Cooperative interaction between Cra and Fnr in the regulation of the cydAB operon of Escherichia coli.

In vivo and in vitro experiments are reported demonstrating that the catabolite repressor-activator (Cra) protein (formerly designated FruR) regulates expression of the cydAB operon of Escherichia coli encoding cytochrome d oxidase. The Fnr protein is required for Cra-mediated transcriptional control, but the ArcA protein antagonizes the response to Cra. The results establish that Fnr, ArcA, and Cra exert their effects in an interdependent fashion.

Cra and the control of carbon flux via metabolic pathways.

The catabolite repressor-activator (Cra) protein controls the direction of carbon flux through metabolic pathways in enteric bacteria. Cra binds to the control regions of target genes and exerts a negative effect on the expression of genes encoding glycolytic and Entner-Doudoroff enzymes, while exerting a positive effect on genes encoding Krebs cycle, glyoxylate shunt and gluconeogenic enzymes. Cra mediates cyclic AMP-independent catabolite repression of positively Cra-regulated genes and catabolite activation of negatively Cra-controlled genes.

Frur mediates catabolite activation of pyruvate kinase (pykF) gene expression in Escherichia coli.

Expression of a pykF-lacZ fusion was studied as a function of the carbon source in wild-type strains and strains lacking or overproducing the FruR protein of Escherichia coli. FruR controls the response to the carbon source by repressing pykF expression more strongly under gluconeogenic than under glycolytic conditions, a phenomenon we term catabolite activation.

In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of gram-positive bacteria.

Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the cis-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.

The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli.

The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator-containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR-binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.

Effect of the FruR regulator on transcription of the pts operon in Escherichia coli.

The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRP.cAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRP.cAMP-binding sites. The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRP.cAMP, and restore P1a transcription, which is repressed by CRP.cAMP. FruR can displace CRP.cAMP from its binding site in the presence of RNA polymerase even though FruR and CRP.cAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR- mutant cells as in cells grown in the presence of glucose. These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.

In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis.

The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues. By using a one-step chromatographic procedure, FruR was purified to near-homogeneity. Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons. The positions of interference between FruR and the operator of the acetate operon were examined. The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting. It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon.

The mannitol repressor (MtlR) of Escherichia coli.

The mannitol operon of Escherichia coli, encoding the mannitol-specific enzyme II of the phosphotransferase system (Mt1A) and mannitol phosphate dehydrogenase (Mt1D), is here shown to contain a single additional downstream open reading frame which encodes the mannitol repressor (Mt1R). Mt1R contains 195 amino acids and has a calculated molecular weight of 21,990 and a calculated pI of 4.5. It is homologous to the product of an open reading frame (URF2D) upstream of the E. coli gapB gene but represents a novel type of transcriptional regulatory protein.

DNA sequence of a gene in Escherichia coli encoding a putative tripartite transcription factor with receiver, ATPase and DNA binding domains.

We have sequenced downstream of the last previously sequenced gene of the glucitol operon (gutABDMRQ) in E. coli and have found that gutQ is the last gene of this operon. Downstream of the gutQ gene is found a palindromic unit (PU or REP sequence), followed by a large open reading frame of 1515 (or possibly 1590) bps transcribed in the direction opposite to that of the gut operon. This open reading frame encodes a protein of 504 (or possibly 529) amino acids with a tripartite structure. The N-terminal "receiver" domain of 187 (or possibly 212) residues is homologous to the FhlA protein of E. coli, a transcriptional activator of formate hydrogen lyase. It may possess a short domain at its extreme N-terminus exhibiting sequence similarity to carbohydrate binding proteins. The central ATPase domain (236 residues) exhibits greatest sequence similarity to the HydG protein of E. coli, a transcriptional activator of labile hydrogenase. The C-terminal DNA binding domain (81 residues) is homologous to NtrX of Azorhizobium caulinodans, a protein involved in transcriptional regulation of nitrogen fixation. Sequence comparisons with well-characterized transcription factors suggest that ORF504 encodes a protein that hydrolyzes ATP to generate the open transcriptional initiation complex of sigma 54-dependent promoters, possibly in response to redox conditions and/or ligand binding. We propose that this tripartite transcription factor arose by fusion of gene fragments encoding its three constituent modules.

In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium.

Evidence has been presented suggesting that the fructose repressor, FruR, is a pleiotropic transcriptional regulatory protein controlling the expression of numerous operons concerned with carbon metabolism in Escherichia coli and Salmonella typhimurium. We have conducted in vitro DNA binding studies to ascertain the nature of the DNA sequences to which FruR binds. Employing both DNA band migration retardation and DNase I footprint analyses, FruR was found to bind to two operators within the regulatory region preceding the structural genes of the fructose operon, fruB(MH)KA. These two operators, O1 and O2, comprise nearly identical palindromes of 12 bp with a half-site of TGAAAC. The binding of FruR to these inverted repeats was found to be reversed by inclusion of micromolar concentrations of fructose-1-phosphate. The two operators are located between the single putative promoter of the fructose operon and the translational initiation site of the fruB gene. Other regulated operons were shown to bind FruR to a single site upstream of the first structural gene as follows: (1) ppsA (positive regulation); (2) icd (positive regulation); (3) aceB (positive regulation); and (4) pts (negative regulation). In all cases, low concentrations of fructose-1-phosphate displaced the protein from the DNA. The binding sites were determined, and a FruR consensus sequence was established. Computer searches revealed the presence of this sequence in numerous functionally diverse operons, implying that FruR is a global transcriptional regulatory protein in enteric bacteria.

Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes.

We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.

Codon usage and G + C content in Bradyrhizobium japonicum genes are not uniform.

To date, the sequences of 45 Bradyrhizobium japonicum genes are known. This provides sufficient information to determine their codon usage and G + C content. Surprisingly, B. japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G + C content than genes not belonging to these two groups. Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes. The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj alpha 9 sequence which is an insertion sequence (IS)-like element. The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors. These were now corrected. The deduced gene product of ORF355 in RSRj alpha 9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes. The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements.