Department of Cell Biology, Graduate school of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan1
Author for correspondence: Naotake Ogasawara. Tel: +743 72 5430. Fax: +743 72 5439. e-mail: nogasawa{at}bs.aist-nara.ac.jp
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ABSTRACT |
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Keywords: C4-dicarboxylate, Bacillus subtilis, two-component regulatory system, transporter, solute-binding protein
Abbreviations: Dct, dicarboxylic acid transport
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INTRODUCTION |
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Several classes of C4-dicarboxylate transport (Dct) systems have been reported in Gram-negative bacteria. In Escherichia coli, uptake of C4-dicarboxylates is achieved by the aerobic DctA system and the anaerobic DcuA, DcuB and DcuC systems, where DcuA and DcuB are homologous proteins (Six et al., 1994 ; Zientz et al., 1999
). In Rhizobium leguminosarum and Rhizobium meliloti, C4-dicarboxylates are required for symbiotic nitrogen fixation and are transported by the DctA permease homologous to E. coli DctA (Finan et al., 1983
; Engelke et al., 1987
; Ronson et al., 1987
; Six et al., 1994
; Sofia et al., 1994
). The purple photosynthetic bacterium Rhodobacter capsulatus has a Dct system which consists of three proteins: C4-dicarboxylate periplasmic binding protein, DctP, and two integral membrane proteins, DctQ and DctM (Shaw et al., 1991
; Forward et al., 1997
). Furthermore, it has been shown that expression of many of the genes encoding Dct systems is positively regulated by two-component systems in response to exogenous C4-dicarboxylates. The Rhi. leguminosarum and Rhi. meliloti dctA genes are activated by the adjacent two-component gene sets dctB and dctD (Jording et al., 1992
; Reid & Poole, 1998
). In Rho. capsulatus, the dctP, dctQ and dctM genes form an operon and are regulated by the adjacent dctS and dstR genes, which code for a two-component sensorregulator pair (Hamblin et al., 1993
). Recently, it was also reported that a two-component regulatory system, DcuS and DcuR, is responsible for the activation of the E. coli dcuB and fumB genes encoding the anaerobic fumarate carrier and fumarase, which are located next to the dcuSR genes (Zientz et al., 1998
; Golby et al., 1999
).
In the framework of the B. subtilis genome-sequencing project (Kunst et al., 1997 ), our group had sequenced a 150 kb region between the pbpC and phoB genes, and identified two genes, ydbE and ydbH, whose products were similar to DctP in Rho. capsulatus and DctA in Rhi. leguminosarum, respectively (Fig. 1
). The deduced products of ydbE and ydbH exhibited 41·2 and 53·9% amino acid sequence identity to the corresponding proteins, respectively. In addition, as in the case of Gram-negative bacteria, ydbF and ydbG, which were thought to code for a sensor kinase and response regulator, respectively, were located between ydbE and ydbH (Fig. 1
). The deduced products, the YdbF and YdbG proteins, have significant similarity to E. coli DcuS and DcuR (30·6% and 35·3% identity, respectively). Furthermore, no other genes encoding close homologues of the reported genes involved in C4-dicarboxylate transport in Gram-negative bacteria could be identified in the whole B. subtilis genome sequence. These analyses led us to hypothesize that the B. subtilis ydbEFGH genes encode the C4-dicarboxylate transport and its regulatory systems. Indeed, the studies described here reveal that the ydbH gene encodes a C4-dicarboxylate transporter and the ydbFG two-component genes are necessary for the activation of ydbH expression. An unexpected finding is that the YdbE protein is not directly involved in C4-dicarboxylate uptake, but is necessary for ydbH expression in concert with the YdbFG two-component system.
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METHODS |
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Transformation.
Transformation of B. subtilis was performed as described by Kawamura et al. (1980) .
Construction of pMUTINT3 insertion mutants.
Oligonucleotide primers dbEF (5'-CGAGGCAGATCTGTCTCGCACTGATGATCG-3') and dbER (5'-CGAGGCAGATCTCTCGAGCTTCGATGTGGAGGG-3'), dbFF (5'-CGAGGCAGATCTGCTCTCAATCCGTTGGAAAATCACG-3') and dbFR (5'-CGAGGCAGATCTCTCGAGCTTGTGCTGACAGGATGC-3'), dbHF (5'-CGAGGCAGATCTAGTTCAGGTCATAACCGC-3') and dbHR (5'-CGAGGCAGATCTCTCGAGCATCAACCATGTTGGACG-3'), and dbGF (5'-CGAGGCGGATCCGCTCGTAAAGAATGGAAGGTTCTGCTCATTG-3') and dbGR (5'-CGAGGCGGATCCCTCGAGCTGATCTCCTGCAAGGTC-3') were used to amplify 320, 304, 398 and 218 bp segments of ydbE, ydbF, ydbH and ydbG internal regions, respectively, from B. subtilis 168 genomic DNA. Each PCR product was restricted at the BglII or BamHI sites in the primers and inserted into BamHI-restricted pMUTINT3 (Moriya et al., 1998 ; Vagner et al., 1998
). The resulting plasmids were introduced into the B. subtilis genome and integrated by a single crossover, with selection of the transformants by means of erythromycin resistance (0·5 µg erythromycin ml-1), to give the pMUTINT3-inserted mutants. The correct integration of pMUTINT3 was confirmed by Southern hybridization.
