Abstract
Respiration is a fundamental process in living cells, resulting in ATP production following electron transfer from low-redox- potential electron donors to high-redox-potential electron acceptors such as O2. In prokaryotes, enhanced respiratory flexibility allows the use of alternative electron acceptors, including nitrogen oxides, which contributes to their ability to colonize microaerobic or anaerobic environments. So far, Brucella spp. have been classified as strictly aerobic bacteria, defining O2 as the sole electron acceptor. Use of nitrate as alternative electron acceptor has been suggested, as all genes involved in the denitrification pathway are present in Brucella, but data on possible growth under anaerobic conditions in the presence of nitrates have not yet been available. Comparative RNA-Seq analysis of zoonotic Brucella suis 1330 and the atypical species B. microti grown to mid-log in screw-cap tubes containing Tryptic Soy broth revealed specific upregulation of nor and nos denitrification genes in B. microti. Under anoxic conditions, both species grew in broth, but in the presence of NaNO3 only, and in a NO3-concentration-dependent manner for B. microti. Growth of B. suis was limited, as compared to classical conditions, whereas B. microti, as well as other atypical species such as B. inopinata BO1 and strains isolated from Australian rodents and African bullfrogs, were well-adapted to anaerobic conditions in the presence of NaNO3 as electron acceptor. narG mutants of B. microti and B. suis, lacking nitrate reductase, lost growth capacities under anoxic conditions. Remarkably, denitrification kinetics in the absence of oxygen revealed significantly earlier and faster nitrite production and consumption in B. microti, correlating with RT-qPCR-monitored higher expression of nir, nor, nos genes encoding the corresponding reductases of the denitrification pathway. To our knowledge, this is the first experimental evidence of NO3-dependent anoxic growth of Brucella spp., showing that, in contrast to classical Brucellae, atypical species and strains grow equally well under oxic and anoxic conditions. At least the novel Brucella spp. should therefore definitely be considered as facultative anaerobic bacteria.
