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PubMed2000

Expression of different-size transcripts from the clpP-clpX operon of Escherichia coli during carbon deprivation.

Transcription of the clpP-clpX operon of Escherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entire clpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpX coding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the sigma(32)-dependent promoter of the clpP-clpX operon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of the clpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoB mutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpX transcript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2000

The TorR high-affinity binding site plays a key role in both torR autoregulation and torCAD operon expression in Escherichia coli.

In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces the torCAD operon, which encodes the TMAO respiratory system of Escherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torR gene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers the torR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that the torR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a torS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

Bacterial Proteins↗

PubMed2001

Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function.

Agmatine↗

PubMed2001

Characterization of the sat operon in Streptococcus mutans: evidence for a role of Ffh in acid tolerance.

An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bona fide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3' to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans.

Bacterial Proteins↗

PubMed2001

Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element.

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.

Bacterial Outer Membrane Proteins↗

PubMed2001

The pyrimidine operon pyrRPB-carA from Lactococcus lactis.

The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.

Amino Acid Sequence↗

PubMed2002

Regulation of the metC-cysK operon, involved in sulfur metabolism in Lactococcus lactis.

Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine beta-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.

Bacterial Proteins↗

PubMed2002

Identification of Bacillus subtilis CysL, a regulator of the cysJI operon, which encodes sulfite reductase.

The way in which the genes involved in cysteine biosynthesis are regulated is poorly characterized in Bacillus subtilis. We showed that CysL (formerly YwfK), a LysR-type transcriptional regulator, activates the transcription of the cysJI operon, which encodes sulfite reductase. We demonstrated that a cysL mutant and a cysJI mutant have similar phenotypes. Both are unable to grow using sulfate or sulfite as the sulfur source. The level of expression of the cysJI operon is higher in the presence of sulfate, sulfite, or thiosulfate than in the presence of cysteine. Conversely, the transcription of the cysH and cysK genes is not regulated by these sulfur sources. In the presence of thiosulfate, the expression of the cysJI operon was reduced 11-fold, whereas the expression of the cysH and cysK genes was increased, in a cysL mutant. A cis-acting DNA sequence located upstream of the transcriptional start site of the cysJI operon (positions -76 to -70) was shown to be necessary for sulfur source- and CysL-dependent regulation. CysL also negatively regulates its own transcription, a common characteristic of the LysR-type regulators. Gel mobility shift assays and DNase I footprint experiments showed that the CysL protein specifically binds to cysJ and cysL promoter regions. This is the first report of a regulator of some of the genes involved in cysteine biosynthesis in B. subtilis.

Amino Acid Sequence↗

PubMed2002

Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae.

The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-type C. diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

Bacterial Proteins↗

PubMed2002

Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin.

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

Bacterial Proteins↗

PubMed2002

Newly identified cytochrome c oxidase operon in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 specifically induced in heterocysts.

Two operons have been cloned from Anabaena sp. strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases. The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit. One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation. Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts. The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells. Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen. It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp. strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts.

Anabaena↗

PubMed2003

Open reading frame all0601 from Anabaena sp. strain PCC 7120 represents a novel gene, cnaT, required for expression of the nitrate assimilation nir operon.

Expression of the nitrate assimilation nir operon in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the ntcB gene is found in a cluster of genes located in the complementary strand, upstream from the nir operon. Just downstream of ntcB, there is an open reading frame, all0601 (previously designated orf356 and now designated the cnaT gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with ntcB homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of cnaT was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions, nir operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of cnaT did not impair expression of ntcB, and expression of cnaT itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the nir operon in Anabaena sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.

Anabaena↗

PubMed2003

A second and unusual pucBA operon of Rhodobacter sphaeroides 2.4.1: genetics and function of the encoded polypeptides.

A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically. The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original beta-apoprotein. The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide. In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide. We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL. Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation. Thus, the sole source of alpha-polypeptides for the LH2 complex is puc1A. The role of the puc1C-encoded polypeptide was also determined. We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon. Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation. Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded beta-polypeptide.

