An ABC-Type Cobalt Transport System Is Essential for Growth of Sinorhizobium melilotiat Trace Metal Concentrations

Abstract

ABSTRACT We report expression and mutant phenotypes for a gene cluster in Sinorhizobium meliloti , designated cbtJKL , that has been shown to encode an ABC-type c o b alt t ransport system. Transcription of cbtJKL initiated 384 nucleotides upstream from the cbtJ translation start codon, and the resulting 5′ region contained a putative B 12 riboswitch. Expression of the cbtJKL genes appeared to be controlled by (cobalt-loaded) cobalamin interacting at the B 12 riboswitch, since (i) a putative B 12 riboswitch was located within this large upstream region, (ii) cbtJ transcription was repressed upon addition of cobalt or vitamin B 12 , and (iii) deletions in the B 12 riboswitch resulted in constitutive cbtJKL transcription. Insertion mutants in cbtJKL failed to grow in LB medium, and growth was restored through the addition of cobalt but not other metals. This growth phenotype appeared to be due to the chelation of cobalt present in LB, and cbtJKL mutants also failed to grow in minimal medium containing the chelating agent EDTA unless the medium was supplemented with additional or excess cobalt. In uptake experiments, 57 Co 2+ accumulation was high in wild-type cells expressing the cbtJKL genes, whereas wild-type cells in which cbtJKL expression was repressed showed reduced accumulation. In cbtJKL mutant cells, 57 Co 2+ accumulation was reduced relative to that of the wild type, and presumably, this residual cobalt transport occurred via an alternate ion uptake system(s) that is not specific to cobalt. In symbiosis, the alternate system(s) appeared to mediate cobalt transport into bacteroid cells, as low cbtJKL expression was detected in bacteroids and cbtJKL mutants formed N 2 -fixing nodules on alfalfa.