Acid stress in the food pathogen Bacillus cereus.

AIMS: The pathogen Bacillus cereus, which is associated with a number of foods including dairy products, was studied for its response to acid stress during the exponential phase. METHODS AND RESULTS: Bacillus cereus was found to adapt to acid stress (pH 4.6) when pre-exposed to a non-lethal, inducing pH of 6.3 or to inducing concentrations of heat, ethanol, salt or hydrogen peroxide. Cells were found to maintain their internal pH at a higher level than the external acid pH and adapted cells had a higher internal pH than unadapted cells. A constitutive acid-sensitive mutant that was also heat- and ethanol-sensitive was found to be capable of high levels of adaptation despite its lack of induction of proteins induced in the wild type by exposure to moderate pH (6.3) values. CONCLUSIONS: A number of proteins were found to be underexpressed in the mutant compared with the wild type at pH 6.3, including some with homology to ribosomal proteins and to the sporulation regulator RapK, while one differentially expressed band contained two proteins, one of which was homologous to the competence regulator CodY. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the processing of B. cereus-associated foods by acidification. The linked developmental processes of stationary phase, sporulation and possibly competence appear to be involved in the response to acid stress.

Heat and salt stress in the food pathogen Bacillus cereus.

AIMS: The effects of stresses imposed on bacterial contaminants during food processing and treatment of packaging material were evaluated on the food pathogen Bacillus cereus. METHODS AND RESULTS: Conditions were established which allowed the cells to adapt to heat, ethanol and hydrogen peroxide stresses, but not to osmotic shock. Cross protection between stresses indicated a clear hierarchy of resistance with salt protecting against hydrogen peroxide, which protected against ethanol, which protected against heat shock. The cultures were shown to be most sensitive to heat, ethanol and oxidative stress at mid-exponential phase and to become resistant at stationary phase. Adaptive levels of stressor were found to induce synthesis of general stress and stress-specific proteins and differential accumulation of proteins was demonstrated between heat- or salt-stressed and unstressed cells. CONCLUSIONS: Sequencing revealed that a number of glycolytic enzymes were regulated by heat and osmotic shocks and that the chaperone GroEL was induced by heat shock. SIGNIFICANCE AND IMPACT OF THE STUDY: The implications of the physiological data in designing storage and processing conditions for food are discussed. The identification of stress-regulated proteins reveals a clear role for glycolysis in adaptation to heat shock and osmotic stress.

The oxidative stress response in Bacillus subtilis.

Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10-30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.

Gene structure and amino acid sequences of alcohol dehydrogenases of Bacillus stearothermophilus.

Partial amino acid sequences of the two alcohol dehydrogenases of Bacillus stearothermophilus and the oligonucleotide sequence of a cloned fragment containing the gene for ADH 2334 were determined and compared with the known, derived ADH 1503 amino acid sequence. The two proteins are identical at 244 of 349 positions. ADH 2334 is encoded in a transcription unit containing an aldehyde dehydrogenase.

The gene coding for polynucleotide phosphorylase in Photorhabdus sp. strain K122 is induced at low temperatures.

Photorhabdus sp. strain K122 was found to produce higher levels of the protein CAP87K when cultured at 9 degrees C than when cultured at 28 degrees C. NH2-terminal sequencing of this protein revealed homology with the NH2 terminus of Escherichia coli polynucleotide phosphorylase. A 4.5-kb DNA fragment from strain K122 was cloned and sequenced and found to have 75% identity to the E. coli rpsO-pnp operon coding for ribosomal protein S15 and polynucleotide phosphorylase, respectively. Predicted proteins encoded by this sequence were found to have 86% identity with ribosomal protein S15 and polynucleotide phosphorylase from E. coli, and the genes were called rpsO and pnp, respectively. Quantitation of rpsO and pnp mRNA transcripts from K122 revealed that there was a 2.4-fold increase in the level of pnp mRNA and a 1.9-fold decrease in the level of rpsO mRNA at 9 degrees C relative to 28 degrees C. Primer extension analysis revealed the positions of possible promoters controlling the expression of rpsO and pnp in K122, suggesting that the genes are expressed independently. The increase in the level of pnp mRNA at 9 degrees C was not due to any relative increase in its stability compared with that of the rpsO transcript. However, there was evidence to suggest that it may be a result of a cold-inducible promoter, P2, in the intergenic region between rpsO and pnp. Several features of P2 support the suggestion that it may be cold inducible.

Isolation and characterization of a hydrogen peroxide resistant mutant of Bacillus subtilis.

A mutant of Bacillus subtilis has been isolated by continuous selection in increasing concentrations of H2O2. It grew with a doubling time of 85 min in minimal medium containing 150 mM H2O2, whereas the wild-type parent lysed in 100 mM H2O2. The mutant was also more resistant to organic peroxides than the wild-type. Further resistance to H2O2 could not be induced by pretreatment with low concentrations of the oxidant. The mutant synthesized a number of proteins at a much higher rate than the wild-type, including constitutive synthesis of all of the proteins which were induced by H2O2 in the wild-type. Four of these proteins were sequenced; three were identified as catalase and two subunits of alkyl hydroperoxide reductase. Two proteins whose synthesis was repressed in the mutant were sequenced, and one was identified as flagellin. The mutant grew as non-flagellated, partially septate, filaments of cells, and fragments of flagella were seen in the surrounding medium.

