Citrate Utilization by Corynebacterium glutamicum Is Controlled by the CitAB Two-Component System through Positive Regulation of the Citrate Transport Genes citH and tctCBA

Abstract

In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacterium Corynebacterium glutamicum was studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded by citH belongs to the citrate-Mg²⁺:H⁺/citrate-Ca²⁺:H⁺ symporter family, whereas the permease encoded by the tctCBA operon is a member of the tripartite tricarboxylate transporter family. The expression of citH or tctCBA in Escherichia coli enabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinant E. coli strains indicated that CitH is active with Ca²⁺ or Sr²⁺ but not with Mg²⁺ and that TctABC is active with Ca²⁺ or Mg²⁺ but not with Sr²⁺. We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, the C. glutamicum wild type grew best when the minimal medium was supplemented with CaCl₂ but that MgCl₂ and SrCl₂ also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression of citH and tctCBA was activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates. C. glutamicum CitAB is the first member of this family studied in Actinobacteria.

Citrate is a ubiquitous natural compound which can be utilized as a carbon and energy source by many bacterial species. The anaerobic catabolism of citrate, which occurs, e.g., in enterobacteria like Klebsiella pneumoniae and Escherichia coli (7) and in lactic acid bacteria (14), usually involves the key enzyme citrate lyase (EC 4.1.3.6), which catalyzes the cleavage of citrate into acetate (the end product) and oxaloacetate (8, 47, 48). The subsequent catabolism of oxaloacetate can occur via different pathways, leading to, e.g., acetate and succinate as end products. Aerobic citrate utilization by bacteria possessing a complete tricarboxylic acid cycle usually requires only a citrate uptake system. Presently, at least five families of citrate transporters in bacteria have been characterized according to the classification system introduced by Saier (44): (i) the metabolite:H⁺ symporter family (transporter classification [TC] no. 2.A.1.6) within the major facilitator superfamily (TC no. 2.A.1), represented by CitH of K. pneumoniae (53, 54); (ii) the citrate-Mg²⁺:H⁺/citrate-Ca²⁺:H⁺ symporter (CitMHS) family (TC no. 2.A.11), represented by CitM and CitH of Bacillus subtilis (6, 30, 31); (iii) the citrate:cation symporter family (TC no. 2.A.24), represented by CitS and CitW of K. pneumoniae (24, 41); (iv) the divalent anion:Na⁺ symporter family (TC no. 2.A.47), represented by CitT of E. coli (42); and (v) the tripartite tricarboxylate transporter (TTT) family (TC no. 2.A.80), represented by TctABC of Salmonella enterica serovar Typhimurium (63). Whereas the transporters of the former four families consist of a single protein, the TctABC system is composed of three different subunits: two integral membrane proteins with presumably 12 (TctA) and 4 (TctB) transmembrane helices, plus a periplasmic citrate binding protein (TctC).

For most citrate transporter genes studied so far with respect to regulation, expression is induced in the presence of the substrate. In many bacteria, the transcription of genes for citrate uptake and catabolism is activated by two-component signal transduction systems (TCS) consisting of a membrane-bound histidine kinase which controls the phosphorylation status of a soluble response regulator and thereby its activity as a transcriptional regulator. Examples are the CitA-CitB TCS of K. pneumoniae and E. coli (9, 34) and the CitS-CitT TCS of B. subtilis (64), which belong to the CitAB/DcuSR family of TCS (23). The periplasmic domains of the CitA histidine kinases from K. pneumoniae and E. coli were shown to bind citrate with high specificities and affinities (between 0.1 and 10 μM at neutral pH) (22, 23). A previous mutagenesis study identified several amino acid residues critical for citrate binding, three of which are highly conserved within the CitAB/DcuSR subfamily (18). The direct involvement of these residues in citrate binding was confirmed by the first crystal structure of a periplasmic domain in a histidine kinase, i.e., that of K. pneumoniae CitA in a complex with citrate (43). More recently, a structural comparison between citrate-free and citrate-bound states of the periplasmic CitA domain became possible, revealing that ligand binding causes considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor (49).

