A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress.

The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so-called secretion stress. A two-component regulatory system, named CssR-CssS, was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha-amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface. This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane-bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.

Bacillus subtilis NhaC, an Na+/H+ antiporter, influences expression of the phoPR operon and production of alkaline phosphatases.

When Bacillus subtilis is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a beta-galactosidase gene transcriptionally fused to the inactivated target gene, was used to identify genes that influence the operation of the Pho regulon. One such gene was nhaC (previously yheL). In the absence of NhaC, growth and APase production were enhanced, while the production of other non-Pho-regulon secretory proteins (proteases and alpha-amylase) did not change. The influence of NhaC on growth, APase synthesis, and its own expression was dependent on the external Na+ concentration. Other monovalent cations such as Li+ or K+ had no effect. We propose a role for NhaC in the uptake of Na+. nhaC appears to be encoded by a monocistronic operon and, contrary to previous reports, is not in the same transcriptional unit as yheK, the gene immediately upstream. The increase in APase production was dependent on an active PhoR, the sensor kinase of the two-component system primarily responsible for controlling the Pho regulon. Transcriptional fusions showed that the phoPR operon and both phoA (encoding APaseA) and phoB (encoding APaseB) were hyperinduced in the absence of NhaC and repressed when this protein was overproduced. This suggests that NhaC effects APase production via phoPR.

YsxC, a putative GTP-binding protein essential for growth of Bacillus subtilis 168.

YsxC is a member of a family of GTP-binding proteins carried by a diverse range of organisms from bacteria to yeasts, plants, and humans. To resolve the issue of whether ysxC of Bacillus subtilis is essential for growth, we attempted to construct mutants in which ysxC was either inactivated or placed under the control of an inducible promoter. Viable mutants were obtained only in the latter case, and these were inducer dependent, demonstrating unambiguously that ysxC is an essential gene.

The value of pyrolysis mass spectrometry to investigate nosocomial outbreaks caused by Serratia marcescens.

Simultaneous outbreaks of S. marcescens infection going on in the Neonatal Intensive Care Unit and the Surgical Department of the same hospital were investigated by pyrolysis mass spectrometry (PyMS). The PyMS analysis of the strains clearly demonstrated that the two outbreaks were caused by different strains. The 14 S. marcescens isolates from the first outbreak were closely related, with the exception of one environmental isolate, which did not harbour the ESBL plasmid, which was present in all other isolates. However, the phage type of all 14 isolates was the same. Among the 9 S. marcescens isolates from the second outbreak, PyMS clearly distinguished 3 that exhibited gentamicin resistance from the remaining 6 gentamicin-susceptible isolates. Phage typing was unhelpful in this case, as none of the isolates were typable. The PyMS typing of nosocomial outbreak strains can reach the level of discrimination approaching that achieved by molecular genetic analysis.

Expression of a new operon from Bacillus subtilis, ykzB-ykoL, under the control of the TnrA and PhoP-phoR global regulators.

The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The ykzB and tnrA genes are contiguous and transcribed divergently. Expression of ykzB and ykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter. In addition, transcription of tnrA depends on the presence of the TnrAB1 binding site. The ykzB and ykoL genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension. ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between ykoL and ykzB. Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter. These boxes are probably recognized by PhoP approximately P during the activation of transcription in phosphate limitation conditions.

The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium bacillus subtilis. Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein.

Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing by signal peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II. The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions. Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II. Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins. These forms of PrsA seem to have a reduced activity, as the secretion of alpha-amylase was strongly impaired. Unexpectedly, type I signal peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II. In conclusion, processing of lipoproteins by SPase II in B. subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria.

Investigation of the presence of different broad-spectrum beta-lactamases among clinical isolates of Enterobacteriacae.

Chromosomal or plasmid-encoded beta-lactamases are the most frequent causes of resistance to broad-spectrum beta-lactam antibiotics in clinical isolates of Gram-negative bacteria. Different screening methods can be used for their detection during routine laboratory work, while molecular biological methods may help in the detection of the genetic background of the phenotypic resistance. Clinical isolates of Klebsiella pneumoniae (170) and Enterobacter cloaceae (82) were obtained from different parts of Hungary, whereas those of Serratia marcescens (15) were isolated in our Department from a nosocomial outbreak. Disk diffusion and the Etest were used to screen inducible Class C beta-lactamase and plasmid-mediated extended spectrum beta-lactamases (ESBLs) among clinical isolates of Enterobacteriaceae. Single-strand conformation polymorphism (SSCP) analysis of the PCR products obtained after using SHV-specific primers revealed the presence of SHV-2 and SHV-5 ESBL among 170 K. pneumoniae strains in 12 and 3 cases, respectively. The results of the screening methods and the PCR-SSCP analysis suggested that 14 of the 15 S. marcescens strains not only produced the Class C, inducible chromosomal beta-lactamase, but also acquired a plasmid-mediated SHV-2-type ESBL. One strain isolated from the environment during the outbreak was genetically related to the other isolates, as demonstrated by the different typing methods, but it did not produce ESBL. The in vivo transfer of SHV-2 gene was assumed from an SHV-2 positive K. pneumoniae strain present in the same ward, in the same patient and at the same time. A very high prevalence of the stable derepressed mutants of E. cloaceae was confirmed among the Hungarian isolates. Seventy seven per cent of the strains produced high amounts of beta-lactamase without induction being responsible for their resistance to third-generation cephalosporins. Nineteen per cent of the strains were inducible when cefoxitin or imipenem was used, as confirmed by direct measurement of the MICs with the Etest.

In-vitro selection of porin-deficient mutants of two strains of Klebsiella pneumoniae with reduced susceptibilities to meropenem, but not to imipenem.

We have evaluated the ability of imipenem and meropenem to select, in vitro, resistant mutants of two clinical isolates of Klebsiella pneumoniae producing both SHV and TEM beta-lactamases. Only meropenem selected mutants of both isolates for which the MICs of meropenem, but not imipenem, were markedly higher than those for the parent strains; the MICs of several other beta-lactam antibiotics, including beta-lactam/beta-lactamase inhibitor combinations, for these mutants were also higher than those for the parent strains. In contrast, the MICs for the imipenem-selected mutants were the same as, or similar to, those for the parent strains. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed that an outer membrane protein in both parent strains was absent in the meropenem-selected mutants, but not in the imipenem-selected mutants. This protein is likely to be a porin, the absence of which is presumably associated with impaired beta-lactam permeability and, therefore, the reduced susceptibilities to these antibiotics exhibited by the mutant strains. We believe that this is the first report of the in-vitro selection of porin-deficient mutants of K. pneumoniae following exposure to meropenem.

Cloning and expression of a beta-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme.

A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA, was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 degrees C) optima were determined.

Transposon Tn917PF1 mutagenesis in Bacillus licheniformis.

The plasmid pTnPFl containing the transposon Tn917PF1 was introduced into the protoplasts of two Bacillus licheniformis strains in the presence of polyethylene glycol. Transpositions were produced at high temperature which inhibited plasmid replication and kanamycin was used for selection.Transposon Tn917PF1 was inserted randomly into the bacterial chromosome,producing different auxotrophic, prophage BLF and bacitracin-non-producing mutants. The auxotrophic mutant phenotypes were characterized by the Holliday-test and some mutations by hybridization with a transposon DNA probe. Insertions for the entire chromosome or for the prophage genophore were found at random, but preferred target sites were detected within limited regions, like the bacitracin synthetase or sulphate reductase genes. The partial physical map of the chromosomal region of bacitracin synthetase was constructed based on the hybridization patterns of insertion mutants.