Complex structure of the membrane nuclease of Streptococcus pneumoniae revealed by two-dimensional electrophoresis

Close contacts between Escherichia coli RNA polymerase and specific purine residues in the tryptophan (trp) operon promoter of Salmonella typhimurium were revealed using the methylating agent dimethyl sulfate. RNA polymerase bound to trp promoter DNA caused alterations in the rate of methylation at seven specific sites; in the anti-sense strand, guanine residues at positions ?37, ?34 and ?2 showed enhanced methylation, while those at positions ?14, ?6 and +3 showed reduced methylation. In the sense strand, only the guanine residue at ?32 showed reduced methylation. No RNA polymerase contacts with adenine residues were observed. Using the same method, close interactions between E. coli trp repressor and purine residues in the trp operator of S. typhimurium were examined. Bound trp repressor alters the methylation rates of both guanine and adenine residues from positions ?25 to +3. The points of contact are distributed rather symmetrically on both DNA strands. Three points of close contact are shared by RNA polymerase and trp repressor, supporting previous models of trp repressor action.     

Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31

Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli σ⁷⁰ and Bacillus subtilis σ⁴³ showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.     

Discrimination of promoters by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RNA polymerase σ<Superscript>A</Superscript> and σ<Superscript>H</Superscript> holoenzymes is associated with periodic patterns in promoter nucleotide sequences

Bacillus subtilis promoters recognized by σ^A and σ^H RNA polymerases were found to have different periodic patterns of nucleotide disposition. Using a special version of Fourier analysis for symbolic sequences, Fourier spectra were obtained for the primary structure of promoters used by the two holoenzymes. Stepwise discriminant analysis with jackknife testing was performed for two promoter data sets (small and large). Based on the spectral patterns of the nucleotide sequences, the data sets could be sorted with 100% accuracy into two classes: promoters recognized by Eσ^A and by Eσ^H. Correlations were obtained between the promoter strength and the characteristics of their Fourier spectra. Moreover, perfect separation was achieved even when the “consensus” ?35 and ?10 sites were replaced by sequences of “blank symbols.” Thus, the periodicity in nucleotide distribution along the DNA chain is itself an attribute sufficient for selective recognition of the cognate promoter by RNA polymerase.     

Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation

Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly-peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an Eσ^E-controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation-independent synthesis of glycogen occurred after integration of an Eσ^A controlled promoter upstream of the operon.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.