A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin.Clostridium beijerinckii (formerly Clostridium acetobutylicum) is an obligately anaerobic spore-forming bacterium that produces acetone, butanol, and small amounts of ethanol when grown on sugary substrates. This process, known as ABE fermentation, has been used extensively for the industrial scale production of acetone and butanol (28). Several different strains are currently under investigation in various laboratories worldwide. They have been separated into three distinct groups on the basis of their biochemical and physiological characteristics, the extent of hybridization between their DNAs, and macrorestriction fragment profiles of their genomes (54, 57). The NCIMB 8052 strain, which belongs to the group that has the largest chromosome, is amenable to genetic manipulation (59, 60). It lacks the active restriction system that is found in the ATCC 824 strain (38). The NCIMB 8052 strain has no indigenous plasmids, but it supports replication of a variety of vectors based on plasmids from Clostridium butyricum, Enterococcus faecalis, and Streptococcus cremoris (41). They may be introduced by either electroporation (46) or intergeneric conjugation with Escherichia coli donors (56). This strain is also susceptible to Tn1545 mutagenesis (58). Homologous recombination between incoming sequences and their counterparts in the bacterial chromosome has recently been demonstrated (55).As an additional aid to genetic analysis of this organism, we have constructed a physical map of the 6.7-Mbp chromosome using pulsed-field gel electrophoresis (PFGE) (50). The application of PFGE to the analysis of bacterial genomes has revealed that the majority of bacteria have single circular chromosomes. However, several exceptions to this general rule have been described. Some bacteria, such as Borrelia burgdorferi and Rhodococcus fascians, have linear chromosomes (4,20,24), whereas Streptomyces lividans and Streptomyces coelicolor have linear chromosomes that may become circular as a result of deletions spanning both telomeres (34,35). Still others, such as Brucella melitensis and Leptospira interrogans, have segmented genomes (40, 63). The segmented genome of Agrobacterium tumefaciens has one circular and one linear component (1). The results reported here indicate that the NCIMB 8052 strain of C. beijerinckii, like most othe...