Presence of two independent chromosomes in the Brucella melitensis 16M genome

Abstract

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

Completion of the Genome Sequence of Brucella abortus and Comparison to the Highly Similar Genomes of Brucella melitensis and Brucella suis

“…Similarity among the Brucella orthologs was high, with an average amino acid sequence identity of greater than 99%. The B. abortus sequence confirmed PFGE maps (27) with regard to genome size, chromosome number, and presence of an inversion described in Chr II relative to other genomes.…”

“…genomes are composed of two circular chromosomes of approximately 2.1 and 1.2 Mbp (22,27,28), with the exception of B. suis biovar 3, which has a single chromosome of 3.1 Mbp. PFGE studies revealed other differences, including a 640-kb inversion in the small chromosome of B. abortus 544 and a deletion in the small chromosome of B. ovis.…”

Equine brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control

“…Brucella genus is classified phylogenetically within the α2 subdivision of Proteobacteria which comprises of Agrobacterium, Bartonella, Ochrobactrum, Rhizobium, Rhodobacter, and Rickettsia (Moreno et al, 1990). Genome of B. abortus has two circular chromosomes of 2.1 Mb and 1.5 Mb size without plasmids (Michaux et al, 1993;MichauxCharachon et al, 1997). Recent years has seen the completion of genome of B. abortus (Sanchez et al, 2001), B. melitensis (GenBank NC 003317 and NC 003318) (DelVecchio et al, 2002) and B. suis (GenBank NC 002969) thus increasing the opportunity for understanding the pathogenicity of Brucella.…”

Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome

“…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).…”