Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis

Kei Asai1, Sang-Hoon Baik1, Yasuhiro Kasahara1, Shigeki Moriya1 and Naotake Ogasawara1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transport systems for C4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Gram-positive bacteria. The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C4-dicarboxylates in Gram-negative bacteria. Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor–regulator pair, were located. Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate. Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0·05% yeast extract but repressed by the addition of malate to the medium. Inactivation of the putative sensor–regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced. Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C4-dicarboxylate-transport protein and its expression is regulated by a C4-dicarboxylate sensor kinase–regulator pair, YdbF and YdbG. Furthermore, it is suggested that YdbE does not directly participate in transport of C4-dicarboxylates, but plays a sensory role in the ydbFydbG two-component system, giving rise to specificity or increased efficiency to the system. Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein.

Keywords: C4-dicarboxylate, Bacillus subtilis, two-component regulatory system, transporter, solute-binding protein

Abbreviations: Dct, dicarboxylic acid transport


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacillus subtilis has a complete set of enzymes for the TCA cycle and can grow on most of the TCA cycle intermediates as sole carbon sources (Fortnagel & Freese, 1968 ; Kunst et al., 1997 ). However, genes for uptake of the carboxylic acid intermediates and the regulation of their expression are not known in B. subtilis, except for citrate. B. subtilis has two transporters for citrate, CitM and CitH (encoded by the yxiQ gene) (Carlsson & Hederstedt, 1987 ; Boorsma et al., 1996 ); the expression of CitM is regulated by a two-component sensor–regulator pair, CitS and CitT (Yamamoto et al., 1999 ). As for C4-dicarboxylates such as malate, fumarate and succinate, previous studies suggested the existence of active transporters (Fournier & Pardee, 1974 ; Willecke & Lange, 1974 ; Ghei & Kay, 1975 ). 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 transport of malate, fumarate and succinate was induced in NYE medium (yeast extract in the medium was supposed to contain a small amount of malate). The second system, probably specific for malate, was further induced by the addition of a large amount of malate. However, C4-dicarboxylate transport systems in B. subtilis have not been studied genetically.

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 sensor–regulator 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.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. (a) Arrangement of genes related to the C4-dicarboxylic acid transport system in B. subtilis. Transcripts detected around the ydbDH genes are shown by arrows, and designated as Tx. See detail in the text. (b) Northern blots using gene-specific probes. Total RNA was prepared from a B. subtilis 168 culture growing in DS medium when the OD600 reached 0·1 (lanes 1), 0·3 (lanes 2) or 0·6 (lanes 3). The approximate positions of transcripts are indicated as Tx. The size of RNA is shown on the left of the blots.

 

   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth media.
Bacillus subtilis 168 (trpC2) was used as a parental strain to construct mutants. A sporulation medium (DS medium, Schaeffer et al., 1965 ) and a minimal salts medium containing 0·05% (w/v) yeast extract (NYE medium, Willecke & Lange, 1974 ) were used as described previously. To monitor the utilization of C4-dicarboxylates as carbon sources by B. subtilis, malate, fumarate or succinate was added at a final concentration of 0·5% (w/v) when the OD600 of cells growing in NYE medium reached 0·1.

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 PydbH–bgaB 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 PDH94–bgaB 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 manufacturer’s 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 manufacturer’s 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).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Operonic structure of the ydbEFGH region
Arrangement of the ydbEFGH genes on the B. subtilis genome is schematically shown in Fig. 1a. The nucleotide sequence of the region suggested that the ydbFGH genes constitute an operon and are transcribed divergently to ydbE, which is co-transcribed with the downstream ydbD encoding a 30 kDa general stress protein (Antelmann et al., 1997 ). Indeed, Northern blot analysis of total RNA from B. subtilis cells growing in a sporulation medium using RNA probes encompassing each gene detected 2·0 kb (T1) and 3·8 kb (T2) transcripts covering ydbDE and ydbFGH, respectively (Fig. 1b). Additionally, a 4·6 kb (T2*) transcript was detected by the ydbF, G and H probes, but not by the ydbI probe (data not shown), suggesting the existence of an additional promoter overlapping the ydbE gene. In addition to the T1 transcript, the ydbE probe detected a transcript of approximately 0·3 kb (T1*), which might be prematurely terminated or degraded product. These transcripts were detected at constant levels in the early, middle and late stages of the vegetative-growth phase, although the intensities of the T2 and T2* transcript are much weaker than those of T1 and T1*. Furthermore, a transcript of 1·3 kb (T3), which was complementary to the size of the ydbH gene, was strongly induced at mid-exponential phase and, soon after, reduced. Several other bands detected were suspected to be observed due to non-specific hybridization with 16S (1·5 kb) and 23S (2·9 kb) rRNAs. In conclusion, the ydbFG genes were found to be co-transcribed weakly throughout the vegetative-growth phase. The ydbE gene was also expressed throughout the vegetative-growth phase, but more actively than ydbFG. In contrast, the ydbH gene has its own promoter whose activity seems to be regulated in response to a subtle change in the environmental conditions.