Besides induction by citrate, the expression of citrate permease genes is sometimes also subject to carbon catabolite repression, which can at least partially override citrate induction, if other carbon sources are available. The expression of citH from B. subtilis is subject to control by the central regulator CcpA (56) and by a product of arginine metabolism (57). The expression of the tctCBA genes of S. enterica serovar Typhimurium and of citS from K. pneumoniae is controlled by the cyclic AMP receptor protein-cyclic AMP complex system (33, 62).

Whereas a wealth of information on citrate transporters and their transcriptional regulation in proteobacteria and gram-positive bacteria with low GC contents (Firmicutes) is available, not much is known about these systems in gram-positive bacteria with high GC contents (Actinobacteria). Here, we describe citrate utilization and its regulation in Corynebacterium glutamicum, a predominantly aerobic soil bacterium belonging to the Corynebacterium-Mycobacterium-Nocardia group of actinomycetes. This species is of interest due to its biotechnological importance as a producer of l-glutamate and l-lysine and its potential to serve as a nonpathogenic model organism for studying selected features common to corynebacteria and pathogenic mycobacteria. Overviews of C. glutamicum biology, genetics, physiology, and biotechnology can be found in two recent monographs (11, 15).

In an early study, Kimura (26) reported that C. glutamicum cannot grow on citrate unless the medium is supplemented with corn steep liquor. Later, Birnbaum and Demain (3) presented evidence that the function of corn steep liquor is to supply energy for the induction of a citrate transport system. They showed that resting cells of C. glutamicum pregrown on glucose with citrate take up citrate and convert it to extracellular glutamate but that cells pregrown on glucose without citrate do not or do so very slowly. Quite recently, we characterized citrate utilization in C. glutamicum by transcriptome and proteome analyses (39). In that study, it was shown that genes for two putative citrate uptake systems (citM, which has been renamed citH based on the studies presented here, and tctCBA) are strongly activated during growth on citrate and that several genes for the transport of alternative carbon sources, such as glucose, fructose, saccharose, and gluconate, show decreased mRNA levels in the presence of citrate. In addition, genes of the tricarboxylic acid cycle, of the respiratory chain, of F₁F_o ATP synthase, and of gluconeogenesis show increased expression during growth on citrate (39).

In the present study, we have analyzed the molecular basis of citrate utilization by C. glutamicum in more detail. We provide evidence that both CitH (previously named CitM) and TctABC are in fact functional citrate transporters but with differing cation specificities. Citrate-dependent activation of citH and tctCBA was found to be dependent on the CitAB two-component system.

MATERIALS AND METHODS

Bacterial strains, media, and growth conditions.

