Calcium signalling in Bacillus subtilis.

Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

Integrated mapping and sequencing of a 115 kb DNA fragment from Bacillus subtilis: sequence analysis of a 21 kb segment containing the sigL locus.

A sequence strategy which combines a low redundancy shotgun approach and directed sequencing has been elaborated. Essentially, the sequences, as well as the size of the fragments utilized for a low coverage shotgun approach, were exploited for the construction of a physical map of the region to be sequenced. The latter considerably simplified the subsequent directed sequencing steps. We report the physical mapping of a 115 kb segment which covers nearly 100 kb of the hisA-cysB region of the Bacillus subtilis chromosome and contains previously sequenced genes sigL and sacB. Sequencing and analysis of a 21305 bp segment, which includes the sigL locus, revealed 21 ORFs, apparently belonging to at least seven transcription units. This segment has a G + C content greater than 47%, compared to 43% characteristic of the flanking regions, and mainly consists of genes whose products seem to be involved in the synthesis of an exopolysaccharide. These observations leave open the possibility that the analysed fragment has been acquired through horizontal transfer.

Analysis of errors in finished DNA sequences: the surfactin operon of Bacillus subtilis as an example.

Increased productivity in DNA sequencing would not be valid without a straightforward detection and estimation of errors in finished sequences. The sequence of the surfactin operon from Bacillus subtilis was obtained by two different groups and by chance we were also working on the same chromosome region. Taking advantage of this situation we report in this paper, the number and nature of errors found in the overlapping part of the DNA sequences obtained by the three laboratories. The coincidence of some of the errors with compression in sequence ladders and with secondary DNA structures as well as the detection of frameshift errors using computer programs, are demonstrated. Finally we discuss the definition of a new sequencing strategy that might minimize both the error rate and the cost of sequencing.

Sequence analysis of the cellulase-encoding celY gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer.

The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced. The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme. No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er. chrysanthemi major endoglucanase, EGZ, or the Er. carotovora CelS endoglucanase. In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er. chrysanthemi. By comparing the molar G + C% of the cellulase-encoding genes and that of Er. chrysanthemi and C. uda chromosomal DNAs, we speculate that celY was transferred from Er. chrysanthemi to C. uda.

Mapping and regulation of the cel genes in Erwinia chrysanthemi.

Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange between the plasmidic mutated and the wild-type chromosomal alleles. These mutations allowed mapping of celZ between ura and pan and celY between xyl and met on the linkage map of E. chrysanthemi. The beta-galactosidase activity of these strains indicated that celZ is expressed in the late exponential and stationary growth phases, while celY expression is almost undetectable.

Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus.

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2-terminal end. These regions may be involved in substrate binding and/or catalytic sites.