Microbiology (1997), 143, 3431-3441 Printed in Great Britain Sequencing and functional annotation of the Bacillus subtilis genes in the 200 kb rrnB-dnaB region Alla Lapidus, Nathalie Galleron, Alexei Sorokin and 5. Dusko Ehrlich Author for correspondence: Alexei Sorokin. Tel: +33 1 34 65 25 33. Fax: +33 1 34 65 25 21. e-mail: sorokine@biotec.jouy.inra.fr Laboratoire de Genetique Microbienne, lnstitut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en- Josas cedex, France The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275O on the genetic map was sequenced. The strategy applied, based on use of yeast artificial chromosomes and multiplex Long Accurate PCR, proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (iii) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20,28,20,15 and 17, respectively. In the sequenced region, a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140" in the chromosome was also observed. Keywords : Bacillus subtilis genome sequencing, yeast artificial chromosome, Long Accurate PCR INTRODUCTION The chromosome region between rrnB and dnaB genetic locus was assigned to us for sequencing in the European Bacillus subtilis genome sequencing project (Kunst et al., 1995). Our approach to the sequencing of this genome is essentially based on using a yeast artificial chromosome (YAC) collection of ordered chromosomal segments (Azevedo et al., 1993). Sequencing of two YACs covering about 140 kb of contigous area was recently reported (Sorokin et al., 1996a; Capuano et al., 1996). This paper reports further development of this approach and its application to another two YACs, containing in total about 200 kb of new sequence. We report functional annotation of genes found according to the results of homology search in databases. Abbreviations: LR PCR, Long Range PCR; MLA PCR, multiplex Long Accurate PCR; YAC, yeast artificial chromosome. The GenBank accession number for the sequence reported in this paper is AF008220. METHODS Strains and growth conditions. Collection of yeast clones containing ordered segments of the B. subtilis genome cloned in YAC was described earlier (Azevedo et al., 1993). Yeast cells containing artificial chromosomes were grown as de- scribed (Azevedo et al., 1993). Escherichia coli JJC 128F', araD139 A(aru-leu)7696 galE15 galK16 A(lac)X74 hsdR hsdM' StrR F'[lacIq D(lacZ)M15 truD361 which is repro- ducibly electrotransformed by M13 DNA with an efficiency of 5 x 10' p.f.u. (pg DNA)-l, was used in YAC DNA cloning experiments. Electrocompetent cells of J Jl28F' were prepared according to the protocol described by Dower et al. (1988) and stored at -80 "C. E. coli TG1 K-12 A(1uc-pro) supE thi hsdR/F' truD36 proAB ladq AlacZ AM15 was used for propagation of M13 phages and for plasmid cloning experi- ments. Competent cells were prepared by 0.1 M CaCl, treatment of early exponential culture. B. subtilis 168 trpC2 strain was provided by C. Anagnostopoulos (INRA, Jouy-en- Josas, France). This strain, considered as standard for systematic B. subtilis genome sequencing, was used for chromosomal DNA preparations. The standard medium for E. coli and B. subtilis was 2YT (Sambrook et al., 1989). Isolation of bacterial chromosomal DNA. B. subtilis chromo- 0002-1947 0 1997 SGM 343 1