Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1?% of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8-534?bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515-1541?bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia.     

Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter

A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Organization of transfer and ribosomal ribonucleic acid genes in Bacillus subtilis.

The structural relationship between the transfer ribonucleic acid (tRNA) and the ribosomal RNA (rRNA) genes of Bacillus subtilis has been studied by restriction endonuclease analysis of total chromosomal deoxyribonucleic acid (DNA) and characterization of DNA fragments cloned in Escherichia coli. The DNA sequences encoding rRNA and tRNA were assayed by hybridization to radioactive RNA. The results support the conclusion that the tRNA genes are interspersed between and closely linked to the rRNA genes of B. subtilis. They probably do not appear between the 16S and 23S rRNA genes as in E. coli.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

tRNA genes are found between 16S and 23S rRNA genes in Bacillus subtilis.

There are at least nine, and probably ten, ribosomal RNA gene sets in the genome of Bacillus subtilis. Each gene set contains sequences complementary to 16S, 23S and 5S rRNAs. We have determined the nucleotide sequences of two DNA fragments which each contain 165 base pairs of the 16S rRNA gene, 191 base pairs of the 23S rRNA gene, and the spacer region between them. The smaller space region is 164 base pairs in length and the larger one includes an additional 180 base pairs. The extra nucleotides could be transcribed in tRNAIIe and tRNA Ala sequences. Evidence is also presented for the existence of a second spacer region which also contains tRNAIIe and tRNA Ala sequences. No other tRNAs appear to be encoded in the spacer regions between the 16S and 23S rRNA genes. Whereas the nucleotide sequences corresponding to the 16S rRNA, 23S rRNA and the spacer tRNAs are very similar to those of E. coli, the sequences between these structural genes are very different.