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PubMed1990

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli. Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon. Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene. The slt-IIv promoter was demonstrated to be identical to the slt-II promoter. The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters. The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter. In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E. coli strains with wild-type or mutant ferric uptake regulation (fur) alleles. Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene. A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB. These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron. Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.

Animals↗

PubMed1990

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions.

Five of the genes for the biosynthesis of isoleucine and valine form the ilvGMEDA operon of Escherichia coli K-12. Expression of the operon responds to changes in the availability of isoleucine, leucine, and valine (ILV). Addition of an excess of all three amino acids results in reduced expression of the operon, whereas limitation for one of the three amino acids causes an increase in expression. The operon is preceded by a leader-attenuator which clearly regulates the increased expression that occurs due to reduced aminoacylation of tRNA. To assess the factors that result in the reduced expression of this operon upon the addition of ILV, a series of plasmids were constructed in which the ilv regulatory region was fused to galK. In response to addition of the amino acids, expression of the galK gene fused to the leader-attenuator decreased five- to sevenfold, instead of the twofold observed for the chromosomal operon. A deletion analysis with these plasmids indicated that the ILV-specific decrease in expression required an intact leader-attenuator but not ilvGp2 or the DNA that precedes this promoter. This conclusion was supported by both S1 nuclease analysis of transcription initiation and determination of galK mRNA levels by RNA-RNA hybridization.

Cloning, Molecular↗

PubMed1990

Repression and catabolite repression of the lactose operon of Staphylococcus aureus.

The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.

Amino Acid Sequence↗

PubMed1990

The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators.

The expression of the Bacillus subtilis sacPA operon is induced by sucrose. A DNA fragment containing the upstream region of this operon was cloned. This fragment contains a promoter from which the operon is expressed. This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B. subtilis sacB gene. Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene. A similar motif is also present in the bgl operon of Escherichia coli. The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon. DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced. The sacT gene product is very similar to the B. subtilis sacY and to the E. coli bglG gene products. The constitutive sacT30 mutation was identified. It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY. These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.

Amino Acid Sequence↗

PubMed1990

Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons.

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.

Amino Acid Sequence↗

PubMed1990

The ilvIH operon of Escherichia coli is positively regulated.

The ilvIH operon of Escherichia coli (located near min 2) encodes acetohydroxyacid synthase III, an isozyme involved in branched-chain amino acid biosynthesis. A strain with lacZ fused to the ilvIH promoter was constructed. Transposon Tn10 was introduced into this strain, and tetracycline-resistant derivatives were screened for those in which ilvIH promoter expression was markedly reduced. In one such derivative, strain CV1008, beta-galactosidase expression was reduced more than 30-fold. The transposon giving rise to this phenotype inserted near min 20 on the E. coli chromosome. Extract from a wild-type strain contains a protein, the IHB protein, that binds to two sites upstream of the ilvIH promoter (E. Ricca, D. A. Aker, and J. M. Calvo, J. Bacteriol. 171:1658-1664, 1989). Extract from strain CV1008 lacks IHB-binding activity. These results indicate that the IHB protein is a positive regulator of ilvIH operon expression. The gene that encodes the IHB protein, ihb, was cloned by complementing the transposon-induced mutation. Definitive evidence that the cloned DNA encodes the IHB protein was provided by determining the sequence of more than 17 amino acids at the N terminus of the IHB protein and comparing it with the nucleotide sequence. A mutation that prevents repression of the ilvIH operon by leucine in vivo and that alters the DNA-binding characteristics of the IHB protein in vitro was shown to be an allele of the ihb gene. The ihb gene is identical to oppI, a gene that regulates the oppABCDF operon (E. A. Austin, J. C. Andrews, and S. A. Short, Abstr. Mol. Genet. Bacteria Phages, p. 153, 1989). Thus, oppI/ihb encodes a protein that regulates both ilvIH, an operon that is repressed by leucine, and oppABCDF, an operon involved in peptide transport that is induced by leucine. We propose that the designation lrp be used in the future instead of oppI or ihb and that Lrp (leucine-responsive regulatory protein) be used in place of IHB.

Acetolactate Synthase↗

PubMed1991

Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV.

Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca2(+)-regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order lcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the lcrV and lcrH gene products were found to be regulatory proteins having important roles in the Ca2(+)-controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.

Amino Acid Sequence↗

PubMed1991

Transcription attenuation-mediated control of leu operon expression: influence of the number of Leu control codons.