The C. glutamicum wild-type strain ATCC 13032 and the ΔcitAB, ΔcitH, ΔtctCBA, and ΔcitH ΔtctCBA deletion mutants (Table 1) were cultivated aerobically on a rotary shaker (120 rpm) at 30°C in CGXII minimal medium (25) containing 30 mg/liter 3,4-dihydroxybenzoate as an iron chelator and carbon sources and salts as described in Results. For strain construction, plates of BHIS agar (brain heart infusion agar [Difco, Detroit, MI] with 0.5 M sorbitol) were used. Precultivation for growth experiments was usually performed in CGXII medium containing 50 mM sodium citrate, 50 mM glucose, and either 5 mM CaCl₂ or 100 mM MgCl₂. For cloning purposes, E. coli DHα (Bethesda Research Laboratories) was cultivated aerobically on a rotary shaker (120 rpm) at 37°C in Luria-Bertani medium (45). In order to test E. coli strains for aerobic growth on citrate as the sole carbon source, the strains were streaked onto Simmons citrate agar plates (Merck). For testing the cation specificity of the citrate transport systems CitH and TctABC, E. coli DH5α carrying either pXMJ19-citH or pXMJ19-tctCBA was cultivated in M9 minimal medium (45) supplemented with 1 g/liter Casamino Acids. For selection purposes, chloramphenicol (10 μg/ml for C. glutamicum and E. coli) and kanamycin (25 μg/ml for C. glutamicum and 50 μg/ml for E. coli) were used.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Description and/or genotypea	Source or reference
C. glutamicum strains
ATCC 13032	Biotin-auxotrophic wild-type strain	1
ATCC 13032 ΔcitAB	Derivative of ATCC 13032 with a citAB deletion	27
ATCC 13032 ΔcitH	Derivative of ATCC 13032 with an in-frame deletion of the citH gene	This work
ATCC 13032 ΔtctCBA	Derivative of ATCC 13032 with an in-frame deletion of the tctCBA operon	This work
ATCC 13032 ΔcitH ΔtctCBA	Derivative of ATCC 13032 with an in-frame deletion of the citH gene and in the tctCBA operon	This work
E. coli strains
MG1655	Wild-type strain	5
DH5α	F− φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK− mK+) deoR thi-1 phoA supE44 λ−gyrA96 relA1	Invitrogen
BL21(DE3)	ompT hsdSB(rB− mB−) gal dcm (DE3)	Novagen
Plasmids
pXMJ19	Cmr; C. glutamicum/E. coli shuttle vector for IPTG-inducible gene expression (pBL1 oriVC.g. pK18 oriVE.c. lacI Ptac)	20
pXMJ19-citAB	Cmr; pXMJ19 carrying the C. glutamicum citAB genes	This work
pXMJ19-citH	Cmr; pXMJ19 carrying the C. glutamicum citH gene	This work
pXMJ19-tctCBA	Cmr; pXMJ19 carrying the C. glutamicum tctCBA operon	This work
pET28b	Kmr; vector for overexpression of genes in E. coli under the control of a T7 promoter and for addition of an N-terminal or C-terminal hexahistidine tag to the synthesized protein (pBR322 oriVE.c. PT7lacI)	Novagen
pET28b-CgCitB-NHis6	Kmr; pET28b derivative for overproduction of the C. glutamicum response regulator CitB with an N-terminal hexahistidine tag	This work
pK19mobsacB	Kmr; vector for allelic exchange in C. glutamicum (pK18 oriVE.c. sacB lacZα)	46
pK19mobsacB-ΔcitH	Kmr; pK19mobsacB derivative containing a crossover PCR product covering the regions up- and downstream of citH	This work
pK19mobsacB-ΔtctCBA	Kmr; pK19mobsacB derivative containing a crossover PCR product covering the region upstream of tctC and regions downstream of tctA	This work
pK19mobsacB-ΔcitAB	Kmr; pK19mobsacB derivative containing a crossover PCR product covering the region upstream of citA and regions downstream of citB	27

^aoriV_C.g., C. glutamicum oriV; oriV_E.c., E. coli oriV.

Construction of C. glutamicum deletion mutants.

C. glutamicum mutants with in-frame deletions of citAB (ΔcitAB), citH (ΔcitH), tctCBA (ΔtctCBA), and citH and tctCBA (ΔcitH ΔtctCBA) were constructed via a two-step homologous recombination procedure as described previously (37). The regions up- and downstream (approximately 450 bp each) of the gene/operon to be deleted were amplified using pairs of oligonucleotides designated Delta-gene/operon-1 and Delta-gene/operon-2 and Delta-gene/operon-3 and Delta-gene/operon-4 (Table 2), respectively, and the products served as templates for crossover PCR with oligonucleotides designated Delta-gene/operon-1 and Delta-gene/operon-4. The resulting PCR product of ∼0.9 kb was digested with the restriction enzymes indicated in the primer table (Table 2) and cloned into pK19mobsacB cut with the same enzymes (46). DNA sequence analysis confirmed that the cloned PCR products did not contain spurious mutations. The transfer of the resulting plasmids, designated pK19mobsacB-Δgene/operon, into C. glutamicum and selection for the first and second recombination events were performed as described previously (37). Kanamycin-sensitive and saccharose-resistant clones were tested by colony PCR analysis with a primer pair designated Delta-gene/operon-out-fw and Delta-gene/operon-out-rv (Table 2). Clones which had the desired in-frame deletion of the gene/operon revealed a 1-kb fragment in which all nucleotides except for the first 6 codons and the last 12 codons were replaced by a 21-bp tag.

