Purine regulon of gamma-proteobacteria: a detailed description

The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhi, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well retained of genes encoding enzymes that participate in the synthesis of inosinemonophosphate from phosphoribozylpyrophosphate and in transfer of unicarbon groups, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream of nonorthologous but functionally replaceable genes. However, the PurR site is poorly retained in front of orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and protein genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.     

Purine Regulon of Gamma-Proteobacteria: A Detailed Description

The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhimurium, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well conserved upstream genes encoding enzymes that participate in the synthesis of inosine monophosphate from phosphoribozylpyrophosphate and in transfer of one-carbon units, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream nonorthologous but functionally replaceable genes. However, the PurR site is poorly conserved upstream orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.     

The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival

Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild-type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.     

Enzymatic ability of Bifidobacterium animalis subsp. lactis to hydrolyze milk proteins: identification and characterization of endopeptidase O

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide alpha s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.     

The phosphate-starvation response in Vibrio cholerae O1 and phoB mutant under proteomic analysis: disclosing functions involved in adaptation, survival and virulence

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.     

Analysis of a region from the bacteriophage resistance plasmid pCI528 involved in its conjugative mobilization between Lactococcus strains.

A 10-kb HindIII fragment of pCI528 cloned into the nonconjugative shuttle vector pCI3340 could be transferred by conjugative mobilization from Lactococcus lactis subsp. lactis MG1363, whereas other HindIII fragments of pCI528 or the vector alone were nonmobilizable. Subcloning of this 10-kb region identified a 4.4-kb BglII-EcoRI fragment which contained all the DNA essential for transfer. Sequence analysis of a 2-kb region within this 4.4 kb-segment revealed a region rich in inverted repeats and two potential overlapping open reading frames, one of which demonstrated homology to mobilization proteins of two nonconjugative staphylococcal plasmids.     

Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P(i)) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2',3' cyclic nucleotide 2'-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3' or 5' phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.     

Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.     

Crystallization and preliminary X-ray studies on the co-repressor binding domain of the Escherichia coli purine repressor.

The purine repressor is a putative helix-turn-helix DNA-binding protein that regulates several genetic loci important in purine and pyrimidine metabolism in Escherichia coli. The protein is composed of two domains, an N-terminal DNA-binding domain and a C-terminal core that binds the purine co-repressors, guanine and hypoxanthine. The co-repressor binding domain (residues 53 to 341) has been crystallized from polyethylene glycol 600-MgCl2 solutions. They are of the monoclinic form, space group P2(1), with a = 38.2 A, b = 125.7 A, c = 61.8 A and beta = 100.2 degrees. They diffract to a resolution of at least 2.2 A and contain two monomers per asymmetric unit. The importance of the structural determination of this domain is underscored by the high degree of sequence homology displayed within the effector binding sites among a sub-class of helix-turn-helix proteins, of which LacI and GalR are members. The structure of the PurR co-repressor binding domain will provide a high resolution view of one such domain and could serve as a possible model for future effector site structural determinations. Perhaps more important will be this structure's contribution to the further understanding of how protein-DNA interactions are modulated.     

Proteomic signature of Lactococcus lactis NCDO763 cultivated in milk

We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.