Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli

The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.     

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.     

Structure and expression of genes coding for xylan-degrading enzymes of Bacillus pumilus

The complete nucleotide sequence of the beta-xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617-bp open reading frame for beta-xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N-terminal region and the molecular mass 62607 Da) of the beta-xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine-Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG. The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3' end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197-201]. The results of the Northern hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5' and 3' ends of the xynA and xynB gene were mapped with nuclease S1. The '-10' regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the '-35' regions were different from all the known promoters for B. subtilis RNA polymerases.     

Expression of a Streptomyces plasmid promoter in Escherichia coli

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.