Amino Acid Sequence↗

PubMed2003

Involvement of an inducible fructose phosphotransferase operon in Streptococcus gordonii biofilm formation.

Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3' end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation. Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm. Expression of fruK, as measured by the beta-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in beta-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms.

Base Sequence↗

PubMed2003

Involvement of the adc operon and manganese homeostasis in Streptococcus gordonii biofilm formation.

Pioneer oral bacteria, including Streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues. An S. gordonii::Tn917-lac biofilm-defective mutant was isolated by using an in vitro biofilm formation assay. Subsequent inverse PCR and sequence analyses identified the transposon insertion to be near the 3' end of an open reading frame (ORF) encoding a protein homologous to a Streptococcus pneumoniae repressor, AdcR. The S. gordonii adc operon, consisting of the four ORFs adcR, adcC, adcB, and adcA, is homologous to the adc operon of S. pneumoniae, which plays a role in zinc and/or manganese transport and genetic competence in S. pneumoniae. AdcR is a metal-dependent repressor protein containing a putative metal-binding site, AdcC contains a consensus-binding site for ATP, AdcB is a hydrophobic protein with seven hydrophobic membrane-spanning regions, and AdcA is a lipoprotein permease with a putative metal-binding site. The three proteins (AdcC through -A) are similar to those of the binding-lipoprotein-dependent transport system of gram-positive bacteria. Reverse transcriptase PCR confirmed that adcRCBA are cotranscribed as an operon in S. gordonii and that the transposon insertion in S. gordonii adcR::Tn917-lac had resulted in a polar mutation. Expression of adcR, measured by the beta-galactosidase activity of the adcR::Tn917-lac mutant, was growth phase dependent and increased when the mutant was grown in media with high levels of manganese (>1 mM) and to a lesser extent in media with zinc, indicating that AdcR may be a regulator at high levels of extracellular manganese. A nonpolar inactivation of adcR generated by allelic replacement resulted in a biofilm- and competence-defective phenotype. The biofilm-defective phenotype observed suggests that AdcR is an active repressor when synthesized and acts at a distant site(s) on the chromosome. Thus, the adc operon is involved in manganese acquisition in S. gordonii and manganese homeostasis and appears to modulate sessile growth in this bacterium.

ATP-Binding Cassette Transporters↗

PubMed2004

Nitrite-responsive activation of the nitrate assimilation operon in Cyanobacteria plays an essential role in up-regulation of nitrate assimilation activities under nitrate-limited growth conditions.

NtcB of the cyanobacterium Synechococcus elongatus strain PCC 7942 is a LysR family protein that enhances expression of the nitrate assimilation operon (nirA operon) in response to the presence of nitrite, an intermediate of assimilatory nitrate reduction. Inactivation of ntcB in this cyanobacterium specifically abolishes the nitrite responsiveness of nirA operon expression, but under nitrate-replete conditions (wherein negative feedback by intracellularly generated ammonium prevails over the positive effect of nitrite) activity levels of the nitrate assimilation enzymes are marginally higher in the wild-type cells than in the mutant cells, raising the issue of whether the nitrite-promoted regulation has physiological importance. On the other hand, the strains carrying ntcB expressed much higher nitrate assimilation enzyme activities under nitrate-limited growth conditions than under nitrate-replete conditions whereas the ntcB-deficient strains showed levels of the enzyme activities lower than those seen under the nitrate-replete conditions. Although the ntcB mutant maintained a constant cell population in a nitrate-limited chemostat when grown as a single culture, it was diluted at a rate expected for nondividing cells when mixed with the wild-type cells and subjected to nitrate limitation in the chemostat culture system. These results demonstrated that the nitrite-promoted activation of the nitrate assimilation operon is essential for up-regulation of the nitrate assimilation activities under the conditions of nitrate limitation and for competitive utilization of nitrate.

Bacterial Proteins↗

PubMed2004

The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter.

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.

Bacterial Proteins↗

PubMed2004

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa.

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.

Ammonia↗