Mutations upstream of the ribosome-binding site affect translational efficiency

The DNA coding for the major outer membrane lipoprotein of Escherichia coli has been fused to the coding region of the beta-galactosidase gene to measure the effect of various mutations on the efficiency of translation initiation. The various mutants were made by either inserting or deleting a small number of nucleotides into or from a region just upstream of the ribosome-binding site. These small mutations dramatically affect translation initiation as measured by the production of beta-galactosidase. We postulate that these mutations affect translation initiation by altering the secondary structure of the messenger RNA. In one case, we predict that a stem and loop just upstream of the Shine-Dalgarno sequence sterically hinders the binding of the ribosome to the mRNA.     

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB

The ale gene specifying the subtilisin YaB produced by alkalophilic Bacillus YaB, has an unusual start codon UUG. Changing this codon to AUG and GUG increasedexpression of the ale gene in B. subtilis DB104 and in an ale deficient mutant strain YaB-DEC4. The relative translational efficiency order of the threeinitiation codons is AU G > GU G > UUG in B. subtilis DB104 and in YaB-DEC4. These data suggest that the preferred initiation codon is AUG for ale gene expression in Bacillus .     

The nucleotide sequence of Bacillus subtilis tRNA genes

Clones carring Bacillus subtilis tRNA genes were isolated from a lambda 816 library. A recombinant phage lambda 816-BS83 which was hybridized effectively with unfractionated tRNA probes contained a 3-kb fragment. By a Southern's blot analysis, it was found that tRNA genes were located in Eco RI-Hinc II region of this fragment. Sequence determination revealed the presence of a cluster of four tRNA genes in this region. The gene organization was as follows: tDNALys-9bp-tDNAGlu-81bp-tDNAAsp-30bp-tDNAPhe. The RNA sequences expected from tDNALys and tDNAPhe were identical with the reported RNA sequences. Two tRNA genes, tDNALys and tDNAAsp encoded the CCA sequence of 3'-terminal region, but the other two, tDNAGlu and tDNAPhe did not. A promoter-like sequence which corresponds to the sigma 55-recognition site was found in a region about 100bp upstream from tDNALys.     

枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

The nucleotide sequence of the rodC operon of Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature-sensitive phenotype of the rodC mutant is the result of a single base-pair change. A cytosine to thymine transition in the non-coding strand results in the replacement of a serine residue in the wild-type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate-glucose poly-(glycerol phosphate) alpha-glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild-type and mutant alleles of a morphogene in B. subtilis.     

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.     

The nucleotide sequence of phenylalanine tRNA from Bacillus subtilis

The nucleotide sequence of tRNA(Phe) from Bacillussubtilis W 23 has been determined using (32)P labeled tRNA. This is the second B. subtilis tRNA so far reported. The nucleotide sequence was found to be pG-G-C-U-C-G-G-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-A-C-G-G-A-C-U-Gm-A-A- ms(2)i(6)A-A-psi-C-C-G-U-G-U-m(7)G-U-C-G-G-C-G-G-T-psi- C-G-A-U-U-C-C-G-U-C-C-C-G-A-G-C-C-A-C-C-A(OH).Images