An ABC-Type Cobalt Transport System Is Essential for Growth of Sinorhizobium melilotiat Trace Metal Concentrations
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ABSTRACTWe report expression and mutant phenotypes for a gene cluster inSinorhizobium meliloti, designatedcbtJKL, that has been shown to encode an ABC-typecobalttransport system. Transcription ofcbtJKLinitiated 384 nucleotides upstream from thecbtJtranslation start codon, and the resulting 5′ region contained a putative B12riboswitch. Expression of thecbtJKLgenes appeared to be controlled by (cobalt-loaded) cobalamin interacting at the B12riboswitch, since (i) a putative B12riboswitch was located within this large upstream region, (ii)cbtJtranscription was repressed upon addition of cobalt or vitamin B12, and (iii) deletions in the B12riboswitch resulted in constitutivecbtJKLtranscription. Insertion mutants incbtJKLfailed 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, andcbtJKLmutants 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,57Co2+accumulation was high in wild-type cells expressing thecbtJKLgenes, whereas wild-type cells in whichcbtJKLexpression was repressed showed reduced accumulation. IncbtJKLmutant cells,57Co2+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 lowcbtJKLexpression was detected in bacteroids andcbtJKLmutants formed N2-fixing nodules on alfalfa.
