E1 activator is a heterodimer composed of proteins in the basic helix-loophelix family that are enriched in islets (i.e., BETA2 [42]) and generally distributed (i.e., HEB [51] and E2A [2,10,17,65]). In the adult pancreas, Pax6 (61) and BETA2 (42) are found in all islet cell types, whereas Pdx-1 appears to be found only in ␤ cells, a subset of islet ␦ cells (48,49), and exocrine acinar cells (71,80). These transcription factors are necessary for ma...

Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the family immediately downstream of the phage late promoters (p R ). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p R promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R as well as terminator kilobases distal to p R . To test if this Q-directed processive transcription plays a role in stx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p R and part of Q were deleted but which has an intact pStx, the previously described stx 2 AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R promoter. Since transcription initiating at p R ultimately requires activation of the phage lytic cascade, expression of stx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage in stx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) strains are responsible for outbreaks and sporadic cases of diarrhea. In some patients, exposure to STEC leads to hemorrhagic colitis and hemolytic uremic syndrome that may lead to death (20). Two major classes of Stxs, Stx1 and Stx2, encoded respectively by stx 1 AB and stx 2 AB, have been identified in STEC (2). The severe clinical consequences of STEC infections are thought to be caused by the activities of Stxs, although Stx2 appears to be more closely associated with these sequelae than does Stx1 (7,28,36). Shiga toxins are of the A-B type, with the glycolipid-binding B subunits being involved in the transport of the enzymatic A subunits into the eukaryotic cell where the A subunit, acting as a glycosylase, catalyzes a cleavage at a unique site in the 28S rRNA (2). The resulting inactivation of the ribosome leads to an inhibition of protein synthesis. More than 60 serotypes of STEC have been associated with human disease (1). The stx genes of many, if not all, STEC strains are in the genomes of prophages of the lambdoid family (19,23,27). This fact probably accounts for the wide dissemination of these genes in diverse E. coli serotypes.Comparison of lambdoid phage genomes (9) has revealed a common arrangement of functionally similar genes and a shared strategy governing gene expression (Fig. 1). In the lysogenic state, the repressor silences transcription of most phage genes (30). Removal of repression, which can occur when DNA damage activates the bacterial SOS response causing RecA-mediated cleavage of the repressor (21), leads to a cascade of regulatory events beginning with expression of the N transcription ant...

SummaryAnalysis 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 l Q transcription activator with its site of action, qut at the associated p R 8 late promoter, and upstream of the analogues of l 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 p R 8(qut) by promoting readthrough of transcription terminators. Another toxin-producing phage, 933W, has the identical Q gene and p R 8(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 p R 8 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 signi®cantly from those of other lambdoid phages.

SummaryThe stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the l bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx 1 AB genes are found ª 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

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Expression of the human β amyloid peptide (Aβ) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular Aβ in vivo . Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with Aβ has identified six likely chaperone proteins: two members of the HSP70 family, three αB-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription–PCR analysis shows that the small heat shock proteins are also transcriptionally induced by Aβ expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular Aβ in this model. Transgenic animals expressing a nonaggregating Aβ variant, a single-chain Aβ dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with Aβ expression. These results suggest that chaperone function can play a role in modulating intracellular Aβ metabolism and toxicity.