An insertion sequence (IS) element of Bruceffa ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobactevium tuberculosis. IS6501 is present in all Bruceffa strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. mefitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. mefitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods. As ISs have been shown to be implicated in chromosomal rearrangement, we propose that the chromosomal polymorphism revealed by PFGE and high copy number of IS6501 observed in B. ovis may be related to the presence of an active IS in this species.

Mapping the restriction fragments of the Brucella melitensis 16M genome with a new restriction endonuclease, Pacl, which cut the DNA into only eight fragments, indicated that this species contains two unique and independent replicons of about 2,100 and 1,150 kb. Pulsed-field gel electrophoresis of intact DNA revealed two bands migrating the expected distances. These replicons were identified as two unique and independent chromosomes by the presence of rRNA operons and genes for heat shock proteins mapping to separate replicons.Brucella melitensis is a species of the genus Brucella, members of which are small gram-negative bacteria that are pathogenic in many animals and occasionally in humans. The genetics of Brucella spp. are completely unknown. No nutritional mutants have been isolated and characterized because of the fastidious growth of these species and the difficulty in studying their strictly aerobic metabolism. In addition, chromosome exchange, by transformation, conjugation, or transduction, is completely unknown in these bacteria. Despite numerous attempts, plasmids or temperate bacteriophages have never been demonstrated in these species. Consequently, physical mapping of the Brucella genome was the first step to be undertaken in the study of Brucella genetics. In a previous paper (1), we concluded that this genome is a unique 3.1-megabase circular chromosome on which several genes are located. The map was obtained by the method described for mapping the Haemophilus influenzae genome (11). The Brucella genomic DNA was digested with three restriction endonucleases (SpeI, XhoI, and XbaI) and subjected to pulsed-field gel electrophoresis (PFGE), and the fragments obtained with one endonuclease were used as linking probes to join the fragments generated with the other two enzymes. However, we encountered several difficulties during the mapping, because no restriction endonuclease that cuts Brucella DNA (which has a G+C content of 59%) into less than 25 fragments was then known. It was therefore necessary to order a total of 113 Spel, XhoI, and XbaI fragments, a process resulting in some ambiguities in the map. Two new endonucleases are now available that recognize 8-bp sites on DNA containing only adenine and thymine and expected to be rare in high-G+C-content DNAs: Pacl (5'-TTAATTAA) and Swal (5'-AIT TAAAT). We used PacI in this study to

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...