Acidic Growth Conditions Research Articles

Activation of the Diacetyl/Acetoin Pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by Acidic Growth

Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into alpha-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.

The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed IpiA, have been identified in the gram-negative alpha-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.

Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C(26) was a major fatty acid in this TG, indicating that the TGS1 gene product uses C(26) fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C(26 : 0)-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

Enthalpies and entropies of proton and cadmium adsorption onto Bacillus subtilis bacterial cells from calorimetric measurements

We used titration calorimetry to measure the bulk heats of proton and Cd adsorption onto a common Gram positive soil bacterium Bacillus subtilis at 25.0 °C. Using the 4-site non-electrostatic model of Fein et al. [Fein, J.B., Boily, J.-F., Yee, N., Gorman-Lewis, D., Turner, B.F., 2005. Potentiometric titrations of Bacillus subtilis cells to low pH and a comparison of modeling approaches. Geochim. Cosmochim. Acta 69 (5), 1123–1132.] to describe the bacterial surface reactivity to protons, our bulk enthalpy measurements can be used to determine the following site-specific enthalpies of proton adsorption for Sites 1–4, respectively: −3.5 ± 0.2, −4.2 ± 0.2, −15.4 ± 0.9, and −35 ± 2 kJ/mol, and these values yield the following third law entropies of proton adsorption onto Sites 1–4, respectively: +51 ± 4, +78 ± 4, +79 ± 5, and +60 ± 20 J/mol K. An alternative data analysis using a 2-site Langmuir–Freundlich model to describe proton binding to the bacterial surface (Fein et al., 2005) resulted in the following site-specific enthalpies of proton adsorption for Sites 1 and 2, respectively: −3.6 ± 0.2 and −35.1 ± 0.3 kJ/mol. The thermodynamic values for Sites 1–3 for the non-electrostatic model and Site 1 of the Langmuir–Freundlich model of proton adsorption onto the bacterial surface are similar to those associated with multifunctional organic acid anions, such as citrate, suggesting that the protonation state of a bacterial surface site can influence the energetics of protonation of neighboring sites. Our bulk Cd enthalpy data, interpreted using the 2-site non-electrostatic Cd adsorption model of Borrok et al. [Borrok, D., Fein, J.B., Tischler, M., O’Loughlin, E., Meyer, H., Liss, M., Kemner, K.M., 2004b. The effect of acidic solutions and growth conditions on the adsorptive properties of bacterial surfaces. Chem. Geol. 209 (1–2), 107–119.] to account for Cd adsorption onto B. subtilis, yield the following site-specific enthalpies of Cd adsorption onto bacterial surface Sites 2 and 3, respectively: −0.2 ± 0.4 and +14.4 ± 0.9 kJ/mol, and the following third law entropies of Cd adsorption onto Sites 2 and 3, respectively: +57 ± 4 and +128 ± 5 J/mol K. The calculated enthalpies of Cd adsorption are typical of those associated with Cd complexation with anionic oxygen ligands, and the entropies are indicative of inner sphere complexation by multiple ligands. The experimental approach described in this study not only yields constraints on the molecular-scale mechanisms involved in proton and Cd adsorption reactions, but also provides new thermodynamic data that enable quantitative estimates of the temperature dependence of proton and Cd adsorption reactions.

Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains

The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.

The Carboxyl Terminus of NifK Protein is Involved in Formation of a Stable Nitrogenase Complex under Acidic Growth Conditions in Azotobacter vinelandii

The Carboxyl Terminus of NifK Protein is Involved in Formation of a Stable Nitrogenase Complex under Acidic Growth Conditions in Azotobacter vinelandii

Effect of pH on Isolation and Distribution of Members of Subdivision 1 of the Phylum Acidobacteria Occurring in Soil

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.

Camptothecin analogs with enhanced activity against human breast cancer cells. II. Impact of the tumor pH gradient

Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors.

The effect of acidic solutions and growth conditions on the adsorptive properties of bacterial surfaces

Data must be collected over a wide pH range to accurately model the adsorption of protons and metal onto bacterial surfaces; however, alterations in the structural and chemical properties of bacterial surfaces resulting from exposure to acidic solutions may affect the mechanisms of cation binding. Binding properties of bacteria may also be affected by nutrient and oxygen levels present during their growth. We measured Cd, Co, and Pb adsorption onto bacteria by using (1) bacteria washed with acidic solutions (pH≈1.5), (2) non-acid-washed bacteria exposed to acidic parent solutions, and (3) non-acid-washed bacteria exposed to neutral parent solutions. The purpose was to determine the effect of acidic conditions on the adsorptive properties of Pseudomonas mendocina, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus. We also measured Co adsorption onto bacteria ( Pseudomonas fluorescens) grown under nutrient-rich and nutrient-limited conditions and onto the facultative bacterium Shewanella oneidensis MR-1 grown under aerobic and anaerobic conditions. Bacteria exposed to acidic solutions adsorbed more metals than bacteria not exposed to such solutions. We attribute the increase in adsorption to the irreversible displacement of structurally bound Mg and Ca by protons. After displacement, the protonated sites can participate in reversible metal adsorption reactions. Thermodynamic modeling suggests that concentrations of functional group sites on bacterial surfaces increase by as much as five times in response to acid washing, assuming that stability constants for the bacterial surface complexes remain the same. Although the sizes of the bacteria changed markedly in response to nutrient limits and oxygen content during growth, the mass-normalized extent of Co adsorption onto both P. fluorescens and S. oneidensis MR-1 was independent of growth conditions. We conclude that adsorption constants derived from experiments in which the bacteria are never exposed to acidic conditions probably provide the most accurate estimates of the extent of bacteria–metal adsorption in natural settings.

Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori.

The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H. pylori acid response. Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.

Ambient pH-regulated enzime secretion in endophytic and pathogenic isolates of the fungal genus Colletotrichum

In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit.

Development and application of a quasi-static Langmuir isotherm for modelling selected resin acid fate in pulp mill wastewater treatment

Resin acids are pulp mill effluent contaminants that exhibit significant solubility, diffusivity, and surfactancy changes with pH within the range typically used for biological treatment. Such physical–chemical property changes which can influence removal during biological wastewater treatment, can be characterized by dynamic surface tension measurements. Dynamic surface tension measurements were made by the maximum bubble pressure method during batch treatment of selected resin acids in pulp mill effluent. Interpretation of dynamic surface tension data was made through the framework of a quasi-static Langmuir isotherm model that was derived as part of this investigation. The results suggested that under acidic conditions, resin acids form associations with other dissolved organic matter contained in pulp mill effluent, while under alkaline conditions, they behave as relatively soluble surfactants. A resin acid residuum, or threshold concentration, has been found to increase under acidic growth conditions. This residuum increase corresponded to an inferred reduction in resin acid bioavailability that was suggested from the isotherm modelling. The development of quasi-static isotherm adsorption models has application in computer simulation for design of adsorption based unit processes, and could potentially be utilized as an informative treatment process monitor.

Guaiacol formation in apple juice, effect of selected additives on Alicyclobacillus growth

Disinfectant or card-board off-flavour formation was observed in the batch of pasteurised reconstituted apple juice. Aroma profile of products was analysed using SPME and GC/MS, guaiacol was detected in the apple juice sample as well as apple concentrate. Sporulating bacteria Alicyclobacillus acidoterrestris, which are able to growth in acidic conditions were isolated and identified as the cause of the off flavour formation. The possibilities of flavour formation inhibition using various additives were evaluated. The inhibitory effect of nisin, sulphur dioxide, benzoic acid and EDTA was followed. The best way of prevention is the sufficient washing and disinfection of apples prior the processing e.g. using chlorine dioxide. But when the treatment is insufficient and contaminated apples are to be processed the tested additives can prolong the lag phase and increase product stability. The inhibitory effect of the tested additives decreased in the order: benzoic acid, EDTA, nisin and sulphur dioxide.

A Genome-wide view of the Escherichia coli BasS-BasR two-component system implicated in iron-responses.

Histidine-to-aspartate (His-->Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. Determination of the entire genomic sequence of Escherichia coli revealed that this gram-negative bacterium has 29 His-kinases and 32 response regulators. Of the 29 His-kinases, 23 have already been experimentally characterized at least to some extent in terms of their physiological functions. No physiological stimulus has yet been identified for each of the remaining 6 His-kinases (BasS, CreC, RstB, YfhK, YehU, and YpdA). Here we characterized the BasS-BasR two-component system with reference to its physiological function, taking genetic approaches together with genome-wide transcriptome profiling. First we showed that the hypothetical yfbE operon that appears to be implicated in the modification of lipopolysaccharides is regulated at the level of transcription in response to external iron, and then we showed that the BasS-BasR system is essential for this iron-dependent induction of yfbE. Another PhoQ-PhoP two-component system was also implicated in the full induction of yfbE in response to iron, but it was not essential. To gain more insight into the BasS-BasR system, we conducted genome-wide transcriptome analysis by microarray, finding that many of the uncovered putative iron-induced and BasS-BasR-dependent genes are somehow associated with acidic and/or anaerobic growth conditions. In this respect, it was found that mutant cells defective in the BasS-BasR system were sensitive to mild-acid growth conditions in the presence of a relatively high concentration of iron. These results are discussed with regard to a comprehensive picture of the His-->Asp phosphorelay signaling network in E. coli.

Genes of the GadX-GadW Regulon inEscherichia coli

Acid in the stomach is thought to be a barrier to bacterial colonization of the intestine. Escherichia coli, however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. The gadX gene and a second downstream araC-like transcription factor gene, gadW, were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in a gadX mutant and even more so by combined mutation of gadX and gadW. However, there was no defect in colonization of the streptomycin-treated mouse model by the gadX mutant in competition with the wild type, and the gadX gadW mutant was a better colonizer than the wild type. Thus, E. coli colonization of the mouse does not appear to require glutamate-dependent acid resistance.