Construction of tet-insertion mutants.
The ydbE gene was inactivated by replacing its central portion with a tetracycline-resistance gene. The upstream region of ydbE was amplified with the primers dbEUF (5'-AAGAAGCTTCATTAATCATCCTGAT-3') and dbEUR (5'-TCATAAATATGTATAGCAGAGCTCTCTCTAGAGGCCTTCCTGGTCGTCATC-3'), generating a 484 bp fragment with flanking HindIII and SacI sites. The downstream region of ydbE was amplified with the primers dbEDF (5'-GATGACGACCAGGAAGGCCTCTAGAGAGAGCTCTGCTATACATATTTATGA-3') and dbEDR (5'-GAAGAATTCCTCAGTTGCCGTATCCAT-3') generating a 466 bp fragment with flanking SacI and EcoRI sites. HindIII/SacI-digested upstream fragment and SacI/EcoRI-digested downstream fragment were cloned into HindIII/EcoRI-digested pTZ18R (TOYOBO, Japan). Then, into the SacI site of the resultant plasmid, a SacI fragment containing a tetracycline-resistance gene derived from pBEST307 (Itaya, 1992 ) was inserted in both orientations. The plasmids were transformed into B. subtilis 168 and integrated by a double crossover with selection for tetracycline resistance (10 µg tetracycline ml-1). Proper replacement of the internal region of the ydbE gene by the tetracycline-resistance gene was confirmed by PCR.
Construction of an in-frame deletion mutant of ydbF.
An in-frame deletion mutant of ydbF was created on the basis of the two-step allele-replacement method of Stahl & Ferrari (1984) . Briefly, the genomic regions upstream and downstream of ydbF were amplified by PCR using the primers bFUF (5'-CCGATGCTTCCGTTCAGCCC-3') and bFUR (5'-GCCATAGCCTGTGCCGCCTTTCCAACGGATTGAGAGC-3'), and bFDF (5'-GCTCTCAATCCGTTGGAAAGGCGGCACAGGCTATGGC-3') and bFDR (5'-TCCTGGGACAGCGTATCATTC-3'), respectively. These two fragments were mixed and further amplified using the primers bFUF and bFDR, yielding a fragment containing a deletion in the ydbF structural gene which joins codon 11 to codon 488. Two copies of this PCR-generated fragment were tandemly cloned into pCP112 (Price et al., 1983
). The resultant plasmid was used for creating an in-frame deletion mutant of ydbF. Proper deletion of the internal region of the ydbF gene was confirmed by PCR.
Construction of PydbHbgaB fusions.
A 217 bp fragment (PDH94) covering the promoter region of ydbH (PydbH) was amplified by PCR with the primers dbHPF1 (5'-GAAGAATTCGTGTTAAAAGGATAAACC-3') and dbHPR (5'-GGAGGATCCGGCCAGACCAGCCCG-3'), and cloned between the EcoRI and BamHI sites of a plasmid pDLd (Nanamiya et al., 1998 ), harbouring amyE-up and amyE-down sequences. The resulting plasmid was used for integration of the PDH94bgaB fusion into the amyE locus of the B. subtilis genome. Fragments of various length covering PydbH were generated by PCR with the primers dbHPF1.1 (5'-GAAGAATTCGATAAACCTGAGACCAAA-3') for PDH83, dbHPF1.2 (5'-GAAGAATTCAGACCAAAAAGACCAAAA-3') for PDH73, dbHPF1.3 (5'-GAAGAATTCAGACCAAAATGTCCGTTA-3') for PDH64, or dbHPF1.4 (5'-GAAGAATTCTCCGTTATGATCATAAAC-3') for PDH53, in combination with dbHPR. These products were fused to bgaB and integrated into the amyE locus as described above.
ß-Galactosidase assay.
