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PubMed1995

Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli.

Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. phi (hyc'-'lacZ+) mod double mutants produced beta-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate, mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod+ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of beta-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered phi (modA'-'lacZ+) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport system in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis.

Biological Transport↗

PubMed1995

Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis.

The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions. The bglP gene encodes an aryl-beta-glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase. Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism. Distinct deletions of the promoter region were created to determine cis-acting sites for regulation. An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon. The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B. subtilis. Catabolite repression is abolished in both ccpA and ptsH1 mutants. The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP. This structure is very similar to that of transcriptional terminators which regulate the induction of the B. subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon. The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process. Expression of bglP is under the negative control of its own gene product. The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression. Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression.

Arbutin↗

PubMed1995

Adjacent and divergently oriented operons under the control of the sporulation regulatory protein GerE in Bacillus subtilis.

The DNA-binding protein GerE is the latest-acting regulatory protein in the mother cell line of gene expression during sporulation in Bacillus subtilis. GerE directs the transcription of several genes that encode structural components of the protein coat that encases the mature spore. We report on the identification and characterization of a cluster of additional genes whose transcription is dependent on GerE. These genes, which are located in the replication terminus region of the chromosome (181 degrees on the genetic map), are arranged in adjacent and divergently oriented operons called cgeAB and cgeCDE, which consist of two and at least three genes, respectively. CgeD, the product of the second member of the cgeCDE operon, is strikingly similar to the product of a B. subtilis gene (ipa-63d) of unknown function and is similar at its amino terminus to certain glycosyl transferases involved in polysaccharide biosynthesis. Strains with mutations in the cgeAB and cgeCDE operons produce spores with altered surface properties, on which basis we propose that proteins encoded by these operons influence maturation of the outermost layer of the spore, perhaps by glycosylation of coat proteins at the spore surface.

Amino Acid Sequence↗

PubMed1995

trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon.

Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within trp leader RNA determines whether transcription will terminate in the leader region of the operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the trpE ribosome-binding site. To assess the relative importance of these postulated events in trp operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range. The roles played by different trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.

Bacillus subtilis↗

PubMed1995

Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli.

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Elevated levels of tryptophan induce transcription antitermination at one or more Rho factor-dependent termination sites in the leader region of the operon. Induction requires translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. In the present paper, we show that two bacterial species that lack tryptophanase activity, Enterobacter aerogenes and Salmonella typhimurium, allow tryptophanase induction and tna operon regulation when they carry a plasmid containing the E. coli tna operon. The role of tnaC in induction was examined by introducing mutations in a 24-nucleotide segment of tnaC of E. coli surrounding and including the crucial Trp codon 12. Some mutations resulted in a noninducible phenotype; these mostly introduced nonconservative amino acid substitutions in TnaC. Other mutations had little or no effect; these generally were in third positions of codons or introduced conservative amino acid replacements. A tryptophan-inserting, UGA-reading glutamine suppressor tRNA was observed to restore partial regulation when Trp codon 12 of tnaC was changed to UGA. Stop codons introduced downstream of Trp codon 12 in all three reading frames established that induction requires translation in the natural tnaC reading frame. Our findings suggest that the TnaC leader peptide acts in cis to prevent Rho-dependent termination.

Amino Acid Sequence↗

PubMed1995

Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida.

The bkd operon of Pseudomonas putida consists of the structural genes encoding the components of the inducible branched-chain ketoacid dehydrogenase. BkdR, a positive regulator of the bkd operon and a homolog of Lrp of Escherichia coli is encoded by a structural gene adjacent to, and divergently transcribed from, the bkd operon of P. putida. BkdR was purified from E. coli containing bkdR cloned into pCYTEXP1, an expression vector. The molecular weight of BkdR obtained by gel filtration indicates that BkdR is a tetramer, and the abundance of BkdR in P. putida was estimated to be about 25 to 40 copies of the tetramer per cell. BkdR bound specifically to the region between bkdR and bkdA1, the latter being the first gene of the bkd operon. One BkdR-DNA complex was observed in gel mobility shift patterns. Approximately 100 bp was protected from the action of DNase I by BkdR, and the addition of L-branched-chain amino acids enhanced the appearance of hypersensitive sites in the protected region. There are four potential BkdR-DNA binding sequences in this region based on similarity to Lrp-binding consensus sequences. Like many other transcriptional activators, BkdR regulates expression of its structural gene. DNAs from several gram-negative bacteria hybridized to a probe containing bkdR, indicating the presence of bkdR-like genes in these organisms.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1995

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.

Amino Acid Sequence↗

PubMed1996

mgpS, a complex regulatory locus involved in the transcriptional control of the puc and puf operons in Rhodobacter sphaeroides 2.4.1.

