Physical map of the Brucella melitensis 16 M chromosome

Abstract

We present the first restriction map of the BruceUa melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G+C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome.Physical mapping of the bacterial chromosome has been performed for several species. The first was established in Escherichia coli by two methods, one using low-frequency cleavage restriction endonucleases (23) and the other by chromosome walking with overlapping recombinant bacteriophages (15). According to the latter method, which involves a comparison of the physical and genetic maps, every genetic marker may be located in a single clone and the corresponding genes are easily isolated and studied. Physical mapping is even more important when the genetics of the bacterial species are poorly understood. In such cases, physical mapping can be used as an initial approach to their genetic study, as demonstrated recently with mycoplasma (3,22,29) and Haemophilus influenzae (14,16). The genetics of Brucella, like that of these two bacteria, are completely unknown for two reasons. First, the growth of Brucella is fastidious and, moreover, the only means of studying its strictly aerobic metabolism consists of complex and timeconsuming methods, so the isolation and characterization of nutritional mutants are difficult. Second, chromosome exchanges, which have been so useful for the genetic mapping of E. coli and Salmonella typhimurium, are unknown in Brucella. Transformation has never been described in this species, and conjugation or transduction is certainly impossible since we, and other authors (6), have failed to demonstrate the presence of plasmids or temperate bacteriophages in these bacteria. Consequently, physical mapping of the Brucella chromosome may be a first step in their genetic study, since several genes corresponding to proteins of the outer membrane (10), or playing a major role in the immunological response (19), and to the 16S rRNA (7) have been cloned and sequenced.In an initial study, we analyzed the genomes of several Brucella species by low-frequency cleavage restriction endonucleases and estimated its size as being at least 2,600 kb (1). During this study, three endonucleases (XbaI, Spel, and XhoI) were demonstrated to cut the Brucella DNA into less than 33 fragments. Hence, it was possible to map the chromosome physically by an adaptation of the method described previously by Lee et al. (16) DNA preparation and restriction dige...

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