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J Gowrishankar

Publications and source records attributed to J Gowrishankar.

At least 37 records · Page 2Linked to original sources
PubMed1997

Evidence for transcription attenuation rendering cryptic a sigmaS-dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium.

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the sigmaS- and sigma70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by sigmaS RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers sigmaS-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the sigmaS-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site.

Artificial Gene Fusion↗
PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗
PubMed1996

How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model.

The proU operon in enterobacteria encodes a binding-protein-dependent transporter for the active uptake of glycine betaine and L-proline, and serves an adaptive role during growth of cells in hyperosmolar environments. Transcription of proU is induced 400-fold under these conditions, but the underlying signal transduction mechanisms are incompletely understood. Increased DNA supercoiling and activation by potassium glutamate have each been proposed in alternative models as mediators of proU osmoresponsivity. We review here the available experimental data on proU regulation, and in particular the roles for DNA supercoiling, potassium glutamate, histone-like proteins of the bacterial nucleoid, and alternative sigma factors of RNA polymerase in such regulation. We also propose a new unifying model, in which the pronounced osmotic regulation of proU expression is achieved through the additive effects of at least three separate mechanisms, each comprised of a cis element [two promoters P1 and P2, and negative-regulatory-element (NRE) downstream of both promoters] and distinct trans-acting factors that interact with it: stationary-phase sigma factor RpoS with P1, nucleoid proteins HU and IHF with P2, and nucleoid protein H-NS with the NRE. In this model, potassium glutamate may activate proU expression through each of the three mechanisms whereas DNA supercoiling has a very limited role, if any, in the osmotic induction of proU transcription. We also suggest that proU may be a virulence gene in the pathogenic enterobacteria.

Amino Acid Transport Systems↗
PubMed1996

Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli.

An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity. One (ossB) was an insertion mutation in the gltBD operon, which encodes the enzyme glutamate synthase (GOGAT), involved in ammonia assimilation and L-glutamate biosynthesis. The other (ossA) was in the fnr gene, encoding the regulator protein FNR for anaerobic gene expression. Several missense or deletion mutations in fnr and gltBD behaved like ossA and ossB, respectively, in conferring osmosensitivity. A mutation affecting the DNA-binding domain of FNR was recessive to fnr+ with respect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration. Our results may most simply be interpreted as suggesting the requirement for monomeric FNR during aerobic growth of E. coli in high-osmolarity media, presumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase [GDH]), but the mechanism of FNR action is not known. We also found that the spoT gene (encoding guanosine 3',5'-bispyrophosphate [ppGpp] synthetase II/ppGpp-3' pyrophosphohydrolase), in multiple copies, overcomes the defect in NH4+ assimilation associated with GOGAT deficiency and thereby suppresses osmosensitivity in gltBD fnr strains. Enhancement of GDH activity in these derivatives appears to be responsible for the observed suppression. Its likely physiological relevance was established by the demonstration that growth of gltBD mutants (that are haploid for spoT+) on moderately low [NH4+] was restored with the use of C sources poorer than glucose in the medium. Our results raise the possibility that SpoT-mediated accumulation of ppGpp during C-limited growth leads to GDH activation and that the latter enzyme plays an important role in N assimilation in situ hitherto unrecognized from studies on laboratory-grown cultures.

Aerobiosis↗
PubMed1996

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli.

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.

Amino Acid Transport Systems↗
PubMed1995

A new phenotype for sbcB mutations in Escherichia coli: RecA-dependent increase in plasmid-borne gene expression.

A new chromosomal mutation (cpeA), that causes increased expression of plasmid-borne genes in Escherichia coli, was identified and mapped to the sbcB locus. The effect of the mutation on plasmid transcription was non-specific with respect to the various promoters that were studied, but was more pronounced for an IncW low-copy-number plasmid than for ColE1- or p15A-based, high-copy-number plasmids. The mutant phenotype was observed even in recB+C+D+ strains, but not in recA mutants. The increased-expression phenotype was also observed in sbcB15 but not in xonA1 (another sbcB allele) mutants, suggesting that the expression of this phenotype is mediated by genes of the so-called RecF pathway family. Consistent with this interpretation was the observation that the cpeA mutant phenotype was less pronounced in recF, recJ and recO mutants. The increased-expression phenotype was also correlated with increased recovery of plasmid DNA from the cpeA/sbcB mutant strains, but there was no evidence for the occurrence of linear plasmid multimers in these strains.

