DnaK chaperone machine and trigger factor are only partially required for normal growth of Bacillus subtilis

While dnaK and tig are the essential components for nascent polypeptide folding in E. coli, deletion did not confer synthetic lethality in B. subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails. Likewise, survival at high temperature suffered dramatically, resulting from deletion of several sets of heat shock genes, thus during sudden stress various heat shock genes act synergistically to protect the proteins.     

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.     

Changes of sensitivity to heat shock during logarithmic growth of Bacillus subtilis

The effect of heat shock on B. subtilis was found to vary within the logarithmic growth phase. Depending on the age of the culture, all cells, or as little as less than 1% of the population, may survive heating for 6 min at 54 degrees C. These characteristic changes in sensitivity to heat shock were observed with B. subtilis grown on various media, as well as with E. coli. The increased sensitivity of B. subtilis to heat shock was observed within a rather narrow time span in the log phase. Preheating at 45 degrees C had a protective effect on the samples collected at the time of greatest heat sensitivity. It is suggested that besides heat shock proteins other factors are also involved in the processes leading to survival after heat shock.     

The use of phage lambda promotors for expressing genes of secreted proteins in Bacillus subtilis cells

At was shown with the help of promoterless alpha-amylase and staphylokinase genes that lambda PR and lambda PL promoters could be used in Bacillus subtilis. Promoters strength was compared to promoter of alpha-amylase gene, this enabled to order the promoters in a row: PAA greater than lambda PR greater than lambda PL. The lambda PR promoter region was controlled by temperature in E. coli cells only, but not in B. subtilis, therefore, the active lambda C1857 gene product was not produced in B. subtilis cells. The lambda PR promoter is used by B. subtilis at a later growth stage than PAA and the lambda PL promoter at a still later stage than lambda PR. The data enables lambda PR to be considered as quite useful for Bacilli.     

Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis]

Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B. subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli. The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B. subtilis and E. coli was studied by using mutant strains. It was established that the expression of these genes in recombinant B. subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E. coli cells is not controlled by the regulatory proteins PhoB or PhoR. Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B. subtilis, also function in other representatives of the genus Bacillus.     

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.     

Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.     

Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i). the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii). heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii). basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

The role of antioxidant systems in response of bacteria Escherichia coli to heat shock]

Shifting the temperature from 30 to 45 degrees C in an aerobic Escherichia coli culture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor. The expression was evaluated by measuring beta-galactosidase activity in E. coli strains that contained fusions of the antioxidant gene promoters with the lacZ operon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type and sodA sodB mutant cells to heat shock was almost the same. In the E. coli culture adapted to growth at 42 degrees C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30 degrees C. The temperature-adapted cells grown aerobically at 42 degrees C showed an increased ability to express the fused katG-lacZ genes.