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Journal of Bacteriology, September 1999, p. 5539-5541, Vol. 181, No. 17
Department of Biology, University of Utah,
Salt Lake City, Utah 84112,1 and
Department of Biochemistry and Molecular Genetics, University
of Virginia Health Sciences Center, Charlottesville, Virginia
229082
Received 3 May 1999/Accepted 18 June 1999
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.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A New Class of Cobalamin Transport Mutants
(btuF) Provides Genetic Evidence for a Periplasmic Binding
Protein in Salmonella typhimurium
ABSTRACT
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TABLE 1.
Effect of btu mutations on minimum level of
CN-B12 required to support methionine synthesis
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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.
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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
-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).
Nucleotide sequence accession number. The Salmonella btuF sequence is GenBank accession no. AF096877.
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ACKNOWLEDGMENTS |
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This work was supported in part by NIH grant GM34804.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, University of Utah, Salt Lake City, UT 84112. Phone: (801) 581-3412. Fax: (801) 585-6207. E-mail: Roth{at}Bioscience.utah.edu.
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