Amino Acid Transport Systems↗
PubMed1994

Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli.

Transcription of the proU operon of Escherichia coli is induced several hundred-fold upon growth at elevated osmolarity, but the underlying mechanisms are incompletely understood. Three cis elements appear to act additively to mediate proU osmoresponsivity: (i) sequences around a promoter, P1, which is situated 250 bp upstream of the first structural gene proV; (ii) sequences around another (sigma 70-dependent) promoter, P2, which is situated 60 bp upstream of proV; and (iii) a negative regulatory element present within the proV coding region. These three cis elements are designated, respectively, P1R, P2R, and NRE. trans-acting mutants with partially derepressed proU expression have been obtained earlier, and a vast majority of the mutations affect the gene encoding the nucleoid protein HNS. In this study we employed a selection for trans-acting mutants with reduced proU+ expression, and we obtained a derivative that had suffered mutations in two separate loci designated dpeA and dpeB. The dpeB mutation caused a marked reduction in promoter P1 expression and was allelic to rpoS, the structural gene for the stationary-phase-specific sigma factor of RNA polymerase. Expression from P1 was markedly induced, in an RpoS-dependent manner, in stationary-phase cultures. In contrast to the behavior of the isolated P1 promoter, transcription from a construct carrying the entire proU cis-regulatory region (P1R plus P2R plus NRE) was not significantly affected by either growth phase or RpoS. The dpeA locus was allelic to hupB, which along with hupA encodes the nucleoid protein HU. hupA hupB double mutants exhibited a pronounced reduction in proU osmotic inducibility. HU appears to affect proU regulation through the P2R mechanism, whereas the effect of HNS is mediated through the NRE.

Alleles↗
PubMed1993

Regulation of kdp operon expression in Escherichia coli: evidence against turgor as signal for transcriptional control.

Kdp, an inducible high-affinity K+ transporter in Escherichia coli, is encoded by genes of the kdpABC operon, and its expression is regulated by the products of kdpD and kdpE. Loss of cell turgor has been proposed to be the signal which induces kdp expression (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464-468, 1981). We reexamined kdp expression during steady-state growth under a variety of conditions and were able to confirm earlier observations which had indicated that it is primarily affected by the concentration of K+ in the medium and by mutations in genes encoding various K+ transporters in E. coli. Changes in pH of the culture also altered kdp expression. In all of these cases, an increase in [K+] of the medium repressed the operon. Several ionic solutes induced steady-state kdp expression (but to differing extents), whereas nonionic solutes had no effect, indicating that kdp expression is not determined by osmolarity of the growth medium. kdp expression during steady-state growth was shown also to be unaffected by the accumulation of other intracellular compatible solutes such as trehalose or glycine betaine, which would be expected to restore cell turgor during growth in high-osmolarity media. Two mutants that are defective in perception of the signal regulating kdp were isolated, and the mutation in each of them was mapped to the kdpDE regulatory locus. Analysis of kdp expression in one of these mutants provided additional evidence against the turgor regulation model. On the basis of these data, we discuss alternative candidates that might serve as the signal for control of kdp operon transcription.

Adenosine Triphosphatases↗
PubMed1991

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene.

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.

Base Sequence↗
PubMed1990

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.

Blotting, Southern↗
PubMed1989

Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus.

The proU locus in Escherichia coli encodes an osmotically inducible transport system for two substrates, glycine betaine and L-proline, whose intracellular accumulation represents an important component in the physiology of osmoregulation. Several osmoresponsive proU::lac mutants were isolated and tested for complementation with plasmids carrying different functional regions of proU. Three classes of mutations were identified which were physically mapped to distinct regions of DNA from this locus. Tn1000-insertion mutagenesis of cloned proU DNA also yielded three phenotypic classes of mutations whose physical distribution approximately corresponded with those of the chromosomal mutants above. Three proteins, of Mr 44,000, 35,000, and 33,000, were shown to be products of proU, and the last of these was localized to the periplasmic space. The data indicate that proU is an operon with three genes, designated in order proV, proW, and proX, encoding respectively the gene products above. All three genes were shown to be necessary for exhibition of the proU-mediated osmoprotective effects of both glycine betaine and L-proline in E. coli.

Bacterial Proteins↗
PubMed1989

Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli.

