A New Class of Cobalamin Transport Mutants (btuF) Provides Genetic Evidence for a Periplasmic Binding Protein in Salmonella typhimurium

Abstract

No periplasmic binding protein has been demonstrated for the ATP-binding cassette (ABC)-type cobalamin transporter BtuCD. New mutations (btuF) are described that affect inner-membrane transport. The BtuF protein has a signal sequence and resembles the periplasmic binding proteins of several other ABC transporters.

Cobalamin is actively transported by Salmonella typhimurium and Escherichia coli (8, 24). The BtuB protein (in concert with the TonB protein) transports cobalamin across the outer membrane. The btuCED operon encodes two membrane proteins (BtuC and BtuD) that provide transport across the inner membrane (4, 5, 17, 22). The inner-membrane BtuCD system is an ATP-binding cassette (ABC) or “traffic ATPase” transporter (2, 7), a type that generally includes a periplasmic binding protein in addition to membrane-spanning components. None of the E. coli transport mutants is defective for a periplasmic binding protein. Direct assays of osmotic shock fluid revealed a protein able to bind cobalamin (8, 15, 27, 28), but no role in transport has been demonstrated (14, 22). It was suggested that the outer-membrane transport system (BtuB-TonB) concentrates cobalamin in the periplasm sufficiently to reduce the need for a binding protein (7, 8, 22). We describe a new class of Salmonella cobalamin transport mutations that may eliminate a periplasmic vitamin B₁₂ binding protein.

New transport mutants.

Under aerobic conditions, a metE mutant, which, under aerobic conditions, requires methionine unless exogenous vitamin B₁₂ is provided (24) at a minimum concentration of about 0.1 nM. Known cobalamin transport mutants (btuB or btuCED mutants) require a higher level of exogenous cobalamin (Table 1). The btuF insertion mutants described here were isolated in the metE deletion strain, TT15696. Their phenotype resembled that of previously known btuCD mutants (Table 1). The btuF gene maps near 5 min of the S. typhimurium chromosome (Fig. 1), far from btuB and btuCED.

TABLE 1.

Effect of btu mutations on minimum level of CN–B₁₂ required to support methionine synthesis

Strain	Relevant genotypea	Ability to grow on minimal medium containing the indicated supplementb
		Nothing	Met	CN–B12 (Concn)
				0.1 nM	1 nM	10 nM	100 nM	1 μM	10 μM	100 μM	500 mM
LT2	WT	+	+	+	+	+	+	+	+	+	+
TT15696	metE	−	+	+	+	+	+	+	+	+	+
TT20702	btuB12::Tn10dCm	−	+	−	−	−	−	±	+	+	+
TT20703	btuCED9::Tn10dCm	−	+	−	±	+	+	+	+	+	+
TT20698	btuF21::Tn10dTc	−	+	−	±	+	+	+	+	+	+
TT20704	btuF21::Tn10dTc butCED9::Tn10dCm	−	+	−	±	+	+	+	+	+	+
TT20706	btuF21::Tn10dTc btuB12::Tn10dCm	−	+	−	−	−	−	−	−	−	+
TT20705	btuB20::Tn10dTc btuCED9::Tn10dCm	−	+	−	−	−	−	−	−	−	+

^aAll strains except LT2 carry a metE deletion mutation (Del1077) which renders methionine synthesis dependent on the B₁₂-dependent MetH enzyme. Three other btuF mutations (TT20699 through TT20701) showed the same phenotype as strain TT20698. WT, wild type. 

^bAll tests were performed with cells grown overnight in E-glucose, methionine, and histidine. Cells were diluted in NaCl, and approximately 10 to 100 cells (in 5 μl) were dropped onto an E-glucose–histidine plate with the indicated concentration of CN–B₁₂ or methionine (Met). Growth was scored as the appearance of colonies after 3 days. 

FIG. 1.

Genetic map of the btuF region. This region is at min 5 of the latest Salmonella map; the homologous region of the E. coli at min 3.7 includes the yadT gene, which is homologous to the btuF gene of Salmonella. Linkages are presented as percent cotransduction in P22-mediated transduction crosses. The arrowheads point to the selective donor marker used in the cross. Transduction methods have been described previously (12).

Epistasis tests show that BtuF and BtuCD act together.

The transport defect of a btuF btuCED double mutant was indistinguishable from that of either single mutant, suggesting that the BtuF and BtuCD proteins contribute to the same function (Table 1). That is, the residual transport ability in each mutant type does not require the function encoded by the other gene. In contrast, the combination of a btuF and a btuB mutation causes a much more severe defect than either single mutation. This synergistic effect is also seen for a btuCD btuB double mutant. This suggests that BtuB acts in both btuCD and btuF single mutants to provide the residual transport seen. The BtuF protein does not seem to be needed for outer-membrane transport but appears to act with BtuCD to provide inner-membrane transport.

Repression of the cob operon in btuF insertion mutants.

Transcription of the cob operon is induced by propanediol (6, 23) and repressed by adenosylcobalamin (Ado–B₁₂) (1). Repression by exogenous CN–B₁₂ requires transport and internal adenosylation (13, 24). If the btuF mutation impairs cobalamin transport, it should also impair repression of the cob operon by exogenous CN–B₁₂.