Four adjacent Leu codons within the leu leader RNA are critically important in transcription attenuation-mediated control of leu operon expression in Salmonella typhimurium and Escherichia coli (P. W. Carter, D. L. Weiss, H. L. Weith, and J. M. Calvo, J. Bacteriol. 162:943-949, 1985). The leader region from S. typhimurium was altered by site-directed mutagenesis to produce constructs having between one and seven adjacent Leu codons, all CUA. leu operon expression was measured in strains containing six of these constructs, each integrated into the chromosome in a single copy. Operon expression was sufficiently high that all strains grew in minimal medium unsupplemented by leucine. Expression of the operon was measured in strains cultured in such a way that their growth was limited by the intracellular concentration of either leucine or of leucyl-tRNA. In general, the leu operon for each construct responded similarly to the parent construct in terms of the degree of expression as a function of the degree of limitation. However, a strain containing (CUA)1 and, to a certain extent, a strain having (CUA)2 responded somewhat more sluggishly and strains containing (CUA)6 and (CUA)7 responded more sensitively to limitations than did the parent construct. In addition, DNA fragments containing the leu promoter and leader region were used as templates in in vitro transcription reactions employing purified RNA polymerase. With nucleoside triphosphate concentrations of 200 microM, RNA polymerase paused during transcription of the leu leader region at a site about 95 bp downstream from the site of transcription initiation. The halftimes of the pause were 1 min at 37 degrees C and 3 min at 22 degrees C. The pause was lengthened substantially when the GTP concentration was lowered to 20 micromoles. Our results are interpreted most easily in terms of an all-or-none model. Given two Leu control codons, the operon responds with nearly maximum output over a wide range of leucine limitation, and that outcome does not change much with increasing numbers of control codons.

Base Sequence↗

PubMed1992

The Zymomonas mobilis glf, zwf, edd, and glk genes form an operon: localization of the promoter and identification of a conserved sequence in the regulatory region.

The Zymomonas mobilis genes that encode the glucose-facilitated diffusion transporter (glf), glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) are clustered on the genome. The data presented here firmly establish that the glf, zwf, edd, and glk genes form an operon, in that order. The four genes of the operon are cotranscribed on a 6.14-kb mRNA. The site of transcriptional initiation for the polycistronic message was mapped by primer extension and nuclease S1 protection analysis. The glf operon promoter region showed significant homology to other highly expressed Z. mobilis promoters, but not to consensus promoters from other bacteria. The highly expressed Z. mobilis promoter set contains two independent, overlapping, conserved sequences that extend from approximately bp -100 to +15 with respect to the transcriptional start sites. Expression of the glf operon was shown to be subject to carbon source-dependent regulation. The mRNA level was threefold higher in cells grown on fructose than in cells grown on glucose. This increase was not the result of differential mRNA processing when cells were grown on the different carbon sources, nor was it the result of differential transcript stability. Degradation of the 6.14-kb glf operon mRNA was biphasic, with initial half-lives of 11.5 min in fructose-grown cells and 12.0 min in glucose-grown cells. Thus, the higher level of glf operon mRNA in fructose-grown cells is the result of an increased rate of transcription. The importance of increasing glf expression in cells growing on fructose is discussed.

Base Sequence↗

PubMed1993

Nucleotide sequence and functional analysis of cbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides.

Structural genes encoding Calvin cycle enzymes in Rhodobacter sphaeroides are duplicated and organized within two physically distinct transcriptional units, the form I and form II cbb operons. Nucleotide sequence determination of the region upstream of the form I operon revealed a divergently transcribed open reading frame, cbbR, that showed significant similarity to the LysR family of transcriptional regulatory proteins. Mutants containing an insertionally inactivated cbbR gene were impaired in photoheterotrophic growth and completely unable to grow photolithoautotrophically with CO2 as the sole carbon source. In the cbbR strain, expression of genes within the form I operon was completely abolished and that of the form II operon was reduced to about 30% of the wild-type level. The cloned cbbR gene complemented the mutant for wild-type growth characteristics, and normal levels of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) were observed. However, rocket immunoelectrophoresis revealed that the wild-type level of RubisCO was due to overexpression of the form II enzyme, whereas expression of the form I RubisCO was 10% of that of the wild-type strain. The cbbR insertional inactivation did not appear to affect aerobic expression of either CO2 fixation operon, but preliminary evidence suggests that the constitutive expression of the form II operon observed in the cbbR strain may be subject to repression during aerobic growth.