TABLE 2.

Oligonucleotides used in this study

Oligonucleotide	Sequence (5′→3′)a	Restriction endonuclease
citAB-OE-fw-SalI	TAT AGT CGA CCT TCA CGC TCT GCT GAT AAT CGC	SalI
citAB-OE-rv-EcoRI	TAT AGA ATT CAT GCT GAC CGA GGT GGA CAT CC	EcoRI
citH-OE-fw-PstI	TAT ACT GCA GCA GAA CAC TCG GGA TCT CAA AGT TTC G	PstI
citH-OE-rv-EcoRI	TAT AGA ATT CGG GGT TTG GCG TCG AAA AGC	EcoRI
tct-OE-fw	TAT ATC TAG ACC CAA GCA CTT AGG CAT CAA ACA TTC	XbaI
tct-OE-rv	TAT ACC CGG GAT GGT CTC TAA GCG ACT TCG AGG	XmaI
cg-citB-for	CGC CGC CAT ATG GAT CAA ACA CTT AAA GTT TTA GTA	NdeI
cg-citB-rev	ATA ATA CTC GAG CTA GAG CAG TGG CTT TGA ATA	XhoI
citH-PE-1	GTG GCT GAG CGT GTA GTC AGC GGC TAA TGC CG
citH-PE-2	CTT TCT GTG TTT CTC GAA ACT TTG AGA TCC CGA G
citH-PE-4	TAA TGC CGT GAC TTG TCC TGA AG
tct-PE-2	TGC GGG TTC TGC TCC TAC TTC GGC C
tct-PE-3	AAT CCC TCT CGG TCG GAC TTT TG
citAB-PE-1	AAA TAG CTA CCA CCA ACG CGA CGG TAG CCA C
citAB-PE-2	TCC ATC ATC AAA ACT GCG AAA ATT CCG GTG C
Delta-citAB-1	TAT ATC TAG AGA GCG TGT AGT CAG CGG CTA ATG	XbaI
Delta-citAB-2	CCC ATC CAC TAA ACT TAA ACA CAA AAC TGC GAA AAT TCC GGT GC
Delta-citAB-3	TGT TTA AGT TTA GTG GAT GGG GGG CGA CCA GAA CAT CTA TAT TC
Delta-citAB-4	ATA TGA ATT CAC CAT GTG GTG TTG TGC GTA CC	EcoRI
Delta-citAB-out-fw	GAC TAC CAG GAT GCC ATC TGA G
Delta-citAB-out-rv	TAT ATC CAA CCT GCA CCA AGT AC
Delta-citM-1	TAT ACT GCA GAT GAA GAT AAG CCC GAC TCC CC	XbaI
Delta-citM-2	CCC ATC CAC TAA ACT TAA ACA GTA GCC CTC TTC ATC GGC GTC G
Delta-citM-3	TGT TTA AGT TTA GTG GAT GGG GAG GAT CAT GGC GAA GCC AAG
Delta-citM-4	ATA TGA ATT CAC CAT GTG GTG TTG TGC GTA CC	EcoRI
Delta-citM-out-fw	CGC CCG CAT ATG AAC CAA TAG C
Delta-citM-out-rv	TGA ATC ACC AGT ATT CGG GTA GC
Delta-tct-1	TAT ATC TAG ACG AGC GCT ACG TGA CTT CTT TGA	XbaI
Delta-tct-2	CCC ATC CAC TAA ACT TAA ACA TGC TCC TAC TTC GGC CAT TTT GGG GG
Delta-tct-3	TGT TTA AGT TTA GTG GAT GGG AAG CAC CTG ACT TCT CAG CTC GAA ACC
Delta-tct-4	TAT ACC CGG GGG TTT AGC AAG CGG GAC GCT TTC G	XmaI
Delta-tct-out-fw	GGG TTT GAT TTT GAG GTG ACA ACG G
Delta-tct-out-rv	TTC CTA GGC TGC CCG CTT GGG CT
P_citH_fw	TTT CCA GTC GGA TCC ACC AAC AG
P_citH_rv	GTT TCT CGA AAC TTT GAG ATC CCG
P_citH_control_fw	CGG GAT CTC AAA GTT TCG AGA AAC
P_citH_control_rv	CGA AGA TGG TGG GGA CCA ACA G
P_tct_fw	GAC AAC AAG GGG GAG TCC TAG C
P_tct_rv	GGT TCT GCT CCT ACT TCG GCC
P_cg2836_control_fw	GGT TAG CGC GCC GCC CTG TC
P_cg2836_control_rv	CGA CTT CTC AAA GGC GAC TTC AC

