Sequence Analysis of the Bacillus subtilis 168 Chromosome Region between the sspC and odhA Loci (184{degrees}-180{degrees})

The nucleotide sequence of 45,389 bp in the 184°-;180°region of the Bacillus subtilis chromosome, containing the cgecluster, which is controlled by the sporulation regulatory proteinGerE, was determined. Fifty-four putative ORFs with putativeribosome-binding sites were recognized. Seven of them correspondto previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE,ctpA, and odhA. The deduced products of 25 ORFs were found todisplay significant similarities to proteins in the data banks.We have identified genes involved in detoxification, cell walls,and in the metabolism of biotins, purines, fatty acids, carbohydratesand amino acids. The remaining 22 ORFs showed no similarityto known proteins. Both an attachment site of the SPßprophage and 2 new putative DNA replication terminators wereidentified in this region.     

Sequence Analysis of a 25-kb Segment in the 17{degrees}-19{degrees} Region of the Bacillus subtilis Chromosome Containing ada Locus

As a part of the Bacillus subtilis genome sequencing project,we have determined a 25-kb sequence covering the 17°–19°region. This region contains 26 complete open reading frames(ORFs) including the alkA and adaA/B operon, which encode genesfor adaptive response to DNA alkylation. A homology search forthe newly identified 21 ORFs revealed that 4 of them exhibita significant similarity to known proteins, e.g., methicillin-resistantStaphylococcus aureus (MRSA) protein homolog, proteins involvedin chloramphenicol resistance, glucosamine synthase and an ABCtransporter protein. The remaining 17 ORFs did not show anysignificant sequence similarities to known gene products inthe database.     

Cloning and Sequencing of a 27.8-kb Nucleotide Sequence of the 79{degrees}-81{degrees}. Region of the Bacillus subtilis Genome Containing the sspE Locus

The nucleotide sequence of a 27830-bp DNA segment in the 79°–81°.region of the Bacillus subtilis genome has been determined.This region contains 29 complete ORFs including the sspE gene,which encodes a small acid-soluble spore protein gamma and locateson the one side terminal of our assigned region. A homologysearch for the products deduced from the 29 ORFs revealed thatnine of them exhibit significant similarity to known proteins,e.g. proteins involved in an iron uptake system, a multidrugresistance protein, a chloramphenicol resistance protein, epoxidehydrolase, adenine glycosylase, and a glucose-1-dehydrogenasehomolog.     

Sequence Analysis of the groESL-cotA Region of the Bacillus subtilis Genome, Containing the Restriction/Modification System Genes

We have determined a 35-kb sequence of the groESL-gutR-cotA(45°–52°) region of the Bacillus subtilis genome.In addition to the groESL, gutRB and cotA genes reported previously,we have newly identified 24 ORFs including gutA and fruC genes,encoding glucitol permease and fructokinase, respectively. Theinherent restriction/modification system genes, hsdMR and hsdMM,were mapped between groESL and gutRB, and we have identifiedtwo open reading frames (ORFs) encoding 5-methylcytosine formingDNA methyl transferase and an operon probably encoding a restrictionenzyme complex. The unusual genome structure of few ORFs andlower GC content around the restriction/modification genes stronglysuggests that the region originated from a bacteriophage integratedduring evolution.     

Systematic Sequencing of the 180 Kilobase Region of the Bacillus Subtilis Chromosome Containing the Replication Origin

We have determined a 180 kb contiguous sequence in the replicationorigin region of the Bacillus subtilis chromosome. Open readingframes (ORF) in this region were unambiguously identified fromthe determined sequence, using criteria characteristic for theB. subtilis gene structure, i.e., starting with an ATG, GTGor TTG codon preceded by sequences complementary to the 3' endof the 16S rRNA. Four rRNA gene sets, 7 individual tRNA genesand 1 scRNA gene were identified, occupying 20 kb in total.In the remaining 160 kb region, 158 ORFs were identified, suggestingthat 1 ORF is coded on average by 1 kb of DNA of the B. subtilisgenome. Among the 158 ORFs, the functions of 48 ORFs were assignedand those of 11 ORFs are suggested through significant similaritiesto known proteins present in data banks. However, the functionsof more than half of the ORFs (63%) remain to be determined.     

Sequential Insertion of Multiple I-SceI Recognition Sites at Designed Loci of the Bacillus subtilis 168 Genome

Bacillus subtilis 168 is the only bacterium-based host serving for the cloning of giant DNA above 1.000 kbp. As rapid verification of the genome structure is crucial during the cloning process, six of 18-base sequence recognized by endonuclease I-SceI were sequentially created in the B. subtilis 168 genome. The established method and materials should be of use for other B. subtilis derivatives.     

Insertion of Unmarked DNA Sequences in Multiple Loci of the Bacillus subtilis 168 Genome: an Efficient Selection Method

The construction process of Bacillus subtilis strain with multiple mutations is accelerated by simultaneous use of a positive selection method. All the strains selected by neomycin acquired two simultaneous mutations at different genomic loci. The extremely low rate of false positives was accounted for by employing the improved method.     

Sequence Analysis of a 32-kb Region Including the Major Ribosomal Protein Gene Clusters from Alkaliphilic Bacillus sp. Strain C-125

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.     

Structure and Expression of the Tobacco Nuclear Gene Encoding RNA-binding Protein RZ-1: The Existence of an Intron in the 3'-Untranslated Region

We have previously characterized a tobacco cDNA encoding a noveltype RNA-binding protein (RZ-1), which contains a zinc fingermotif in addition to a consensus sequence-type RNA-binding domainand is localized in the nucleus. Here we isolated its genomicclone from a Nicotiana sylvestris genomic library. Southernblot analysis suggested that RZ-1 is coded for by a single locusper haploid genome. Comparison of the cDNA and genomic sequencesindicated that the RZ-1 gene contains two introns, one in thecoding region and another in the 3'-untranslated region. RT-PCRand ribonuclease protection analyses showed that splicing ofRZ-1 pre-mRNA occurs efficiently. The RZ-1 protein is activelysynthesized in rapidly dividing tobacco cells, as demonstratedby immunoblot analysis.