An ABC-type cobalt transport system is essential for growth of Sinorhizobium meliloti at trace metal concentrations

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 cobalt transport 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.

Fig. 1.

Schematic of the cbtJ-smb20060region on the pSymB megaplasmid of S. meliloti. (A) DNA fragments were PCR amplified and cloned upstream of the promoterless gfp⁺-lacZgenes in pTH1703 to obtain pTH1968, pTH1969, pTH2030, pTH1970, and pTH2270. Plasmid pFL3108 was a pTH1522 derivative carrying a genomic DNA fragment (8). Upon integration of the reporter fusion clones into the S. melilotigenome, transconjugant colonies grew (+) or did not grow (−) when selected on LB or M9-succinate containing streptomycin and gentamicin (Gm). Strains RmP831, RmP833, SmFL3108, RmP889, RmP835, and RmP1485 are representative transconjugant colonies selected for each integrated plasmid. (B) Diagram showing the genome organization of strains RmP831, RmP833, SmFL3108, RmP889, RmP835, and RmP1485 following recombination of the reporter plasmids. X's indicate the genes whose transcription was disrupted in each strain.

Fig. 2.

Growth of S. melilotiwild-type RmP110 and plasmid integration mutants in LB or LB with Co²⁺, Zn²⁺, Ni²⁺, or Fe³⁺added. S. melilotistrains were cultured in M9-succinate medium, washed with 0.85% NaCl, and then subcultured into LB. After growing for 16 h, the precultures were inoculated into fresh LB or LB supplemented with CoCl₂(0 to 5 μM) (A) or CoCl₂(5 μM), ZnSO₄(2 μM), NiSO₄(2 μM), or FeCl₃(1 mM) (B). The OD₆₀₀was measured after 16 h of incubation.

Fig. 3.

Growth of S. melilotiRmP110 (wild type), RmP833 (cbtJKL), and RmP1477 (cobT) in LB medium supplemented with AdoCbl or left unsupplemented. The strains were initially grown in LB containing 5 μM AdoCbl, washed with 0.85% NaCl, and then inoculated into LB medium supplemented with different concentrations of AdoCbl. The OD₆₀₀was recorded after incubation for 24 h. Assays were performed in triplicate, and values represent the means ± standard deviations (SD).

Fig. 4.

Characterization of the region upstream of cbtJ. The sequences of the cbtJ−35 and −10 promoter recognition sequences and the transcription start site (+1) are indicated. The primers used for mapping the transcription start site are indicated by arrows under the sequences. The sequence of the 201-nt B₁₂riboswitch is shown in bold. The B₁₂box is framed by a dashed-line box. The stem-loop sequences P0/P0′ to P6/P6′ are indicated by arrows above the respective sequences, based on the conserved structure of the B₁₂riboswitch (67). The ribosome binding site (RBS) and start codon of cbtJare shown in bold. The autoradiograph image shows a sequencing gel and the ³²P-labeled ML18490 extension products obtained with RNA from RmP110 grown in LB (lane 1). Data obtained with primer ML18489 are not shown but were consistent with those obtained with ML18490. The arrow indicates the primer extension product.

Fig. 5.

Nanomolar concentrations of CoCl₂and micromolar concentrations of AdoCbl affect cbtJexpression in S. meliloti. Cells were grown in M9-succinate medium, washed with 0.85% NaCl, and then inoculated into M9-succinate with 0 to 50 nM CoCl₂(A) or M9-succinate with 0 to 100 μM AdoCbl and no CoCl₂(B). After growth for 36 h, LacZ (β-galactosidase) activity was determined. The fusion strains were RmP110 (wild type), RmP831 (cbtJp::gfp-lacZfusion), and RmP833 (cbtJKLmutant with cbtJ::gfp-lacZfusion).

Fig. 6.

Regulation of cbtJand cobPpromoter (cbtJpand cobPp) activities by CoCl₂and AdoCbl. (A and B) Transcriptional fusions in S. melilotiRmP110 (wild type) and RmP1477 (cobT) grown in M9-succinate without exogenous CoCl₂(none) or with 10 nM CoCl₂, 10 μM AdoCbl, or 10 nM CoCl₂and 10 μM AdoCbl. (A) The DNA fragments upstream of cbtJwere cloned as transcriptional gfp-lacZfusions in the promoterless, broad-host-range replicating plasmid pTH2224. The location of the transcription start site is marked +1, and the dashed line indicates the deleted B₁₂riboswitch. (B) The region upstream of cobPwas inserted upstream of the gfp-lacZgenes and 5′ of the putative B₁₂riboswitch, designated +1. (C) GFP expression (fluorescence emission [OD₆₀₀]) given as averages for three independent assays, with SD of <10%. The relative GFP fluorescence was <1,400 units for RmP110 and RmP1477 carrying the empty plasmid pTH2224. ND, not determined.

Fig. 7.

Cobalt (⁵⁷Co²⁺) accumulation by the wild type (RmP110) and a cbtJKLmutant (RmP833) of S. meliloti. Cells were grown in LB or LB supplemented with 5 μM CoCl₂, washed, and then resuspended in transport buffer (50 mM MOPS, pH 7.5, 10 mM MgSO₄, and 15 mM succinate). Transport assays were initiated by the addition of CoCl₂(⁵⁷Co²⁺; specific activity, 265 mCi/mmol) to cell suspensions at a final concentration of 0.5 μM. (A) ⁵⁷Co²⁺accumulation by RmP110 and RmP833 cells grown in LB or LB with 5 μM CoCl₂. (B) ⁵⁷Co²⁺accumulation by RmP110 cells grown in LB or with the addition of 50 μM unlabeled CoCl₂at 10 min. Each data point represents the average for three independent measurements. The error bars represent SD. Green fluorescence readings (mean emission at 510 nm/OD₆₀₀± SD) for the cells used in this experiment were 92 ± 11 (RmP110), 80 ± 8 (RmP110 plus CoCl₂), 4,927 ± 282 (RmP833), and 1,400 ± 43 (RmP833 plus CoCl₂).