Genetic characterization and molecular cloning of the tripeptide permease (tpp) genes of Salmonella typhimurium.

Of the three bacterial peptide transport systems only one, the oligopeptide permease, has been characterized in any detail. We have now isolated Salmonella typhimurium mutants deficient in a second transport system, the tripeptide permease (Tpp), using the toxic peptide alafosfalin. Alafosfalin resistance mutations map at three loci, the gene encoding peptidase A (pepA) and two transport-defective loci, tppA and tppB. Locus tppA has been mapped to 74 min on the S. typhimurium chromosome, cotransducible with aroB, and is a positive regulator of tppB. Locus tppB maps at 27 min in the cotransduction gap between purB and pyrF. We cloned tppB, the structural locus for the tripeptide permease. Two simple methods are described for mapping the location of cloned DNA fragments on the chromosome of S. typhimurium.     

Two genetically distinct pathways for transcriptional regulation of anaerobic gene expression in Salmonella typhimurium

Expression of the tripeptide permease gene tppB is anaerobically induced. This induction is independent of the fnr (oxrA) gene product, which is known to be required for the anaerobic induction of several respiratory enzymes. We isolated, characterized, and mapped mutations in two genes, oxrC and tppR, which prevent the anaerobic induction of tppB expression. Mutations in oxrC were highly pleiotropic, preventing the anaerobic expression of the formate dehydrogenase component of formate hydrogen lyase (fhl), a tripeptidase (pepT), and two of the three known hydrogenase isoenzymes (hydrogenases 1 and 3). On the other hand, expression of nitrate reductase, fumarate reductase, and a number of other fnr (oxrA)-dependent enzymes was not affected by mutations in oxrC. Thus, there appeared to be at least two distinct classes of anaerobically induced genes, those which required fnr for their expression and those which required oxrC. It seems that fnr-dependent enzymes perform primarily respiratory functions, whereas oxrC-dependent enzymes served fermentative or biosynthetic roles. We found the primary defect of oxrC mutants to be a deficiency in phosphoglucose isomerase activity, implying that a product of glycolysis functions as an anaerobic regulatory signal. Mutations in tppR were specific for tppB and did not affect expression of other oxrC-dependent genes. However, tppR did exhibit phenotypes other than the regulation of tppB. Both oxrC and tppR mutants were hypersensitive to the toxic NAD analog 6-aminonicotinic acid. This suggests that oxrC and tppR may play a role in the regulation of NAD biosynthesis or, alternatively, that NAD or a related nucleotide serves as the anaerobic signal for oxrC-dependent enzymes.     

An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression

The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmotically regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology. We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.     

Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae

The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.     

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.     

Substrate specificity of the AmpG permease required for recycling of cell wall anhydro-muropeptides

AmpG was originally identified as a gene required for induction of beta-lactamase. Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides. We have now studied the specificity of the AmpG permease. The principal requirement is for the presence of the disaccharide, N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc). These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth. AmpG permease is sensitive to carbonylcyanide m-chlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force.     

The Isolation and Characterization of Saccharomyces Cerevisiae Mutants That Constitutively Express Purine Biosynthetic Genes

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.     

PTR3, a novel gene mediating amino acid-inducible regulation of peptide transport in Saccharomyces cerevisiae

We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid-inducible peptide transport. PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database. Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-induced levels of peptide transport. PTR3 was required for amino acid-induced expression of PTR2 , the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression. It was determined that PTR3 also modulates expression of BAP2 , the gene encoding the branched-amino acid permease. Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system.     

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium

Summary The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.     

Monitoring global gene expression of proteases and improvement of human lysozyme production in the nptB gene disruptant of Aspergillus oryzae

Aspergillus oryzae has numerous protease genes that might cause proteolytic degradation of heterologously-produced proteins. The productivity of the heterologous protein can be improved by protease gene disruption, but it is difficult to select disruption targets efficiently. In this study, we monitored the expression of 132 protease genes by DNA microarray. A group of protease genes up-regulated during cultivation was identified by clustering analysis. In this protease group, the nptB gene encoding neutral protease II was included as well as the alpA, tppA, and pepA genes, disruption of which has improved human lysozyme (HLY) production. The nptB gene was disrupted to investigate its involvement in HLY production, and nptB disruptants showed an improvement in the production. These observations suggest that monitoring the expression of protease genes is an efficient strategy in screening potential disruption targets for heterologous protein production in A. oryzae.     

Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Deltafnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.     

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

The Pseudomonas aeruginosa hemA promoter is regulated by Anr,Dnr, NarL and Integration Host Factor

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.