A new method has been developed in order to select mutants showing decreased puc operon transcription in Rhodobacter sphaeroides 2.4.1. A transcriptional fusion of a promoterless fragment derived from the sacB gene, encoding the levansucrase from Bacillus subtilis, to the upstream regulatory region of the puc operon has been constructed. With appropriate levels of exogenous sucrose, survivors of a sucrose killing challenge have been isolated. Subsequent analysis revealed the presence of both cis- and trans-acting "down" mutations in relation to puc operon expression. One of the trans-acting regulatory mutations was chosen for further study. The original mutation showed less than 2% of the level of puc operon transcription compared with the wild type under aerobic conditions and an 86% reduction under dark dimethyl sulfoxide conditions. This mutation can be complemented by a 3.9-kb BamHI DNA fragment derived from a cosmid contained within a genomic cosmid bank. DNA sequence analysis of this fragment revealed the presence of a 2.8-kb open reading frame, designated mgpS, which would encode a 930-amino-acid protein. The N-terminal portion of the putative protein product presents homologies to proteins of the RNA helicase family. Disruption of the chromosomal mgpS resulted in decreased transcription of both puc and puf, while the presence of mgpS in multicopy in the wild type, 2.4.1., increased puc expression by a factor of 2 under aerobic conditions. Structural analysis of the mgpS locus revealed that expression of mgpS was likely to be complex. A smaller protein containing the 472 C-terminal amino acids of MgpS is able to act by itself as an activator of puc transcription and is expressed independently of the large open reading frame in which it is contained.

Aerobiosis↗

PubMed1996

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

We studied capsule-defective (Cap-) serogroup B meningococcal mutants created through Tn916 or omega-fragment mutagenesis. The Cap- phenotypes were the results of insertions in three of four linked genes (synX, synC, and synD) involved in CMP-N-acetylneuraminic acid and polysialic acid capsule biosynthesis, and in ctrA the first of four linked genes involved in capsule membrane transport. Mutations in the CMP-N-acetylneuraminic acid biosynthesis genes synX and synC caused defects in lipooligosaccharide sialylation but not mutations in the putative (alpha2 -> 8)-linked polysialyltransferase (synD) or in ctrA. Reverse transcriptase PCR studies indicated that the four biosynthesis genes (synX to -D) and the capsule transport genes (ctr to -D) were separately transcribed as operons. The operons were separated by a 134-bp intergenic region. Primer extension of synX and ctrA demonstrated that transcription of the operons was divergently initiated from adjacent start sites present in the intergenic region. Both transcriptional start sites were preceded by a perfect -10 Pribnow promoter binding region. The synX to -D, but not the ctrA to -D, transcriptional start site was preceded by a sequence bearing strong homology to the consensus sigma 70 -35 promoter binding sequence. Both promoters showed transcriptional activity when cloned behind a lacZ reporter gene in Escherichia coli. Our results confirm the intrinsic relationship between polysialic acid capsule biosynthesis and lipooligosaccharide sialylation pathways in group B Neisseria meningitidis. Our study also suggests that the intergenic region separating the synX to -D and ctrA to -D operons is an important control point for the regulation of group B capsule expression through coordinated transcriptional regulation of the synX to -D and drA to -D promoters.

Bacterial Capsules↗

PubMed1996

Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum.

A locus encoding polypeptides associated with flagellar structure and function was identified, sequenced, and characterized in Treponema phagedenis and Treponema pallidum. This locus includes homologs of the FlgD, FlgE, MotA, MOB, FliL, and FliM polypeptides found in Salmonella typhimurium and Bacillus subtilis. These polypeptides are extensively conserved between the two treponemes. Several additional polypeptides or unknown function, including Tapl, located upstream of FlgD, and ORF4, located between FlgE and MotA, were also identified. Transcription analysis using RNA PCR indicated that these genes are likely transcribed as part of a single operon and comprise the 5' region of the treponemal fla operon. Primer extension analysis identified a putative promoter, preceding T. phagedenis tap1 in a region of divergent transcription. Pfla resembles the class II or class III motility-related promoters of S. typhimurium. FlgE and Tap1 were further characterized. Western blotting (immunoblotting) indicated that T. pallidum FlgE exhibited an unusual polypeptide ladder that was similar but not identical to that of T. phagedenis. Triton X-114 phase partitioning of T. phagedenis cells coupled with Western blotting revealed that Tap1 was located in the aqueous phase. Computer analysis indicated that Tap1 had no significant membrane spanning regions, suggesting that it resides primarily in the cytoplasm. The organization and expression of this operon are similar in both treponemes but different from those of previously described motility-related operons. These results indicate that despite extensive amino acid sequence conservation, the expression of spirochete flagellar polypeptides is different from that in other bacteria.

Amino Acid Sequence↗

PubMed1996

Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

Amino Acid Sequence↗

PubMed1996

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Bacillus subtilis↗

PubMed1996

Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro.

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).

Bacillus subtilis↗

PubMed1996

Lrp is a direct repressor of the dad operon in Escherichia coli.

Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presence of D- or L-alanine. Alanine is thought to act as an inducer to block the action of a postulated repressor. This operon is also believed to be regulated by catabolite repression. We have used in vivo and in vitro experiments that show that the dad repressor is the leucine-responsive regulatory protein (Lrp). dad expression in a dad-lacZ operon fusion strain was increased four- to sevenfold when cells were grown in minimal medium containing alanine or leucine. A strain lacking Lrp had high-level constitutive dad expression. Gel retardation and footprinting studies revealed that Lrp binds in vitro to multiple sites over a large area in the dad promoter region. This binding was reduced by alanine or leucine. In vitro transcription assays, using a plasmid template and primer extension analysis, identified three major dad transcripts (Tr1, Tr2, and Tr3). The formation of these transcripts was differentially regulated by cyclic AMP-cyclic AMP receptor protein complex, and each was strongly repressed by Lrp. Alanine or leucine completely (for Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition. Site-directed mutagenesis of an Lrp binding site strongly reduced Lrp binding and prevented Lrp repression of dad transcription in vivo and in vitro. Taken together, these results strongly suggest that Lrp and alanine or leucine act directly to repress and induce, respectively, transcription of the dad operon.

Bacterial Proteins↗

PubMed1996

Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE.

The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa), and the second genes codes for a 490-amino-acid protein. ModF (54 kDa). The role of ModF has not yet been determined; however, mutations in modE depressed modABCD transcription even in the presence of molybdate, suggesting that ModE is a repressor. ModE, in the presence of 1 mM molybdate, repressed the production of plasmid-encoded ModA and ModB' proteins in an in vitro transcription-translation system. DNA mobility shift experiments confirmed that ModE binds to an oligonucleotide derived from the operator region of the modABCD operon. Further experimentation indicated that ModE binding to target DNA minimally requires an 8-bp inverted-repeat sequence, TAAC GITA. A highly conserved amino acid sequence, TSARNOXXG (amino acids 125 to 133), was identified in ModE and homologs from Azotobacter vinelandii, Haemophilus influenzae, Rhodobacter capsulatus, and Clostridium pasterianum. Mutants with mutations in either T or G of this amino acid sequence were isolated as "superrepressor" mutants. These mutant proteins repressed modABCD transcription even in the absence of molybdate, which implies that this stretch of amino acids is essential for the binding of molybdate by the ModE protein. These results show that molybdate transport in E. coli is regulated by ModE, which acts as a repressor when bound to molybdate.

Amino Acid Sequence↗

PubMed1996

Modulation of luminescence operon expression by N-octanoyl-L-homoserine lactone in ainS mutants of Vibrio fischeri.

Population density-dependent expression of luminescence in Vibrio fischeri is controlled by the autoinducer N-3-oxohexanoyl-L-homoserine lactone (autoinducer 1 [AI-1]), which via LuxR activates transcription of the lux operon (luxICDABEG, encoding the putative autoinducer synthase [LuxI] and the luminescence enzymes). We recently identified a novel V. fischeri locus, ainS, necessary for the synthesis of a second autoinducer, N-octanoyl-L-homoserine lactone (AI-2), which via LuxR can activate lux operon transcription in the absence of AI-1. To define the regulatory role of AI-2, a luxI ainS double mutant was constructed; in contrast to the parental strain and a luxI mutant, the luxI ainS mutant exhibited no induction of luminescence and produced no detectable luminescence autoinducer, demonstrating that V. fischeri makes no luminescence autoinducers other than those whose synthesis is directed by luxI and ainS. A mutant defective only in ainS exhibited accelerated luminescence induction compared with that of the parental strain, indicating that AI-2 functions in V. fischeri to delay luminescence induction. Consistent with that observation, the exogenous addition of AI-2 inhibited induction in a dose-dependent manner in V. fischeri and Escherichia coli carrying the lux genes. AI-2 did not mediate luxR negative autoregulation, alone or in the presence of AI-1, and inhibited luminescence induction in E. coli regardless of whether luxR was under the control of its native promoter or a foreign one. Increasing amounts of AI-1 overcame the inhibitory effect of AI-2, and equal activation of luminescence required 25- to 45-fold-more AI-2 than AI-1. We conclude that AI-2 inhibits lux operon transcription. The data are consistent with a model in which AI-2 competitively inhibits the association of AI-1 with LuxR, forming a complex with LuxR which has a markedly lower lux operon-inducing specific activity than that of AI-1-LuxR. AI-2 apparently functions in V. fischeri to suppress or delay induction at low and intermediate population densities.

Bacterial Proteins↗

PubMed1996

Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE.

In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones. The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. This suggests a role for HrcA in negative regulation of heat shock gene expression. We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. C. crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

Amino Acid Sequence↗

PubMed1997

Mutations that render the promoter of the histidine operon of Salmonella typhimurium insensitive to nutrient-rich medium repression and amino acid downshift.

The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.

Amino Acids↗