Genetic mapping of a linked cluster of ribosomal ribonucleic acid genes in Bacillus subtilis.

A ribosomal ribonucleic acid gene set consisting of genes for 16S, 23S, 5S, and 4S ribonucleic acid species has been genetically mapped to a position between the markers recG13 and abrB74 on the Bacillus subtilis chromosome and designated rrnA. A ribosomal mutation, ksgA, was found to be linked to rrnA. This places rrnA in a region of the chromosome where ribosome-related genes occur but that is not directly adjacent to the major cluster of ribosome-related markers.     

A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis.

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.     

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.     

Electron microscope mapping of the distribution of ribosomal genes of the Bacillus subtilis chromosome

When high molecular weight Bacillus subtilis DNA is denatured, renatured and then examined by electron microscopy the main renatured product seen is a long duplex, usually with a single strand at each end, due to in-register renaturation. In addition, structures containing short duplex segments of length 4830 ± 250 base pairs, with two single-strand arms at each end, are seen at a low frequency. Several lines of evidence support the hypothesis that these short duplex segments are formed by out-of-register renaturation of the 16 S + 23 S ribosomal RNA genes (rDNA) of B. subtilis. They are of the correct length. Their formation is inhibited if homologous, but not if heterologous, ribosomal RNA is added to the hybridization mixture. The frequency of occurrence of the duplex structures is consistent with the rDNA hypothesis. Heteroduplex molecules are seen with two or three rDNA duplex segments separated by single-strand substitution loops with specific lengths for each of the two single-strand arms of any one loop. On the basis of these structures, linkage groups containing seven or nine rDNA sets (each set containing one 16 S and one 23 S rDNA gene) separated by spacer DNA's are proposed. All of the 16 S rDNA genes are linked to 23 8 rDNA and vice versa with little or no spacer DNA between a 16 S and 23 S sequence. If 5 S DNA is present in the set, any spacer between it and the other ribosomal RNA gene must also be short. The prophage SPO2 bacterial att site maps at a distance of 6200 bases away from a 16 S + 23 S rDNA set, which is itself separated by a very short spacer (less than 600 bases) from a second rDNA set.     

Order of ribosomal protein genes in the Rif cluster of Bacillus subtilis is identical to that of Escherichia coli

Mutants of Bacillus subtilis with electrophoretic variants of ribosomal protein L1, L5, L9, or L11 were used to determine the order of the genes for these proteins by transformation experiments. The proteins are homologous with Escherichia coli proteins L1, L10, L12, and L11, respectively; using the gene locus designations based on this correspondence, we determined the order of the loci to be cysA-rplK-rplA-rplJ-rplL-rpoB. The order of the last five loci was identical to that of E. coli.     

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster.

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome.     

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.     

Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4

Summary A mutation causing an alteration in Bacillus subtilis ribosomal protein S4 was mapped by transformation and PBS-1 transduction to a site between aroG and argA, a region of the B. subtilis chromosome not previously demonstrated to contain ribosomal protein genes. The S4 mutation conferred a spore-plus phenotype in a streptomycinresistant, spore-minus genetic background. The altered protein was detectable by polyacrylamide gel electrophoresis of ribosomal proteins of recombinants scored for the sporeplus phenotype in genetic crosses.     

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.     

Mapping by interspecies transformation experiments of several ribosomal protein genes near the replication origin of Bacillus subtilis chromosome.

Summary Bacillus subtilis 168 was transformed with DNAs from B. amyloliquefaciens K or B. licheniformis IAM 11054. These two species show a considerable difference in ribosomal proteins from B. subtilis. Analyses of the transformants indicated that the genes for 16 proteins, S3, S5, S8, S12, S17, S19, BL4, BL5, BL6, BL8, BL14, BL16, BL17, BL22, BL23 and BL25 are located in the cysA-str-spc region on B. subtilis chromosome. The genes for 10 proteins, S4, S6, S13, S16, S20, BL15, BL18, BL20, BL24 and BL28 could not be found in this region in the prsent experiments.     

Transformation mapping of the genes controlling tryptophan biosynthesis in Bacillus subtilis

Forty tryptophan auxotrophs of Bacillus subtilis have been placed in six phenotypic classes on the basis of growth responses, accumulation properties, and, in some cases, specific enzymatic defects. Three-point transformation crosses between representative mutants of the six different types have permitted the determination of the orders of the gene loci. In addition, mutational site orders for mutants within each of the classes have been determined by the same techniques. The organization of the cluster of genes controlling tryptophan biosynthesis in B. subtilis appears to be essentially analogous to that of Escherichia coli and Salmonella typhimurium.     

The general stress protein Ctc of Bacillus subtilis is a ribosomal protein

Cells respond to stress conditions by synthesizing general or specific stress proteins. The Ctc protein of Bacillus subtilis belongs to the general stress proteins. The synthesis of Ctc is controlled by an alternative sigma factor of RNA polymerase, sigmaB. Sequence analyses revealed that Ctc is composed of two domains, an N-terminal domain similar to the ribosomal protein L25 of Escherichia coli, and a C-terminal domain. The similarity of the N-terminal domain of Ctc to L25 suggested that Ctc might be a ribosomal protein in B. subtilis. The function of the C-terminal domain is unknown. We purified Ctc to homogeneity and used the pure protein to raise antibodies. Western blot analyses demonstrate that Ctc is induced under stress conditions and can be found in ribosomes of B. subtilis. As observed for its E. coli counterpart L25, Ctc is capable of binding 5S ribosomal RNA in a specific manner. The stress-specific localization of Ctc in B. subtilis ribosomes and the sporulation defect of ctc mutants at high temperatures suggest that Ctc might be required for accurate translation under stress conditions.     

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.     

UGA suppressor that maps within a cluster of ribosomal protein genes.

A suppressor of UGA mutations (supU) maps near or within a cluster of ribosomal protein genes at 72 min on the Salmonella typhimurium genetic map. The suppressor is relatively inefficient, and its activity is abolished by rpsL (formerly strA) mutations. The suppressor is dominant to a wild-type supU allele. The map position of this suppressor suggests that it may owe its activity to an alteration of ribosome structure.