Abstract
Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.
Highlights
The genus Brucella consists of small, non-motile, non-spore forming, gram-negative, facultatively intracellular bacteria capable of infecting a variety of mammals
When the Western blot analysis was performed using the total antigen extracts obtained from 109 Colony forming units (CFU) of B. neotomae, a thin protein band with size corresponding to the superoxide dismutase (SOD) consistently reacted with the SODspecific antibodies (Fig. 1C and D)
Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at substantially low levels in comparison to B. abortus RB51
Summary
The genus Brucella consists of small, non-motile, non-spore forming, gram-negative, facultatively intracellular bacteria capable of infecting a variety of mammals. There are six wellrecognized species of Brucella that show a marked host preference – B. abortus (cattle), B. melitensis (goat and sheep), B. suis (pig), B. canis (dog), B. ovis (sheep, especially ram), and B. neotomae (wood rat). B. suis biovar 2 is known to be incapable of causing significant clinical disease in pigs as well as in humans [3]. In the past few years, Brucella has been recovered from several marine mammals, including cetaceans (dolphin, whale and porpoise) and pinnipeds (seals and otters); these marine isolates belong to two potential new species, B. pinnipedialis and B.ceti [4]. A new species of Brucella, B. microti, was isolated from wild common voles suffering from a systemic disease [5,6]
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