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.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Growth properties of mutants in NYE medium with or without an added carbon source. Optical density at 600 nm of wild-type strain168 (a), pMUTINT3-inactivated ydbE (b), ydbF (c), ydbG (d) and ydbH (e) mutants and in-frame deletion mutant of ydbF (f) was monitored. Time 0 indicates the time when the appropriate carbon source was added. Symbols; no addition ({circ}), with the addition of succinate ({blacksquare}), fumarate ({blacktriangleup}) or malate ({bullet}).

 
The defect in the ydbF::pMUTINT3 mutant could be due to a polar effect of the plasmid insertion on the downstream ydbG expression, although it seemed unlikely as discussed below. We constructed a large, in-frame deletion within ydbF, on the basis of the methods described previously by Stahl & Ferrari (1984) . Such a ydbF mutant, where ydbG expression might not be affected, also showed deficiencies in utilization of succinate and fumarate (Fig. 2f).

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.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Expression profiles of the ydbH::bgaB gene fusion in NYE medium with or without an added carbon source. BgaB activity was measured in wild-type strain 168 (a) and ydbE (b), ydbF (c), ydbG (d) and ydbH (e) mutants. Time 0 indicates the time when the appropriate carbon source was added. Symbols; no addition ({circ}), with the addition of succinate ({blacksquare}), fumarate ({blacktriangleup}) or malate ({bullet}).

 
YdbE is not directly required for uptake of C4-dicarboxylates in B. subtilis
In Rho. capsulatus, DctP is thought to be one of the subunits of the C4-dicarboxylate-transport machinery (Forward et al., 1997 ). However, the results described above suggest that the B. subtilis homologue, YdbE, functioned in the induction of the YdbH transporter. To elucidate this difference, we tested whether fumarate uptake could occur without the YdbE function in B. subtilis. To inactivate ydbE and, at the same time, to induce ydbH artificially, we replaced the internal region of the ydbE gene with a tetracycline-resistance gene which has a constitutive strong promoter and is followed by no effective transcription-termination signal. When the tetracycline-resistance gene was inserted in the same direction as that of ydbE, the resulting mutant showed defects in the utilization of fumarate as the carbon source and in the induction of the PydbH::bgaB expression, as were observed in the ydbE::pMUTINT3 mutant (Fig. 4b). On the contrary, when the tetracycline-resistance gene was inserted in the reverse orientation, the growth of the mutant was prolonged by the addition of fumarate and the expression of ydbH was induced (Fig. 4c). Since the difference between the two ydbE null mutants was only the direction of transcription of the inserted tetracycline-resistance gene, these results indicated that the readthrough transcription from the tetracycline-resistance gene affected the fumarate response in the mutants. A high level of transcription of the ydbFGH genes in the ydbE::tet (reverse) mutant was confirmed by inserting pMUTINT3 into the ydbF locus and monitoring the activity of the promoterless lacZ gene in the plasmid (Fig. 4, right panel). Thus, when ydbFG (and ydbH) were artificially overexpressed, ydbH expression and fumarate uptake could resume without YdbE function, suggesting that ydbE was required, if at all, for the efficient induction of ydbH but not for the uptake of C4-dicarboxylates in B. subtilis. It should be noted that two factors would contribute to ydbH expression in the ydbE::tet (reverse) mutant. One is transcription from the promoter for the ydbH gene induced by the overexpression of the YdbFG two-component system, because the induction of the PydbH activity in the ydbE::tet (reverse) mutant was observed at the amyE locus. The other would be the readthrough transcription from the tet gene in the mutant.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. The effect of the orientation of a tetracycline-resistance gene inserted into the ydbE gene on fumarate response. (a) ydbE+ strain. (b) ydbE mutant with insert in the same orientation as ydbE. (c) ydbE mutant with insert in the opposite orientation to ydbE. The left-hand panels show growth properties measured by optical density at 600 nm. The centre panels show the expression profile of ydbH as measured by BgaB activity. Cells were cultured in NYE medium without ({circ}) and with ({bullet}) the addition of fumarate. Time 0 indicates the time when fumarate was added. In the right-hand panel, the organization of the ydbEFGH genes and the tetracycline-resistance gene are indicated by black and white arrows, respectively. Shaded arrows indicate the lacZ gene introduced by integration of pMUTINT3, with its activity measured in NYE medium with fumarate in four experiments and shown with standard deviation.

