Chapter 30 : Physical Map of the Bacillus subtilis 168 Chromosome

Conversion of nucleotide sequences on the B. subtilis chromosome. Circles represent the B. subtilis chromosome. Two hypothetical genes, pay and tax, are in boxed regions, and cat and neo denote the drug resistance gene cassettes. The DNA segments used in transformation steps I through III are prepared in E. coli and linearized, x x indicates a double crossover followed by homologous recombination. A net change that the last strain acquires is only the pay mutations. Details are given in the text.

Strategy for determination of restriction enzyme sites on the chromosome. Circles represent the B. subtilis chromosome, and short bars around the circles represent recognition sites of restriction enzymes. The sites can be eradicated, i.e., converted to other sequences, from the chromosome by using the gene-directed mutagenesis method described in the legend to Fig. 1 . The method can be sequentially applied, and the number of recognition sites can be decreased progressively from left to right. Patterns of fragments after enzyme digestion and gel electrophoresis are shown at the bottom. Dotted lines indicate changes of fragments resulting from the loss of the recognition site. Details are described in reference 15 .

Separation of B. subtilis chromosome DNA by PFGE after SfiI or NotI digestion. (Left) Lane 1, concatemeric lambda DNA size markers. Chromosomal DNA from CU741, a wild-type B. subtilis, was digested with NotI (lane 2), SfiI (lane 3), or DNA from Saccharomyces cerevisiae (lane 4). (Right) Lanes 1, 5, and 9 contain concatemeric lambda DNA plus lambda DNA digested with HindIII as size standards. Noil-digested chromosomal DNA was from CU741 (lane 2), BEST4041 (lane 3), or BEST4101 (lane 4). SfiI-digested chromosomal DNA was from OA101 (lane 6), BEST3015 (lane 7), or BEST3019 (lane 8). Sizes (in kilobase pairs) are in parentheses. SfiI or NotI generates 26 or more than 80 fragments. Designations for each fragment are given in Table 1 . Derivatives constructed by progressive removal of NotI (BEST4041 and BEST4101) or SfiI (BEST3015 and BEST3019) are described in reference 15.

SfiI and NotI physical map of the B. subtilis 168 chromosome. The map was redrawn on the basis of data in reference 15. Total genome size is 4,165 kb, which is divided into five linearized parts (1,000 kb each). Short horizontal bars across the vertical lines mark SfiI or NotI sites. Physical distances (in kilobases) are indicated on the left. Locations of genes at arrowheads had been precisely determined and were used as landmarks on the physical map ( Table 2 and text). Genes whose locations on both SfiI and NotI fragments have been determined are included beside the NotI segments ( Table 2 ). Open triangles indicate 23-kb deletions found in the standard strain, CU741, used for map construction ( 11 ; see text). Two weakly cleavable SfiI sites are indicated by boldface arrows. The region of the SPβ prophage is boxed.

Correlation between the genetic and physical maps. Genetic map information given in Table 2 is included. Closed circles indicate gene loci whose precise physical locations have been determined. Vertical bars or open circles represent DNA regions to which cloned genes have been assigned by the results of Southern hybridization. Open arrows indicate regions where the physical location is slightly displaced from that on the genetic map.

Stability and asymmetry of the B. subtilis 168 chromosome. Locations of the oriC site (at 39 kb) and the terC site (at kb 2012) and regions for naturally occurring DNA deletions on the B. subtilis 168 chromosome (regions I, II, and III) are indicated by arrows. The genome size for B. subtilis 168 (strain CU741) was estimated to be 4,165 kb (see details in the text and Fig. 4 ). The broken circle indicates the lengths for the clockwise segment (1,973 kb) and the counterclockwise segment (2,192 kb).