An aliquot (1 ml) of cells growing in NYE medium with or without the addition of carbon source was harvested by centrifugation and the activity of ß-galactosidase was assayed according to Youngman et al. (1985) . Incubation for the assay was performed at 28 °C for LacZ (E. coli ß-galactosidase) and at 62 °C for BgaB (Bacillus stearothermophilus ß-galactosidase). Units are expressed as nanomoles of 4-methylumbelliferyl ß-D-galactoside hydrolysed min-1 (mg protein)-1. Protein concentration was determined using a Bio-Rad Protein Assay Kit.
Northern and primer-extension analysis.
Total RNA was extracted from wild-type cells growing in DS medium at exponential phase (OD600 0·1, 0·3 and 0·6) by the method described by Igo & Losick (1986) . In the Northern analysis, aliquots containing 5 µg of total RNA were electrophoresed and blotted onto positively charged nylon membrane (Hybond-N+; Amersham). Hybridization and detection were performed using digoxigenin-labelled RNA probes (10 ng), according to the manufacturers instructions (Boehringer Mannheim). RNA probes were synthesized in vitro with T7 RNA polymerase using PCR products as templates. Templates were prepared using primers in which the T7 promoter sequence was added to the specific sequences for amplification. In the primer-extension analysis, 20 µg of the RNA sample was incubated with the digoxigenin-end-labelled primers (1 pM) specific for the ydbH gene (dbHDig; 5'-GACAATGACCCCGATGATGAC-3') for 60 min at 60 °C, and gradually cooled down for 90 min to the ambient temperature. After the addition of dNTPs (2·5 mM each) and reverse transcriptase (Gibco-BRL), the reaction mixture was incubated for 60 min at 37 °C. The cDNA products were then electrophoresed through an 8% polyacrylamide/urea gel, blotted onto the positively charged nylon membrane and detected according to the manufacturers instructions (Boehringer Mannheim). DNA ladders for use as size markers were created with the same digoxigenin-end-labelled primers and a DIG Taq DNA sequencing kit (Boehringer Mannheim).
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RESULTS |
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Growth of wild-type and ydbEFGH mutants in minimal medium containing yeast extract and C4-dicarboxylate
To examine the function of ydbE, ydbF, ydbG and ydbH in the uptake of C4-dicarboxylates, we constructed knockout mutants of each gene and then tested their ability to utilize C4-dicarboxylates as the sole carbon source for growth. Disruption of the genes was accomplished by integrating a pMUTINT3 plasmid harbouring an internal fragment of each gene into the genome by a Campbell-type recombination (Vagner et al., 1998 ). The ability of the mutants to utilize C4-dicarboxylates was monitored according to Willecke & Lange (1974)
. The growth of the wild-type strain in a minimal salts medium containing 0·05% (w/v) yeast extract as the sole carbon source (NYE medium) ceased when the OD600 of the culture reached around 0·2. Growth was stimulated by the addition of a carbon source, such as C4-dicarboxylates (Fig. 2a
). The wild-type B. subtilis cells can readily grow utilizing malate, succinate or fumarate as an additional carbon source after exhaustion of the nutrients in yeast extract. If the mutants could not utilize the additional carbon sources, their growth would remain arrested regardless of the supplement. Results shown in Fig. 2 (b
e
) clearly show that all the knockout mutants could utilize malate but not succinate or fumarate, indicating that the ydbEFGH genes are essential for the uptake of succinate and fumarate in B. subtilis.
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The presence of at least two transport systems for malate has been suggested in B. subtilis. Therefore, the apparent absence of a defect in the utilization of malate in the mutants might be due to the presence of additional transport system(s). The inactivation of the ydbD gene, which encodes a general stress protein and was co-transcribed with ydbE in the normal growth phase, showed no effect on C4-dicarboxylate utilization (data not shown).
Expression of ydbH is regulated by ydbE, ydbF and ydbG
We next addressed the question of whether the defect of C4-dicarboxylate uptake in the ydbF and ydbG mutants, probably encoding a two-component sensor and regulator pair, was due to a defect in the activation of expression of the ydbH gene which appeared to encode a C4-dicarboxylate transporter. We constructed a transcriptional fusion between the ydbH promoter region and a promoterless B. stearothermophilus bgaB gene, which encodes a thermostable ß-galactosidase, and integrated it into the amyE locus of the B. subtilis genome. In a strain carrying the PydbH::bgaB fusion in an otherwise wild-type genetic background, ß-galactosidase was induced in NYE medium and also to a similar extent in the medium supplemented with succinate. The activity was slightly elevated by the addition of fumarate, but markedly decreased by the addition of malate (Fig. 3a). As expected, when we introduced the PydbH::bgaB fusion into the pMUTINT3-inserted ydbF and ydbG mutants, the ß-galactosidase activity was decreased to background levels in all tested conditions and was not stimulated by succinate or fumarate (Fig. 3c
, d
). In contrast, inactivation of the ydbH gene did not abolish PydbH expression, but rather stimulated it (Fig. 3e
). Unexpectedly, we found that PydbH expression completely disappeared also in the knock-out mutant of the ydbE gene (Fig. 3b
), whose product has a strong similarity to the Rho. capsulatus C4-dicarboxylate periplasmic binding protein, DctP.