The citrulline biosynthetic operon, argC-F, and a ribose transport operon, rbs, from Bacillus subtilis are negatively regulated by Spo0A.

A method is described here that can be used to identify operons whose expression is controlled by any particular regulator protein. This method was used to identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis. Twenty-eight strains were identified, each of which contains an operon-lacZ transcriptional fusion, negatively regulated, either directly or indirectly, by Spo0A. In one of these strains (CSA8), the lacZ gene is fused to the argC-F operon positioned at 100 degrees on the B. subtilis chromosome. The regulated expression of this operon by Spo0A-P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium. In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli. Expression of the ribose transport operon is directed by a single SigA-type promoter. Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late-exponential/transition phase of the growth cycle and this control is not mediated through the transition-state regulator, AbrB.

Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium.

The phenomenon of phase variation in the insect-pathogenic bacterium Xenorhabdus luminescens was investigated. Differential activity of the lipase enzyme (EC 3.1.1.3) was observed between the two phases of the bacteria. The enzyme was found to be secreted into the culture medium, and about five to six times greater specific activity was secreted by the primary phase than by the secondary form. The lipase gene (lip-1) was cloned and sequenced. The data imply that there is only a single Tween 80-utilizing lipase gene in X. luminescens K122. The sequence revealed a translation product of 645 amino acids, from which a hydrophobic leader sequence of 24 amino acids is removed during processing. The structure of the gene was shown to be the same in the primary and secondary forms of X. luminescens. In addition, transcription was found to start at the same position, 169 bp upstream of the translation initiation codon, in the two forms of the bacteria. Equal amounts of lipase RNA accumulated in the two forms, and at least as much lipase protein was secreted by the secondary form as by the primary. This suggests that the difference in specific activity between the enzymes secreted by the two phases probably arises from a posttranslational type of regulation.

Cloning and characterization of genes induced by hydrogen peroxide in Bacillus subtilis.

Transcriptional fusions of Bacillus subtilis DNA to the lacZ gene were screened for induction, initially by ethanol and then by hydrogen peroxide (H2O2). Two fusions were identified which were induced late following treatment with sublethal concentrations of H2O2 (100 microM). The oxy-1 promoter was induced 4-5-fold and mapped to 11 degrees while the oxy-2 promoter was induced 20-fold and mapped close to the right of the defective prophage PBSX, at about 120 degrees. The oxy-2 fusion was induced by mitomycin C as well as H2O2, which correlated with the induction of PBSX by these agents. This was probably not a transcriptional induction, but rather a consequence of the induction of PBSX replication extending into adjacent regions of the chromosome.

Regulation of the oxidative stress response by the hpr gene in Bacillus subtilis.

Bacillus subtilis mutants with null mutations in the spo0 A gene are resistant to oxidative stress during the exponential phase of growth. This resistance phenotype can be suppressed by mutations in the abrB gene, or in the hpr gene. Both of these gene products are negative regulatory proteins which are over-produced in a spo0 A strain, and the over-production of the hpr gene product results from over-production of the abrB gene product. The results suggested that the resistance to oxidative stress in a spo0 A strain is due to the lack of a protein directly controlled by the hpr negative regulator. Other mutations in the spo0 A gene conferring resistance to ethanol stress (eth) or suppressors of sporulation phenotypes (sof) had no effect on the sensitivity to oxidative stress of strains bearing them.

Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases).

Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena. Clones of C. flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus.

The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.

A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay ('e.l.i.s.a.') procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.

Oxidative stress and growth temperature in Bacillus subtilis.

Pretreatment of Bacillus subtilis with low concentrations of hydrogen peroxide protected the cells against the lethal effects of higher levels of oxidative stress. During the period of adaptation, eight proteins were induced, as detected by one-dimensional gel electrophoresis. Four of these proteins were the same size as four of the proteins induced by the temperature upshift. The range of proteins synthesized in response to an elevation in temperature depended both on the starting (lower) temperature and on the temperature to which the cells were shifted. Both catalase and superoxide dismutase were present at high levels in B. subtilis, but neither was induced by oxidative stress or temperature upshift. In fact, catalase activity was reduced after the temperature upshift.

Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis.

The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle. The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase. Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth. The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain. In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type. Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain. By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2. The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered.

Hexapeptide repeat structure in Dictyostelium spore coat protein.

The sequences of the NH2-termini of two spore coat proteins of Dictyostelium discoideum have been determined. One of them (SP60) consists of perfect hexapeptide repeats of the sequence Gly-Asp-Trp-Asn-Asn-Asx-. The sequence has some homology to the parvovirus capsid protein which does not display periodicity. The NH2-terminal sequence of the second protein, SP70, contains a modified amino acid in two positions and like SP60 is highly hydrophilic and acidic.