 
The cis-acting sequence required for the induction of ydbH expression
To further analyse the regulation of expression of ydbH, first we determined the position of the 5' end of the ydbH transcript by primer-extension analysis using total RNA of B. subtilis 168 cells growing in DS medium, in which ydbH expression was strongly induced at mid-exponential phase (Fig. 1b). As shown in Fig. 5a, initiation sites of transcription were detected at 41 and 42 bp upstream of the translation start site, accompanied by a possible promoter sequence for {sigma}A, TTCCCC and TATCAT (Fig. 5b). Then we gradually shortened the upstream sequence of the -35 region in the PydbH::bgaB assay, in order to determine the cis-acting region necessary for the induction of ydbH (Fig. 5b). Although the promoter activity was detected at the same level when the bgaB gene was fused to PDH94, PDH83 or PDH73, it was decreased to the background level when it was fused to PDH64 or PDH53. Therefore, it was suggested that the region from -73 to-65 (AGACCAAAA) was important for the activation of ydbH. Interestingly, this sequence was repeated twice within the region.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 5. (a) Determination of the ydbH transcription-start site by primer extension. Total RNA was prepared from a B. subtilis 168 culture growing in DS medium when the OD600 reached 0·5 (lane 1) or 0·8 (lane 2) and used for primer-extension analyses. Sequencing ladders were generated by using the primer used for the reverse-transcriptase reaction. The sequence and the transcriptional-start sites (indicated by asterisks) are shown. (b) Nucleotide sequence of the ydbH promoter region. The deduced amino acid sequences of the C-terminal portion of YdbG and the N-terminal portion of YdbH are shown. A putative ribosome-binding site is boxed. The experimentally determined transcriptional-start sites are labelled by asterisks and the deduced -10 and -35 regions are underlined. Arrowheads indicate the first nucleotide of fragments fused to the bgaB gene to measure the ydbH promoter activity in DS medium. Black arrowheads indicate that activity was at the same level as that of PDH96 derived activity, whereas a white one indicates that activity was at background level. Arrows show direct repeat sequences. The nucleotide sequence indicated by small letters is the region that shows a similarity with the cre consensus sequence shown below.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The transport system for C4-dicarboxylates, such as malate, fumarate and succinate, was poorly understood in Gram-positive bacteria. The whole genome sequence of B. subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to the C4-dicarboxylate periplasmic binding proteins and transport proteins in Gram-negative bacteria, respectively. Between ydbE and ydbH, two other genes, ydbF and ydbG that were predicted to encode a two-component sensor–regulator pair, were located. Furthermore, the deduced products, YdbF and YdbG proteins, had significant similarities to the E. coli C4-dicarboxylate sensor and regulator pair, DcuS and DcuR, respectively. Indeed, inactivation of each one of the ydbEFGH genes caused deficiency of utilization of fumarate and succinate but not of malate. The expression of ydbH was stimulated in minimal salts medium containing 0·05% yeast extract, but repressed by the addition of malate to the medium. The inactivation of the putative sensor–regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. However, the utilization of fumarate and the stimulation of ydbH expression occurred without the ydbE function under conditions where ydbFG and/or ydbH were artificially overexpressed. These results, together with the sequence similarities, implied that 1) YdbH is a C4-dicarboxylate transport protein and 2) YdbF and YdbG constitute a C4-dicarboxylate sensor kinase and regulator pair, which activates ydbH expression. Furthermore, ydbFG seems to regulate the expression of ydbH in response to external signal(s) as is the case in the Gram-negative bacteria, because the inactivation of ydbH did not affect its own regulation, indicating that the regulation may not depend on cellular metabolism of incorporated C4-dicarboxylates.

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.


