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PubMed1993

Differential decay of RNA of the CFA/I fimbrial operon and control of relative gene expression.

CFA/I fimbriae on human enterotoxigenic Escherichia coli are composed of the CfaB protein, the product of the second gene of the CFA/I operon. We show here that CfaB is expressed at a higher level than other proteins of the CFA/I operon. mRNA encoding the CfaB protein is much more abundant than mRNA encoding CfaA, the first protein, together with CfaB or mRNA encoding CfaA only. Only one promoter, upstream of cfaA, is present. These data indicate that a primary transcript containing cfaA and cfaB is processed into a cfaA-specific mRNA and a cfaB-specific mRNA. The cfaA mRNA is unstable, while the cfaB mRNA is stable and therefore accumulates in CFA/I-producing E. coli. The cfaB mRNA is probably stabilized by a stem-loop structure downstream of the cfaB gene. No distinct mRNA fragments could be detected encoding the other two proteins, CfaC and CfaE, of the CFA/I operon. These results indicate that cfaC- and cfaE-specific mRNAs degrade very rapidly and/or are produced in small amounts.

Bacterial Proteins↗

PubMed1993

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.

Amino Acid Sequence↗

PubMed1994

The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease.

A 3-kb DNA segment of the Bacillus caldolyticus genome including the 5' end end of the pyr cluster has been cloned and sequenced. The sequence revealed the presence of two open reading frames, pyrR and pyrP, located immediately upstream of the previously sequenced pyrB gene encoding the pyrimidine biosynthesis enzyme aspartate transcarbamoylase. The pyrR and pyrP genes encoded polypeptides with calculated molecular masses of 19.9 and 45.2 kDa, respectively. Expression of these ORFs was confirmed by analysis of plasmid-encoded polypeptides in minicells. Sequence alignment and complementation analyses identified the pyrR gene product as a uracil phosphoribosyltransferase and the pyrP gene product as a membrane-bound uracil permease. By using promoter expression vectors, a 650-bp EcoRI-HincII fragment, including the 5' end of pyrR and its upstream region, was found to contain the pyr operon promoter. The transcriptional start point was located by primer extension at a position 153 bp upstream of the pyrR translation initiation codon, 7 bp 3' of a sequence resembling a sigma A-dependent Bacillus subtilis promoter. This established the following organization of the ten cistrons within the pyr operon: promoter-pyrR-pyrP-pyrB-pyrC-pyrAa-pyrA b-orf2-pyrD-pyrF-pyrE. The nucleotide sequences of the region upstream of pyrR and of the pyrR-pyrP and pyrP-pyrB intercistronic regions indicated that the transcript may form two mutually exclusive secondary structures within each of these regions. One of these structures resembled a rho-independent transcriptional terminator. The possible implication of these structures for pyrimidine regulation of the operon is discussed.

Amino Acid Sequence↗

PubMed1994

Role of the transcriptional activator AppY in regulation of the cyx appA operon of Escherichia coli by anaerobiosis, phosphate starvation, and growth phase.

Transcriptional lacZ fusions have been used to analyze the regulation of the appA operon of Escherichia coli. The appA operon contains the genes cyxA and cyxB, coding for the putative third cytochrome oxidase, and appA, encoding acid phosphatase. The analysis showed that the cyxAB and the appA genes are cotranscribed from a potentially strong promoter, Pcyx, located immediately upstream of cyxA and that the operon in addition contains an internal promoter, PappA, contributing significantly to the transcription of the appA gene. The two promoters were both induced by starvation for Pi and by entry into stationary phase. The cyx promoter was in addition found to be activated by anaerobic growth conditions. The product of the previously identified appY gene, which when present on a high-copy-number plasmid stimulates synthesis of acid phosphatase, was shown to activate the cyx promoter. An insertion mutation in the appY gene was constructed in vitro and recombined into the chromosome. The appY mutation eliminated induction of the cyx promoter by anaerobiosis and severely reduced induction of this promoter by phosphate starvation and upon entry into stationary phase but had no effect on induction of the appA promoter. The appY mutation had no effect on survival in stationary phase, nor did it have any effect on growth rate or yield under aerobic or anaerobic conditions. The possibility that AppY is a third global regulator of energy metabolism genes is discussed.

Anaerobiosis↗

PubMed1994

Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport.