The pH signalling transcription factor PacC controls virulence in the plant pathogen Fusarium oxysporum.

Gene expression in fungi by ambient pH is regulated via a conserved signalling cascade whose terminal component is the zinc finger transcription factor PacC/Rim1p. We have identified a pacC orthologue in the vascular wilt pathogen Fusarium oxysporum that binds the consensus 5'-GCCAAG-3' sequence and is proteolytically processed in a similar way to PacC from Aspergillus nidulans. pacC transcript levels were elevated in F. oxysporum grown in alkaline conditions and almost undetectable at extreme acidic growth conditions. PacC+/- loss-of-function mutants displayed an acidity-mimicking phenotype resulting in poor growth at alkaline pH, increased acid protease activity and higher transcript levels of acid-expressed polygalacturonase genes. Reintroduction of a functional pacC copy into a pacC+/- mutant restored the wild-type phenotype. Conversely, F. oxysporum merodiploids carrying a dominant activating pacCc allele had increased pacC transcript and protein levels and displayed an alkalinity-mimicking phenotype with reduced acid phosphatase and increased alkaline protease activities. PacC+/- mutants were more virulent than the wild-type strain in root infection assays with tomato plants, whereas pacCc strains were significantly reduced in virulence. We propose that F. oxysporum PacC acts as a negative regulator of virulence to plants, possibly by preventing transcription of acid-expressed genes important for infection.

Absence of malolactic activity is a characteristic of H+-ATPase-deficient mutants of the lactic acid bacterium Oenococcus oeni.

The lack of malolactic activity in H(+)-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni. Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR. No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found. The effect of a reduction in H(+)-ATPase activity on L-malate metabolism was then investigated by using other malolactic bacteria. Spontaneous H(+)-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance. Two mutants were selected. These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L. lactis and L. mesenteroides parental strains, respectively. These mutants were also acid sensitive. However, in contrast to the ATPase-deficient mutants of O. oeni, activation of L-malate metabolism was observed with the L. lactis and L. mesenteroides mutants under optimal or acidic growth conditions. These data support the suggestion that expression of the genes encoding malolactic enzymes in O. oeni is regulated by the mleR product, as it is in L. lactis. Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O. oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria.

Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae.

Two operons, comAB and comCDE, play a key role in the co-ordination of spontaneous competence development in cultures of Streptococcus pneumoniae. ComAB is required for export of the comC-encoded competence-stimulating peptide (CSP). Upon CSP binding, the histidine kinase ComD activates ComE, its cognate response regulator, required for autoinduction of comCDE and for induction of the late competence genes. To understand better the early control of competence development, mutants upregulating comCDE (ComCDEUP) were isolated using a comC-lacZ transcriptional fusion. Mutants were generated by polymerase chain reaction mutagenesis of the comCDE region and by in vitro transposon mutagenesis of the chromosome. Both types of ComCDEUP mutants exhibited similar phenotypes. They differed from wild type in displaying trypsin-resistant transformation, competence under acid growth conditions and expression of comCDE under microaerobiosis; increased production of CSP in the mutants could account for the various phenotypes. The ComCDEUP transposon mutations included four independent insertions in the ciaR gene, which encodes the response regulator of a two-component system previously found to affect competence, and two immediately upstream of the comAB operon. The latter two resulted in comAB overexpression, indicating that CSP export is rate limiting. Among comDE point mutations, a single amino acid change in ComD (T233I) conferred constitutive, CSP-independent competence and resulted in comAB overexpression, providing support for the hypothesis that ComE regulates comAB; a ComE mutant (R120S) exhibited altered kinetics of competence shut-off. Collectively, these data indicate that pheromone autoinduction, cross-regulation of the comAB and comCDE operons and, possibly, competence shut-off contribute to the early control of competence development in S. pneumoniae. They argue for a metabolic control of competence, mediated directly or indirectly by CiaR, and they suggest that both comAB and comCDE are potential targets for regulation.

Acid resistance of enterohaemorrhagic and generic Escherichia coli associated with foodborne disease and meat

As part of the Australia New Zealand Food Authorities (ANZFA) food standards code, salami manufacturers are required to demonstrate that their process is capable of achieving a 3-log reduction in Escherichia coli. Non-pathogenic E. coli strains with similar or greater acid resistance to enterohaemorrhagic E. coli (EHEC) are needed if industry is to conduct challenge studies to demonstrate compliance with the standard. In the present study, E. coli isolates from sheep and beef carcasses and meat were shown to have wide-ranging acid resistance in broth when preadapted to growth in acidic conditions. Times required for a 3-log reduction in E. coli ranged from less than 1 day to more than 28 days. Variable acid resistance was observed in both EHEC strains associated with foodborne outbreaks and generic E. coli strains. Generic E. coli strains with the greatest acid resistance were assessed for pathogenicity markers and their survival in fermented meat compared with EHEC strains. It was demonstrated that generic E. coli strains could be used for challenge studies to determine compliance with or validate performance standards designed for the control of EHEC.