In wild-type strains, the cob operon is repressed by 0.1 μM CN–B₁₂. Strains with a btuF (or btuCED) mutation required a 10-fold-higher CN–B₁₂ concentration for repression. Strains with a btuB mutation were not fully repressed even by 1 mM CN–B₁₂. These results were obtained with derivatives of strain TT20707, which carries a cobD24::MudJ insertion (forming a cob-lac operon fusion) and a deletion mutation (cobR4) which renders transcription independent of propanediol but still subject to repression by Ado–B₁₂ (3, 10).

Transport assays.

Assays of cyanocobalamin (CN-Cbl) transport were initiated by addition of ⁵⁷Co-labeled CN–B₁₂. After 50 min of incubation, an excess of unlabeled CN–B₁₂ was added to stop uptake and allow observation of loss of previously assimilated cobalamin from cells. The btuF mutants were indistinguishable from btuC mutants (Fig. 2). Both mutant types initially took up vitamin B₁₂ (12.7 nM) as well as wild-type cells, suggesting no defect in outer-membrane transport; this initial transport is eliminated by a btuB mutation. Both btuF and btuC mutants accumulated less CN-Cbl at steady state, suggesting that import is ultimately opposed by leakage of periplasmic CN-Cbl back out through the outer membrane. In wild-type cells, unlabeled CN–B₁₂ stopped the accumulation of vitamin B₁₂, but in btuF and btuC mutants, addition led to loss of labeled vitamin B₁₂ by diffusion out of the periplasm. Four other btuF mutants behaved essentially like those in Fig. 2. In all cases, btuF mutations appear to block inner-membrane transport.

FIG. 2.

Transport of CN-Cbl by btu mutants. Cells were grown to mid-log phase (optical density at 660 nm [OD₆₆₀], 0.4 to 0.8) in the Davis-Mingioli minimal medium (11). Harvested cells were suspended to a final OD₆₆₀ of 6 in 0.1 M potassium phosphate buffer (pH 6.6) containing 20 μM calcium nitrate and 1% glucose. These suspensions were stored on ice and assayed within 3 h of harvest. Transport was assayed by the method of Bradbeer and Woodrow (9). Each 15-ml reaction mixture was 0.1 M potassium phosphate buffer (pH 6.6) containing final concentrations of 20 μM calcium nitrate, 1% glucose, and sufficient cells to give an OD₆₆₀ of 0.2. After 5 min of incubation at 37°C, the reaction was started by adding ⁵⁷Co-labeled CN-Cbl to a final concentration of 12.67 nM. Samples were removed at intervals and filtered (0.45-μm-pore-size Millipore filter HAWP) to collect cells. After 50 min of uptake, unlabeled CN-Cbl was added (final concentration, 5 μM) and sampling was continued. All filters were washed twice with 10 ml of 0.1 M lithium chloride, air dried, placed in Beckman Ready Safe scintillation fluid, and counted in a Beckman LS6500 liquid scintillation counter. Results are expressed as picomoles of CN-Cbl taken up per milliliter of cell suspension with an OD₆₆₀ of 0.6.

Sequencing.

The DNA sequence of the btuF gene was determined following PCR amplification of sequences between genetically characterized insertion mutations as indicated in the genetic map (Fig. 1). Methods and primers used are described elsewhere (18). All sequenced btuF insertion mutations lie within a 801-nucleotide open reading frame (ORF) near the hemL gene. Their positions are btuF23::Tn10dTc (bp 97), btuF22::Tn10dTc (bp 400), and btuF79::Tn10dCm (bp 477). The inferred BtuF sequence includes a highly probable signal sequence with a cleavage site at amino acid 22 (score, 0.97; maximum, 1), a feature expected of a periplasmic binding protein. The BtuF amino acid sequence shows a very strong similarity to the yadT ORF of E. coli. The sequence resembles those of three known ABC-type, periplasmic binding proteins. One transports hemin across in the inner membrane of Yersinia enterocolitica and Yersinia pestis (16, 26). Another (FecB of E. coli) transports citrate-iron chelates (25), and the third transports ferrisiderophores (CbrA of Erwinia chrysanthemi) (21).

Regulation of expression.

Expression of btuF is not regulated, based on tests using a MudJ insertion mutation (btuF80::MudJ), which fuses transcription of the inserted lac operon to the btuF gene. A strain with this fusion (TT20711) was grown on glucose, on glycerol, and on ethanolamine; the fused lacZ gene produced about 45 U of β-galactosidase under all conditions. Addition of CN–B₁₂ (5 nM or 5 μM) had no effect on transcription during growth on any of these three carbon sources. This is interesting in light of the fact that the btuA gene is controlled (by Ado-B₁₂) (19, 20), while the btuCED operon is not (22).

In summary, we infer that the BtuF protein acts as a periplasmic binding protein with BtuCD to transport cobalamins across the inner membrane. Such a protein is expected for a transporter of this type but was not found among previously characterized E. coli cobalamin transport mutants.

Nucleotide sequence accession number.

The Salmonella btuF sequence is GenBank accession no. AF096877.