UV-A-induced expression of GroEL in the UV-A-resistant marine cyanobacterium Oscillatoria sp. NKBG 091600.
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The authors have examined the response to UV-A irradiation of the UV-A-resistant marine cyanobacterium Oscillatoria sp. NKBG 091600, which produces the UV-A-absorbing compound biopterin glucoside. The expression of a 60 kDa protein was markedly induced at 500 min after UV-A irradiation. This protein was identified by N-terminal amino acid sequence analysis as GroEL. Northern blot analysis demonstrated that GroEL synthesis was controlled by UV-A at the transcriptional level. A CIRCE element and a putative SOS consensus sequence were found upstream of the groESL operon, overlapping two putative promoter sequences. Primer extension analysis revealed that groESL transcription in UV-A-induced cells starts from the proximal promoter overlapped by the SOS consensus sequence. This indicates that an SOS response regulation is instrumental in UV-A-induced GroEL expression of Oscillatoria sp. NKBG 091600. Furthermore, this UV-A-inducible GroEL may function to upregulate biopterin glucoside biosynthesis, thereby allowing growth under UV-A irradiation. (+info)
Hypermutation in derepressed operons of Escherichia coli K12.
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This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB- reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB- reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon. (+info)
Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.
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Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria. (+info)
Molecular characterization of a flagellar export locus of Helicobacter pylori.
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Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli sigma70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, and murB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. Engineered fliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both the virB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI). (+info)
Genes for tRNA(Arg) located in the upstream region of the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157:H7.
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We found two genes for tRNA(Arg) in the region upstream of genes for Shiga-like toxin type II (SLT-II) in Escherichia coli O157:H7. The two encoded forms of tRNA(Arg) recognize rare codons in E. coli K12 but these rare codons occur in the toxin genes at high frequency. (+info)
A HilA-independent pathway to Salmonella typhimurium invasion gene transcription.
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Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined. sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype. sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis. sirC expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS. (+info)
Genetic diversity of the streptococcal competence (com) gene locus.
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The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure. (+info)
Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87.
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The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG. (+info)