Aerobiosis↗

PubMed1993

Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus.

The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, is near the top of the flagellar-gene regulatory hierarchy, and it is one of the earliest transcription units to be expressed in the cell cycle. In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo. These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A. Dingwall, A. Zhuang, K. Quon, and L. Shapiro, J. Bacteriol. 174:1760-1768, 1992). We propose that this 18-bp sequence contains all or part of the fliF promoter. We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A 2-bp deletion at -12 and -13 in ftr4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly. We suggest that the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle.

Base Sequence↗

PubMed1993

Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified. These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses. The complete sol operon was transcriptionally induced or derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U. Gerischer and P. Dürre, J. Bacteriol. 174:426-433, 1992). Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats. The distal promoter P1 showed only minor transcription initiation in solventogenic C. acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence. The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C. acetobutylicum but was not recognized in E. coli. The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases. Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases. mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.

Alcohol Dehydrogenase↗

PubMed1993

Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85.

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

Base Sequence↗

PubMed1995

Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli.

The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO. The marRAB operon, transcribed from marOII, controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (S. P. Cohen, H. Hachler, and S. B. Levy, J. Bacteriol. 175:1484-1492, 1993). To clarify the role of the operon in response to environmental signals, its transcription was studied under different conditions, using a marOII-lacZ transcriptional fusion introduced into the chromosome of wild-type or mar-deleted cells. In wild-type cells, uncoupling agents (such as carbonyl cyanide m-chlorophenylhydrazone) and different redox-cycling compounds (e.g., menadione and plumbagin) induced expression from the marOII-lacZ fusion two- to sevenfold. In the mar-deleted strain, LacZ expression from the fusion was 10-fold higher than in wild-type cells. This activity was temperature sensitive (3-fold lower at 42 than at 30 degrees C) and decreased 20-fold with the introduction of the gene for MarR. Structurally different compounds which induce the mar operon in wild-type cells reversed the MarR repression of marOII-lacZ expression. To determine the size of MarR, it was fused to MalE as a MarR fusion protein of 144 amino acids [MarR(144)] or of 125 amino acids (deleted of 19 amino acids at the N terminus) [MarR(125)]. Only the MarR(144) fusion showed repressor ability. The purified MarR(144) fusion, but not the MarR(125) fusion, bound specifically to marO in vitro, as revealed by gel retardation, with an apparent dissociation constant of 5 x 10(-9) M. MarR, therefore, controls expression of the marRAB operon presumably by binding to marO. MarR repression in cells can be reversed by different compounds, facilitating the response of bacteria to multiple environmental stress conditions.

Bacterial Proteins↗

PubMed1995

Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon.

There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.

Bacillus subtilis↗

PubMed1995

The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole.

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.

Amino Acid Sequence↗

PubMed1996

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat.

Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria. These deletions affected the level of expression of the operon and the magnitude of its heat shock activation. The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions. The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR. Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shock-dependent cleavage. However, the groESL operon was found to be heat shock activated even after most of the IR was deleted. This observation, together with the fact that the groESL operon of A. tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon. The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions.

Agrobacterium tumefaciens↗

PubMed1996

Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis including a novel gene, pyrK, involved in pyrimidine biosynthesis.

Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon in Lactococcus lactis. Two of the genes are the well-known pyr genes pyrDb and pyrF, encoding dihydroorotate dehydrogenase and orotidine monophosphate decarboxylase, respectively. The third gene encodes a protein which was shown to be necessary for the activity of the pyrDb-encoded dihydroorotate dehydrogenase; we propose to name the gene pyrK. The pyrK-encoded protein is homologous to a number of proteins which are involved in electron transfer. The lactococcal pyrKDbF operon is highly homologous to the corresponding part of the much-larger pyr operon of Bacillus subtilis. orf2, the pyrK homolog in B. subtilis, has also been shown to be necessary for pyrimidine biosynthesis (A. E. Kahler and R. L. Switzer, J. Bacteriol. 178:5013-5016, 1996). Four genes adjacent to the operon, i.e., orfE, orfA, orfC, and gidB, were also sequenced. Three of these were excluded as members of the pyr operon by insertional analysis (orfA) or by their opposite direction of transcription (orfE and gidB). orfC, however, seems to be the distal gene in the pyrKDbF-orfC operon.

Bacterial Proteins↗