The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.
(49/19133)
Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene. (+info)
BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member.
(50/19133)
A cluster of genes for the anaerobic degradation of benzoate has been described for the phototrophic bacterium Rhodopseudomonas palustris. Here we provide an initial analysis of the regulation of anaerobic benzoate degradation by examining the contributions of two regulators: a new regulator, BadR, encoded by the benzoate degradation gene cluster, and a previously described regulator, AadR, whose gene lies outside the cluster. Strains with single mutations in either badR or aadR grew slowly on benzoate but were relatively unimpaired in growth on succinate and several intermediates of benzoate degradation. A badR aadR double mutant was completely defective in anaerobic growth on benzoate. Effects of the regulators on transcriptional activation were monitored with an R. palustris strain carrying a chromosomal fusion of 'lacZ to the badE gene of the badDEFG operon. This operon encodes benzoyl-coenzyme A (benzoyl-CoA) reductase, an unusual oxygen-sensitive enzyme that catalyzes the benzene ring reduction reaction that is the rate-limiting step in anaerobic benzoate degradation. Expression of badE::'lacZ was induced 100-fold when cells grown aerobically on succinate were shifted to anaerobic growth on succinate plus benzoate. The aadR gene was required for a 20-fold increase in expression that occurred in response to anaerobiosis, and badR was responsible for a further 5-fold increase in expression that occurred in response to benzoate. Further studies with the badE::'lacZ fusion strain grown with various kinds of aromatic acids indicated that BadR probably responds to benzoyl-CoA acting as an effector molecule. Sequence information indicates that BadR is a member of the MarR family of transcriptional regulators. These studies expand the range of functions regulated by MarR family members to include anaerobic aromatic acid degradation and provide an example of a MarR-type protein that acts as a positive regulator rather than as a negative regulator, as do most MarR family members. AadR resembles the Escherichia coli Fnr regulator in sequence and contains cysteine residues that are spaced appropriately to serve in the capacity of a redox-sensing protein. (+info)
RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa.
(51/19133)
As components of a Pseudomonas aeruginosa quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second P. aeruginosa quorum-sensing system. To date, no information exists on negative regulation of the quorum-sensing cascade in P. aeruginosa. Here we describe a novel gene, rsaL, which is located downstream from lasR and transcribed antisense relative to lasR. In P. aeruginosa, overexpression of rsaL results in reduced lasB expression and decreased elastase activity. With the use of a six-His protein fusion system, we demonstrate that rsaL encodes an 11-kDa protein. Direct quantitation of PAI-1 levels in cultures and studies utilizing Escherichia coli lambda lysogens carrying lacZ transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene, lasI. RsaL's repressive effect on lasI and the associated decrease in elastase activity have important implications for the expression of all LasR-PAI-1-dependent virulence genes and the overall pathogenicity of P. aeruginosa. (+info)
Transcriptional activation of ydeA, which encodes a member of the major facilitator superfamily, interferes with arabinose accumulation and induction of the Escherichia coli arabinose PBAD promoter.
(52/19133)
Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes. (+info)
Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB.
(53/19133)
Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation. Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI). Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of orfA. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in S. meliloti and to the consensus PHO box in Escherichia coli. Introduction of a phoB mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the ackA::lacZ fusion. Mutations in either ackA alone or both pta and ackA did not affect the nodulation or nitrogen fixation phenotype of S. meliloti. (+info)
Mutational analysis of the vacA promoter provides insight into gene transcription in Helicobacter pylori.
(54/19133)
Analysis of 12 Helicobacter pylori promoters indicates the existence of a consensus -10 hexamer (TAtaaT) but little conservation of -35 sequences. In this study, mutations in either the H. pylori vacA -10 region or the -35 region resulted in decreased vacA transcription and suggested that an extended -10 motif is utilized. Thus, despite the lack of a -35 consensus sequence for H. pylori promoters, the -35 region plays a functional role in vacA transcription. (+info)
Genetic organization of the Escherichia coli K10 capsule gene cluster: identification and characterization of two conserved regions in group III capsule gene clusters encoding polysaccharide transport functions.
(55/19133)
Analysis of the Escherichia coli K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An rfaH mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS3 element and a phiR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes. (+info)
Characterization of mdcR, a regulatory gene of the malonate catabolic system in Klebsiella pneumoniae.
(56/19133)
The Klebsiella pneumoniae mdcR gene, which encodes a LysR-type regulator, was overexpressed in Escherichia coli. Purified MdcR was found to bind specifically to the control region of either the malonate decarboxylase (mdc) genes or mdcR. We have also demonstrated that MdcR is an activator of the expression of the mdc genes, whereas it represses the transcription of the putative control region of mdcR, PmdcR, indicating a negative autoregulatory control. (+info)