   ACKNOWLEDGEMENTS
 
We are grateful to Drs R. H. Doi and H. Saito for critical reading of the manuscript. We also thank Drs Y. Fujita, Y. Miwa, K. Yoshida and M. Oda for helpful discussions. This work was supported by a grant, JSPS-RFTF96L00105, from the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture Japan.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ankenbauer, R. G. & Nester, E. W. (1990). Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.J Bacteriol 172, 6442-6446.[Medline]

Antelmann, H., Bernhardt, J., Schmid, R., Mach, H., Völker, U. & Hecker, M. (1997). First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis.Electrophoresis 18, 1451-1463.[Medline]

Boorsma, A., van der Rest, M. E., Lolkema, J. S. & Konings, W. N. (1996). Secondary transporters for citrate and the Mg2+–citrate complex in Bacillus subtilis are homologous proteins.J Bacteriol 178, 6216-6222.[Abstract]

Cangelosi, G. A., Ankenbauer, R. G. & Nester, E. W. (1990). Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein.Proc Natl Acad Sci USA 87, 6708-6712.[Abstract]

Carlsson, P. & Hederstedt, L. (1987). Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli.Gene 61, 217-224.[Medline]

Engelke, T., Jagadish, M. N. & Pühler, A. (1987). Biochemical and genetical analysis of Rhizobium meliloti mutants defective in C4-dicarboxylate transport.J Gen Microbiol 133, 3019-3029.

Finan, T. M., Oresnik, I. & Bottacin, A. (1983). Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.J Bacteriol 154, 1403-1413.[Medline]

Fortnagel, P. & Freese, E. (1968). Analysis of sporulation mutants. II. Mutants blocked in the citric acid cycle.J Bacteriol 95, 1431-1438.[Medline]

Forward, J. A., Behrendt, M. C., Wyborn, N. R., Cross, R. & Kelly, D. J. (1997). TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria.J Bacteriol 179, 5482-5493.[Abstract]

Fournier, R. E. & Pardee, A. B. (1974). Evidence for inducible, L-malate binding proteins in the membrane of Bacillus subtilis: identification of presumptive components of the C4-dicarboxylate transport systems.J Biol Chem 249, 5948-5954.[Abstract/Free Full Text]

Fujita, Y., Miwa, Y., Galinier, A. & Deutscher, J. (1995). Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr.Mol Microbiol 17, 953-960.[Medline]

Ghei, O. K. & Kay, W. W. (1975). Regulation of C4-dicarboxylic acid transport in Bacillus subtilis.Can J Microbiol 21, 527-536.[Medline]

Gilson, E., Alloing, G., Schmidt, T., Claverys, J. P., Dudler, R. & Hofnung, M. (1988). Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma.EMBO J 7, 3971-3974.[Abstract]

Golby, P., Davies, S., Kelly, D. J., Guest, J. R. & Andrews, S. C. (1999). Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS–DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli.J Bacteriol 181, 1238-1248.[Abstract/Free Full Text]

Hamblin, M. J., Shaw, J. G. & Kelly, D. J. (1993). Sequence analysis and interposon mutagenesis of a sensor-kinase (DctS) and response-regulator (DctR) controlling synthesis of the high-affinity C4-dicarboxylate transport system in Rhodobacter capsulatus.Mol Gen Genet 237, 215-224.[Medline]

Heijne, G. V. (1989). The structure of signal peptidases from bacterial lipoproteins.Protein Eng 2, 531-534.[Abstract]

Henkin, T. M. (1996). The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis.FEMS Microbiol Lett 135, 9-15.[Medline]

Hueck, C. J. & Hillen, W. (1995). Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria?Mol Microbiol 15, 395-401.[Medline]

Hueck, C. J., Hillen, W. & Saier, M. H.Jr (1994). Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria.Res Microbiol 145, 503-518.[Medline]

Igo, M. M. & Losick, R. (1986). Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis.J Mol Biol 191, 615-624.[Medline]

Itaya, M. (1992). Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome.Biosci Biotechnol Biochem 56, 685-686.[Medline]

Jording, D., Sharma, P. K., Schmidt, R., Engelke, T., Uhde, C. & Pühler, A. (1992). Regulatory aspects of the C4-dicarboxylate transport in Rhizobium meliloti – transcriptional activation and dependence on effective symbiosis.J Plant Physiol 141, 18-27.