Expression of the Bacillus subtilis hut operon is induced by histidine and subject to regulation by carbon catabolite repression and amino acid repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify the cis-acting sites required for histidine induction and amino acid repression. Histidine induction was found to be primarily mediated by transcriptional antitermination at a palindromic sequence located immediately downstream of the first structural gene in the hut operon, hutP. High levels of histidine induction were observed only in hut-lacZ fusions which contained this palindromic sequence. The hutC1 mutation, which results in constitutive expression of the hut operon, was sequenced and found to contain a GC to TA transversion located within the stem-loop structure. Transcription of hut DNA in vitro revealed that the palindromic structure functions as a transcriptional terminator with wild-type hut DNA but not with hutC1 DNA. Two sites were found to be involved in amino acid repression of hut expression: (i) an operator, hutOA, which lies downstream of the hut promoter, and (ii) the hut terminator. The rate of [14C]histidine uptake in amino acid-grown cells was sixfold lower than that seen in cells grown without amino acids. Thus, inhibition of histidine transport in amino acid-grown cells indirectly regulates hut expression by interfering with histidine induction at the hut terminator.

Bacillus subtilis↗

PubMed1994

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.

Base Sequence↗

PubMed1994

The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon.

Synthesis of glutamate can be limited in bacterial strains carrying mutations to loss of function of glutamate synthase (2-oxoglutarate:glutamine aminotransferase) by using low concentrations of NH4+ in the growth medium. By using such gltB/D mutant strains of Salmonella typhimurium, we demonstrated that: (i) a large glutamate pool, previously observed to correlate with growth at high external osmolality, is actually required for optimal growth under these conditions; (ii) the osmoprotectant glycine betaine (N,N,N-trimethylglycine) apparently cannot substitute for glutamate; and (iii) accumulation of glutamate is not necessary for high levels of induction of the proU operon in vivo. Expression of the proU operon, which encodes a transport system for the osmoprotectants proline and glycine betaine, is induced > 100-fold in the wild-type strain under conditions of high external osmolality. Ramirez et al. (R. M. Ramirez, W. S. Prince, E. Bremer, and M. Villarejo, Proc. Natl. Acad. Sci. USA 86:1153-1157, 1989) observed and we confirmed that in vitro expression of the lacZ gene from the wild-type proU promoter is stimulated by 0.2 to 0.3 M K glutamate. However, we observed a very similar stimulation for lacZ expressed from the lacUV5 promoter and from the proU promoter when an important negative regulatory element downstream of this promoter (the silencer) was deleted. Since the lacUV5 promoter is not osmotically regulated in vivo and osmotic regulation of the proU promoter is largely lost as a result of deletion of the silencer, we conclude that stimulation of proU expression by K glutamate in vitro is not a specific osmoregulatory response but probably a manifestation of the optimization of in vitro transcription-translation at high concentrations of this solute. Our in vitro and in vivo results demonstrate that glutamate is not an obligatory component of the transcriptional regulation of the proU operon.

Amino Acid Transport Systems↗

PubMed1994

Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation.

The cynTSX operon enables Escherichia coli K-12 to degrade and use cyanate as a sole nitrogen source. The promoter of this operon is positively regulated by cyanate and the CynR protein. CynR, a member of the LysR family of regulatory proteins, binds specifically to a 136-bp DNA fragment containing both the cynR and the cynTSX promoters. In this study, we report the results of DNase I digestion studies showing that CynR protects a 60-bp region on the cynR coding strand and a 56-bp sequence on the cynTSX coding strand. CynR binding was not affected by cyanate or its structural homolog azide, a gratuitous inducer of the operon. However, CynR-induced bending of two different DNA fragments was detected. The amount of bending was decreased by cyanate.

Azides↗

PubMed1994

Genetic and molecular characterization of the Escherichia coli secD operon and its products.

The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.

Amino Acid Sequence↗

PubMed1994

Expansion of bacteriocin activity and host range upon complementation of two peptides encoded within the lactacin F operon.