^aIn some cases, oligonucleotides were designed to introduce recognition sites for restriction endonucleases (recognition sites are underlined, and the corresponding restriction endonucleases are listed) and complementary 21-mer sequences for generating crossover PCR products (indicated by italics) into the resulting PCR products.

Plasmid construction.

For the construction of pXMJ19-citAB, the entire citAB operon was amplified using oligonucleotides citAB-OE-fw-SalI and citAB-OE-rv-EcoRI. For the construction of pXMJ19-citH and pXMJ19-tctCBA, the citH gene and the tctCBA operon were amplified using the oligonucleotide pairs citH-OE-fw-PstI/citH-OE-rv-EcoRI and tct-OE-fw/tct-OE-rv, respectively. Since the plasmid pXMJ19 does not carry a ribosomal binding site, regions of 81, 52, and 70 bp upstream of citA, citH, and tctC, respectively, were also amplified with these primers. The PCR products were digested using the appropriate restriction enzymes (Table 2) and cloned into pXMJ19 (20). For the overproduction and purification of CitB with an amino-terminal histidine tag, the citB coding region was amplified using oligonucleotides that introduce an NdeI restriction site including the start codon (cg-citB-for) and an XhoI restriction site after the stop codon (cg-citB-rev). The purified 698-bp PCR product was digested with NdeI and XhoI and cloned into the expression vector pET28b cut with the same enzymes, resulting in plasmid pET28b-CgCitB-NHis₆. The CitB protein encoded by this plasmid (238 amino acids; 25.6 kDa) contains a stretch of 20 additional amino acids (MGSSHHHHHHSSGLVPRGSH) at the amino terminus, including a hexahistidine tag and a thrombin cleavage site.

Overproduction and purification of CitB.

E. coli BL21(DE3) carrying plasmid pET28b-CgCitB-NHis₆ was grown at 30°C in 500 ml of Luria-Bertani medium with 50 μg/ml kanamycin to an optical density at 600 nm (OD₆₀₀) of ∼1 before the addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After cultivation for another 3 h, cells were harvested by centrifugation, washed once, and stored at −20°C. For cell extract preparation, thawed cells were resuspended in 10 ml of TNIG5 (20 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole, 5% [vol/vol] glycerol, pH 7.9). After the addition of 1 mM diisopropylfluorophosphate and 1 mM phenylmethylsulfonyl fluoride, the cells were disrupted using a French pressure cell (SLM Aminco; Spectronic Instruments, Rochester, NY). Intact cells and cell debris were removed by centrifugation (20 min at 5,000 × g and 4°C), and the cell extract was subjected to ultracentrifugation (1 h at 150,000 × g and 4°C). CitB, present in the supernatant from the ultracentrifugation step, was purified by Ni²⁺-chelate affinity chromatography using His-Bind resin (Novagen; Merck KGaA, Darmstadt, Germany). After the column was washed with 10 bed volumes of TNIG20 buffer and 10 bed volumes of TNIG100 buffer (the same as TNIG5 but containing 20 and 100 mM imidazole, respectively), CitB protein was eluted with TNIG400 buffer (containing 400 mM imidazole). Fractions containing CitB were pooled, and the elution buffer was exchanged for BS buffer (50 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 0.5 mM EDTA, 10% [vol/vol] glycerol, pH 7.5) by gel filtration using PD-10 columns (Amersham Biosciences). Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as the standard. The purity of the protein preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent protein detection with Gel Code blue staining reagent (Pierce, Rockford, IL).