Kawai, S., Suzuki, H., Yamamoto, K. & Kumagai, H. (1997). Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352.J Bacteriol 179, 4056-4060.[Abstract]

Kawamura, F., Saito, H. & Ikeda, Y. (1980). Bacteriophage {phi}1 as a gene-cloning vector in Bacillus subtilis.Mol Gen Genet 180, 259-266.[Medline]

Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[Medline]

Moriya, S., Tsujikawa, E., Hassan, A. K., Asai, K., Kodama, T. & Ogasawara, N. (1998). A Bacillus subtilis gene encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition.Mol Microbiol 29, 179-187.[Medline]

Nanamiya, H., Ohashi, Y., Asai, K., Moriya, S., Ogasawara, N., Fujita, M., Sadaie, Y. & Kawamura, F. (1998). ClpC regulates the fate of a sporulation initiation sigma factor, {sigma}H protein, in Bacillus subtilis at elevated temperatures.Mol Microbiol 29, 505-513.[Medline]

Price, C. W., Gitt, M. A. & Doi, R. H. (1983). Isolation and physical mapping of the gene encoding the major sigma factor of Bacillus subtilis RNA polymerase.Proc Natl Acad Sci USA 80, 4074-4078.[Abstract]

Reid, C. J. & Poole, P. S. (1998). Roles of DctA and DctB in signal detection by the dicarboxylic acid transport system of Rhizobium leguminosarum.J Bacteriol 180, 2660-2669.[Abstract/Free Full Text]

Ronson, C. W., Astwood, P. M., Nixon, B. T. & Ausubel, F. M. (1987). Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.Nucleic Acids Res 15, 7921-7934.[Abstract]

Schaeffer, P., Millet, J. & Aubert, J.-P. (1965). Catabolite repression of bacterial sporulation.Proc Natl Acad Sci USA 54, 704-711.[Medline]

Shaw, J. G., Hamblin, M. J. & Kelly, D. J. (1991). Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus.Mol Microbiol 5, 3055-3062.[Medline]

Shimoda, N., Toyoda, Y. A., Aoki, S. & Machida, Y. (1993). Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium.J Biol Chem 268, 26552-26558.[Abstract/Free Full Text]

Six, S., Andrews, S. C., Unden, G. & Guest, J. R. (1994). Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct).J Bacteriol 176, 6470-6478.[Abstract]

Sofia, H. J., Burland, V., Daniels, D. L., Plunkett, G.III & Blattner, F. R. (1994). Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76·0 to 81·5 minutes.Nucleic Acids Res 22, 2576-2586.[Abstract]

Stahl, M. L. & Ferrari, E. (1984). Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation.J Bacteriol 158, 411-418.[Medline]

Vagner, V., Dervyn, E. & Ehrlich, S. D. (1998). A vector for systematic gene inactivation in Bacillus subtilis.Microbiology 144, 3097-3104.[Abstract]

Walmsley, A. R., Shaw, J. G. & Kelly, D. J. (1992a). The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.J Biol Chem 267, 8064-8072.[Abstract/Free Full Text]

Walmsley, A. R., Shaw, J. G. & Kelly, D. J. (1992b). Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.Biochemistry 31, 11175-11181.[Medline]

Willecke, K. & Lange, R. (1974). C4-dicarboxylate transport in Bacillus subtilis studied with 3-fluoro-L-erythro-malate as a substrate.J Bacteriol 117, 373-378.[Medline]

Yamamoto, H., Murata, M. & Sekiguchi, J. (1999). CitST two-component system regulates gene expression of citrate transport in Bacillus subtilis. In Abstracts of the 10th International Conference on Bacilli, Baveno, Italy, p. 71.

Youngman, P., Perkins, J. & Sandman, K. (1985). Use of Tn917-mediated transcriptional gene fusions to lacZ and cat-86 for the identification and study of regulated genes in the Bacullus subtilis chromosome. In Molecular Biology of Microbial Differentiation, pp. 47-54. Edited by J. A. Hoch & P. Setlow. Washington, DC: American Society for Microbiology.

Zientz, E., Bongaerts, J. & Unden, G. (1998). Fumarate regulation of gene expression in Escherichia coli by the DcuSR (dcuSR genes) two-component regulatory system.J Bacteriol 180, 5421-5425.[Abstract/Free Full Text]

Zientz, E., Janausch, I. G., Six, S. & Unden, G. (1999). Functioning of DcuC as the C4-dicarboxylate carrier during glucose fermentation by Escherichia coli.J Bacteriol 181, 3716-3720.[Abstract/Free Full Text]

Received 19 July 1999; revised 12 October 1999; accepted 18 October 1999.