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DISCUSSION |
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Elucidation of the exact role of YdbE, similar to Rho. capsulatus C4-dicarboxylate binding periplasmic protein DctP, awaits further characterization. Rho. capsulatus DctP was shown to be able to bind C4-dicarboxylate in vitro (Walmsley et al., 1992a , b
) and thought to constitute a transporter complex together with a membrane translocator, DctQM (Forward et al., 1997
). In our study, fumarate could be utilized without the ydbE function when ydbH was artificially induced by overexpressing ydbFG and/or ydbH. This suggested that transport of fumarate could be achieved by the YdbH transporter alone, and ydbE would play a role mainly in the regulation of ydbH expression together with ydbFG. An external periplasmic solute-binding protein necessary for sensory transduction in two-component systems has been reported for Agrobacterium tumefaciens. A. tumefaciens initiates infection of a wide range of plants with signals at the wound site. In this process, the ChvE protein, which is a periplasmic monosaccharide-binding protein, was found to interact directly with the periplasmic domain of the VirA sensor protein to activate the VirG regulator (Ankenbauer & Nester, 1990
; Cangelosi et al., 1990
; Shimoda et al., 1993
). In Gram-positive bacteria without a defined periplasmic space, external solute-binding proteins are suggested to be expressed at the cell surface as lipoproteins (Gilson et al., 1988
). The amino-terminal part of YdbE has hydrophobic amino acid residues which possess characteristic features of a signal peptide and a weak but significant consensus sequence for the precursors of lipoproteins could be recognized (L-L-A-C-L-A; the consensus sequence is L-Y-Z-cleavage site-C-y-z; where Y is A, S, V, Q or T, Z is G or A, y is S, G, A, N, Q or D and z is S, A, N or Q; Heijne, 1989
). Therefore, it may be assumed that in B. subtilis, ydbE exists at the cell surface and plays a sensory role in the ydbFG two-component system, giving rise to specificity or increased efficiency, although overproduction of YdbFG proteins led to the expression of ydbH in the absence of YdbE.
It is interesting to note that inactivation of the ydbEFGH system in B. subtilis does not cause a defect in malate utilization, in contrast to other known Dct systems essential for transporting all C4-dicarboxylates. Moreover, expression of the transporter protein, YdbH, was repressed by the addition of malate. It was reported that malate was the best inducer for the C4-dicarboxylic acid transport system in B. subtilis. It was also suggested that at least two transport systems for malate were induced in B. subtilis (Willecke & Lange, 1974 ). The first system, which was required for the uptake of malate, fumarate and succinate, was induced in NYE medium. The second system was then induced by the addition of a large amount of malate. In a search of the B. subtilis genome database, we noticed a newly discovered ORF, yufR, whose deduced product was highly homologous (55·0%) to the malate-specific permease in Streptococcus bovis (Kawai et al., 1997
). In our preliminary experiments, the expression of yufR was activated by malate. Therefore, the Dct system in this study would correspond to the first C4-dicarboxylate-transport system reported by Willecke & Lange (1974)
, though the exact chemical nature of the inducer in yeast extract is obscure, and another highly active transporter for malate, such as the YufR protein, could be induced by the addition of malate.
The deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. Furthermore, a catabolite-responsive element (CRE) was found in the -10 region of the promoter (Fig. 5b), suggesting the negative regulation of ydbH expression by a CRE-binding protein, such as CcpA (Catabolite Control Protein) (Hueck et al., 1994
; Fujita et al., 1995
; Hueck & Hillen, 1995
; Henkin, 1996
). In our preliminary experiments, the expression of ydbH was repressed in the presence of glucose, and the induction of ydbH was restored even in the presence of glucose when a ccpA (encoding CcpA) mutation was introduced into the cells. Interestingly, the repression by the addition of malate was also suppressed in the ccpA mutant, although the mechanism of the involvement of CcpA in the repression by malate needs further examination.
Finally, based on the results discussed in this report, we propose to rename the ydbE, F, G and H genes dctB (solute Binding), dctS (Sensor kinase), dctR (response Regulator) and dctP (Permease), respectively.
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ACKNOWLEDGEMENTS |
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Received 19 July 1999;
revised 12 October 1999;
accepted 18 October 1999.