The sequence of 4,362 nucleotides encompassing the proU operon of Escherichia coli was determined. Three open reading frames were identified whose orientation, order, location, and sizes were in close accord with genetic evidence for three cistrons (proV, proW, and proX) in this operon. Similarities in primary structure were observed between (i) the deduced sequence of ProV with membrane-associated components of other binding-protein-dependent transport systems, in the nucleotide-binding region of each of the latter proteins, and (ii) that of ProW with integral membrane components of the transport systems above. The DNA sequence data also conclusively established that ProX represents the periplasmic glycine betaine-binding protein. Two copies of repetitive extragenic palindromic sequences were identified beyond the 3' end of the proX gene. The primer extension technique was used to identify the 5' ends of proU mRNA species that are present in cells grown at high osmolarity; the results suggest that at least some of the osmotically induced proU transcripts have a long leader region, extending as much as 250 base pairs upstream of the proV gene. Evidence was also obtained for the existence of a sequence-directed bend in DNA in the upstream regulatory region of the proU operon.

Amino Acid Sequence↗
PubMed1986

proP-mediated proline transport also plays a role in Escherichia coli osmoregulation.

The growth of Escherichia coli strains in media having elevated osmolarity was promoted in the presence of low concentrations of the L-proline analog 5-hydroxy-L-pipecolic acid. The osmoprotective ability of this compound was correlated with the presence in these strains of a functional proP+ gene. The results suggest that the proP-mediated transport of L-proline (in addition to that by proU) is important in osmoregulation. proP::lac operon fusions were used to demonstrate that this gene shows limited induction of expression upon growth in media having elevated osmolarity and that it is transcribed clockwise on the chromosome.

Biological Transport↗
PubMed1986

Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance.

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU.

Betaine↗
PubMed1985

Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation.

Mu d1(Ap lac)-generated operon fusions were used in the identification of genes in Escherichia coli whose transcriptional expression is altered by changes in the osmolarity of the growth medium. One such osmoresponsive gene, designated osrA, was induced 400-fold when the osmolarity of the medium was increased with the addition of either ionic or neutral impermeable solutes but was not induced with glycerol, which is freely permeable across the cell membrane. osrA was mapped to 57.5 min and was shown to be transcribed clockwise on the E. coli chromosome. The ability of small concentrations of L-proline to promote the growth of E. coli in high-osmolar medium was shown to have been specifically lost in osrA mutants; other lines of evidence were also obtained to support the notion that osrA codes for an osmoresponsive L-proline transport system and is homologus to proU in Salmonella typhimurium. A second osmoresponsive operon identified was kdp, which codes for an inducible K+-transport system in E. coli. kdp expression was elevated 12-fold when the osmolarity of the growth medium was increased with the addition of impermeable ionic solutes but not neutral solutes; furthermore, osmoresponsivity of kdp expression was demonstrable only in K+-limiting media. kdp mutants were able to grow normally in high-osmolar media, but strains defective in both kdp and trkA (a gene for a second major K+-transport system) displayed an osmosensitive phenotype. The results suggest that transport systems for L-proline and K+, specified by osrA (proU) and kdp, respectively, play independent and important roles in osmoregulation in E. coli. A third osmoresponsive gene that was identified was lamB, which codes for an outer membrane protein for maltodextrin transport and lambda phage adsorption; its expression was reduced fourfold with increase in the osmolarity of the growth medium.

Biological Transport↗
PubMed1982

Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.

Bacteriophage lambda↗
PubMed1982

Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon.

Bacteriophage lambda ppheA-lac was used to obtain strains of Escherichia coli K-12 in which pheA and lacZ are each transcribed from a separate pheA promoter. Mutants in which both beta-galactosidase and chorismate mutase P-prephenate dehydratase (the pheA gene product) were derepressed were isolated, and a transacting gene (pheR) was identified. pheR was mapped at min 93 on the E. coli chromosome; pheR mutants acquired the wild-type phenotype when either F117 (which covers the 93-min region) or F116 (which covers min 59 to 65) was introduced into the cell. A rifampin resistance mutation, rpoB366, was found to derepress transcription of the pheA operon. pheR and rpoB366 affected two different systems for the phenylalanine-mediated control of pheA. A mutation in miaA (trpX), a gene known to be involved in attenuation in the tryptophan operon, was also shown to increase transcription of the pheA gene.

Chromosome Mapping↗