Physical and genetic map of the Clostridium saccharobutylicum (formerly Clostridium acetobutylicum) NCP 262 chromosome

A physical and genetic map of the Clostridium saccharobutylicum NCP 262 chr omosome was constructed. The order of macrorestriction fragments was determ ined by analysing fragments generated after single and double digestion wit h the restriction enzymes BssHII, I-Ceul, Sse83871, RsrII and SfiI and sepa ration by PFGE. The I-Ceul backbone of C. saccharobutylicum was constructed by indirect end-labelling with rrs- and 3' rrl-specific probes located on either side of the I-Ceul site in the rm operon, and reciprocal separation of BssHII and I-Ceul digestion products by two-dimensional PFGE. The positi ons of BssHII fragments on the physical map were determined using a library of linking clones containing BssHII cleavage sites. The size of the circul ar genome was estimated to be 5.3 Mb with a mean resolution of approximatel y 140 kb. The chromosome of C. saccharobutylicum contains 12 rm operons, lo cated on 46% of the chromosome, which are transcribed divergently from the deduced origin of replication. The genetic map was constructed by determini ng the location of 28 genes involved in house-keeping, heat-shock response, sporulation, electron transfer and acid- and solvent-formation. Comparison of the C. saccharobutylicum genetic map with those of the spore-forming ba cteria Bacillus subtilis, Clostridium acetobutylicum, Clostridium perfringe ns and Clostridium beijerinckii indicated C. saccharobutylicum to be most s imilar to the latter two Clostridium species, with the order of the genes w ithin the gyrAB and recA loci being conserved.