We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative ...configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4‐related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5′ and 3′ to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP‐like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.
Transcription of the gene
osmE
of
Escherichia coli
is inducible by elevated osmotic pressure and during the decelerating phase of growth.
osmE
expression is directed by a single promoter,
osmE
p
. ...Decelerating phase induction of
osmE
p
is dependent on the σ
s
(RpoS) factor, whereas its osmotic induction is independent of σ
s.
Purified Eσ
s
and Eσ
70
were both able to transcribe
osmE
p
in vitro
on supercoiled templates. In the presence of
rpoD800
, a mutation resulting in a thermosensitive σ
70
factor, a shift to non‐permissive temperature abolished induction of
osmE
p
after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting
osmE
p
activity were isolated. Down‐promoter mutations decreased transcription in both the presence and the absence of σ
s
, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the
osmE
promoter. Three up‐promoter mutations brought
osmE
p
closer to the consensus of Eσ
70
‐dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of σ
s
in vivo
. Our data suggest that Eσ
s
transcribes wild‐type
osmE
p
with a higher efficiency than Eσ
70.
A model in which an intrinsic differential recognition contributes to growth phase‐dependent regulation is proposed. Generalization of this model to other σ
s
‐dependent promoters is discussed.
Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth. osmE expression is directed by a single promoter, osmEp. ...Decelerating phase induction of osmEp is dependent on the σs (RpoS) factor, whereas its osmotic induction is independent of σs. Purified Eσs and Eσ70 were both able to transcribe osmEpin vitro on supercoiled templates. In the presence of rpoD800, a mutation resulting in a thermosensitive σ70 factor, a shift to non‐permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting osmEp activity were isolated. Down‐promoter mutations decreased transcription in both the presence and the absence of σs, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter. Three up‐promoter mutations brought osmEp closer to the consensus of Eσ70‐dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of σsin vivo. Our data suggest that Eσs transcribes wild‐type osmEp with a higher efficiency than Eσ70. A model in which an intrinsic differential recognition contributes to growth phase‐dependent regulation is proposed. Generalization of this model to other σs‐dependent promoters is discussed.
Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth. osmE expression is directed by a single promoter, osmEp. ...Decelerating phase induction of osmEp is dependent on the sigmas (RpoS) factor, whereas its osmotic induction is independent of sigmas. Purified Esigmas and Esigma70 were both able to transcribe osmEp in vitro on supercoiled templates. In the presence of rpoD800, a mutation resulting in a thermosensitive sigma70 factor, a shift to non-permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting osmEp activity were isolated. Down-promoter mutations decreased transcription in both the presence and the absence of sigmas, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter. Three up-promoter mutations brought osmEp closer to the consensus of Esigma70-dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of sigmas in vivo. Our data suggest that Esigmas transcribes wild-type osmEp with a higher efficiency than Esigma70. A model in which an intrinsic differential recognition contributes to growth phase-dependent regulation is proposed. Generalization of this model to other sigmas-dependent promoters is discussed.
The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase. A strain carrying a treA-lacZ transcriptional fusion was constructed. The beta-galactosidase activity produced ...in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible. treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E. coli strains. The treA promoter was identified by S1 nuclease protection. Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start. Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase. treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon.
Polymerase chain reaction analysis of a large collection of bacteriophages with T-even morphology revealed four phages that are distantly related to all the others. The genomes of these pseudo T-even ...phages hybridized under stringent conditions to only a limited portion of the T4 genome that encodes virus head, head-to-tail joining and contractile tail genes. Except for this region, no extensive hybridization was detected between most pairs of the different pseudo T-even genomes. Sequencing of this conserved region of the pseudo T-even phage RB49 revealed substantial nucleotide sequence divergence from T4 (∼30% to 40%), and random genomic sequencing of this phage indicated that more than a third of its sequences had no detectable homology to T4. Among those sequences related to the T-even genes were virion structural components including the constituents of the phage base plate. Only a few sequences had homology to T4 early functions; these included ribonucleotide diphosphatase reductase, DNA ligase and the large subunit of DNA topoisomerase. The genomes of the pseudo T-even phage were digested by restriction enzymes that are unable to digest the T-even DNAs which contain glucosylated hydroxymethyl-cytosine residues. This suggests that only limited nucleotide modifications must be present in the pseudo T-even genomes. Conservation of much of the morphogenetic region of these diverse phage genomes may reflect particularly strong sequence constraints on these gene products. However, other explanations are considered, including the possibility that the various morphogenetic segments were acquired by the pseudo T-even genomes by modular evolution. These results support the notion that phage evolution may proceed within a network of both closely and distantly related genomes.
Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach ...enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organisations. Presented data constitute a significant insight into bacterial RNA landscapes and a step towards the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both ...bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.
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