Lactacin F is a membrane-active bacteriocin produced by Lactobacillus johnsonii VPI11088 (Laf+). The genetic determinants encoding lactacin F are organized in a 1-kb polycistronic operon composed of a promoter (P(laf)), three genes (lafA, lafX, and ORFZ), and a functional rho-independent transcription terminator. Two Laf- derivatives of VPI11088, designated NCK64 and NCK65, were characterized. NCK64 contained a frameshift mutation in the lafA gene causing premature termination of translation. NCK65 harbored a 10-kb chromosomal deletion covering the laf operon. When the lafA gene was cloned independently and expressed in NCK65, bacteriocin activity was limited to L. helveticus 87, only one of the six known lactacin F-sensitive (Lafs) indicators. When lafX was introduced into NCK65, no bacteriocin activity against any of the sensitive strains was detected. Genetic combination of lafA and lafX, in cis or in trans, restored bacteriocin activity against all Lafs indicators. When two NCK65 clones containing either lafA or lafX were plated slightly apart on agar plates, fully active lactacin F was present in the intervening area where the two excreted gene products, LafA and LafX, diffused together. The genetic analysis revealed that the interaction of two bacteriocinogenic peptides encoded within the laf operon is likely to participate in the formation of poration complexes in the membranes of susceptible bacteria.

Amino Acid Sequence↗

PubMed1994

Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis.

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993).

Amino Acid Sequence↗

PubMed1995

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2.

The par region of the broad-host-range, IncP alpha plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the delta parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac-) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra+ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable deltapar derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.

Bacterial Proteins↗

PubMed1995

Point mutations in the leader boxA of a plasmid-encoded Escherichia coli rrnB operon cause defective antitermination in vivo.

We have introduced point mutations into the leader boxA of a plasmid-encoded Escherichia coli rrnB operon to study the in vivo role of this regulatory element in the natural context of rRNA synthesis. The same mutations were previously shown to cause severe antitermination defects in vitro and in the context of a reporter gene assay. The plasmid-encoded rrnB mutant constructs studied here also contained point mutations in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rrn operons by primer extension analysis. Point mutations in boxA reduced the fraction of plasmid-derived rRNA in the cell from 75% to about 50%. The reduction was similar for both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA. The boxA mutations do not affect the amount of transcription initiation, suggesting that a suboptimal leader boxA causes premature transcription termination at an early stage of transcription. Our results are consistent with a role for antitermination in the completion of full-length rrn transcripts but give no indications of posttranscriptional boxA functions.

Base Sequence↗

PubMed1995

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

Aerobiosis↗

PubMed1995

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

Amino Acid Sequence↗

PubMed1995

Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis.

The divergently transcribed nasA gene and nasB operon are required for nitrate and nitrite assimilation in Bacillus subtilis. The beta-galactosidase activity of transcriptional lacZ fusions from the nasA and nasB promoters was high when cells were grown in minimal glucose medium containing poor nitrogen sources such as nitrate, proline, or glutamate. The expression was very low when ammonium or glutamine was used as the sole nitrogen source. The repression of the genes during growth on good sources of nitrogen required wild-type glutamine synthetase (GlnA), but not GlnR, the repressor of the glnRA operon. Primer extension analysis showed that the -10 region of each promoter resembles those of sigma A-recognized promoters. Between the divergently oriented nasA and nasB promoters is a region of dyad symmetry. Mutational analysis led to the conclusion that this sequence is required in cis for the activation of both nasA and nasB. The derepression of these genes in a glnA mutant also required this sequence. These results suggest that an unidentified transcriptional activator and glutamine synthetase function in the regulation of nasA and the nasB operon.

Bacillus subtilis↗

PubMed1995

Regulation of the molybdate transport operon, modABCD, of Escherichia coli in response to molybdate availability.

The mod (chlD) locus at 17 min on the Escherichia coli chromosome encodes a high-affinity molybdate uptake system. To further investigate the structure and regulation of these genes, the DNA region upstream of the previously identified modBC (chlJD) genes was cloned and sequenced. A single open reading frame, designated modA, was identified and appears to encode a periplasmic binding protein for the molybdate uptake system. To determine how the mod genes are regulated in response to molybdate, nitrate, and oxygen, we constructed a series of mod-lacZ operon fusions to the upstream region and introduced them in single copy onto the E. coli chromosome. Whereas molybdate limitation resulted in elevated mod-lacZ expression, neither oxygen nor nitrate had any significant effect on gene expression. A regulatory motif, CATAA, located at the modA promoter was identified and shown to be required for molybdate-dependent control of the modABCD operon. Mutations within this sequence resulted in nearly complete derepression of gene expression and suggest that transcription of the operon is mediated by a molybdenum-responsive regulatory protein.

Alleles↗

PubMed1995

The ars operon of Escherichia coli confers arsenical and antimonial resistance.

The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.

Adenosine Triphosphatases↗