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

Abstract

We have determined a 180 kb contiguous sequence in the replication origin region of the Bacillus subtilis chromosome. Open reading frames (ORF) in this region were unambiguously identified from the determined sequence, using criteria characteristic for the B. subtilis gene structure, i.e., starting with an ATG, GTG or TTG codon preceded by sequences complementary to the 3' end of the 16S rRNA. Four rRNA gene sets, 7 individual tRNA genes and 1 scRNA gene were identified, occupying 20 kb in total. In the remaining 160 kb region, 158 ORFs were identified, suggesting that 1 ORF is coded on average by 1 kb of DNA of the B. subtilis genome. Among the 158 ORFs, the functions of 48 ORFs were assigned and those of 11 ORFs are suggested through significant similarities to known proteins present in data banks. However, the functions of more than half of the ORFs (63%) remain to be determined.

Sequencing and functional analysis of the genome of Bacillus subtilis strain 168

“…Whilst cosmid libraries have been used to create overlapping libraries of individual Saccharomyces cerevisiae [23] and Caenorhabditis elegans [24] chromosomes and for the genome of Mycobacterium leprae [25], and lambda libraries for the E. coli genome [26], it has proved impossible to construct cosmid or lambda libraries that completely cover the B. subtilis chromosome. This is because large fragments of B. subtilis DNA have been found to be unstable in E. coli due to their high AT content (43% GC) and the elevated levels of expression of many B. subtilis genes in this host [27,28].…”

General Structure/Function Properties of Microbial Methionyl‐Trna Synthetases

“…Alignment of MetRS sequences from Bacillus subtilis (Ogasarawa et al, 1994), Thermus thermophilus (Nureki et al, 1991), Haemophilus influenzae (Fleishman et al, 1995), Mycoplasma genitulium (Fraser et al, 1995), yeast cytoplasm (Walter et al, 1983) and the yeast mitochondrion (Tzagoloff et al, 1989) actually shows that the peptide regions studied in the present work are conserved (Fig. 1).…”

Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes

“…The sequencing of the entire genome of Bacillzls szlbtilis is greatly impaired by the difficulty in cloning its DNA in standard Escbericbia coli vectors (Glaser et al, 1993;Ogasawara et al ,1994). This fact specifically hindered the report here data on LA PCR mapping of the 150 kb region between the spoIIIC andpheA loci of B. szlbtilis (for a recent version of the B. szlbtilis genetic map, see Anagnostopoulos et al , 1993).…”

“…B.-szlbtilis genome project and obliged the participants to use PCR for closing uncloneable gaps (Glaser e t al., 1993;Ogasawara et al , 1994 ;Sorokin e t al., 1993). T o provide a general solution to this problem, a strategy based on the use of yeast artificial chromosomes (YACs) was developed in this laboratory (Sorokin et al, 1996a).…”