Electrophoretic mobility shift assays (EMSAs).

For testing the binding of CitB to putative target promoters, purified CitB protein (0 to 1.4 μM) was mixed with 100-ng DNA fragments (219 to 336 bp; final concentrations, 22 to 35 nM) in a total volume of 20 μl. The binding buffer contained 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl₂, 0.5 mM EDTA, and 10% (vol/vol) glycerol. The reaction mixture was incubated at room temperature for 30 min and then loaded onto a native 15% polyacrylamide gel. Electrophoresis and DNA detection with Sybr green I were performed as described before (59). The DNA fragments were generated by PCR, purified with a PCR purification kit (Qiagen, Hilden, Germany), and eluted in EB buffer (10 mM Tris-HCl, pH 8.5).

Analysis of glucose and citrate.

The quantitation of glucose and citrate in the supernatants of cultures was performed by high-performance liquid chromatography (HPLC) using a Merck Hitachi HPLC system equipped with an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad). Isocratic elution was performed with 6 mM H₂SO₄ at 80°C for 20 min at a flow rate of 0.6 ml/min. Citric acid was detected with a LaChrom L-7400 UV detector (Merck Hitachi) at 215 nm. Glucose was detected with a LaChrom L-7490 refractive index detector (Merck Hitachi). Quantification was performed using calibration curves obtained with external standards.

Primer extension analysis.

Nonradioactive primer extension analysis was performed as described previously (16). Each transcriptional start site was determined using at least two different digoxigenin-labeled oligonucleotides: citH-PE-1, citH-PE-2, and citH-PE-4 for citH, tct-PE-2 and tct-PE-3 for tctCBA, and citAB-PE-1 and citAB-PE-2 for citAB (Table 2).

DNA microarray analysis.

The preparation of RNA and the synthesis of fluorescently labeled cDNA were carried out as described previously (35). Custom-made DNA microarrays printed with 70-mer oligonucleotides for C. glutamicum ATCC 13032 were obtained from Operon (Cologne, Germany) and are based on the genome sequence entry with accession no. NC_006958 (21). Hybridization and stringent washing of the microarrays were performed according to the instructions of the supplier. Hybridization was carried out for 16 to 18 h at 42°C by using a microarray user interface hybridization system (BioMicro Systems, Salt Lake City, UT). After being washed, the microarrays were dried by centrifugation (5 min at 1,600 × g) and the levels of fluorescence at 532 nm (for Cy3-dUTP) and 635 nm (for Cy5-dUTP) were determined with 10-μm resolution by using a GenePix 6000 laser scanner (Axon Instruments, Sunnyvale, CA). Quantitative image analysis was carried out using GenePix image analysis software (GenePix Pro 6.0; Axon Instruments), and the results were saved as GenePix results files. For data normalization, GenePix results files were processed using the Bioconductor/R packages limma and marray (http://www.bioconductor.org). Processed and normalized data, as well as experimental details (minimum information about a microarray experiment) (10), were stored in the in-house microarray database for further analysis (40).

RESULTS

Use of E. coli to test whether the C. glutamicum genes citH and tctCBA encode functional citrate transporters.

Our previous study on citrate utilization by C. glutamicum had shown that the genes for two putative citrate transport systems, citH (cg0088; previously designated citM) and tctCBA (cg3127, cg3126, and cg3125), were the ones most strongly induced in the presence of citrate in the medium (39). CitH belongs to the CitMHS family (TC no. 2.A.11), whereas TctABC is a member of the TTT family (TC no. 2.A.80) (63). The CitH protein shows 42 and 43% sequence identity to the B. subtilis citrate transporters CitM and CitH (6, 30). The first of several putative start codons for citH (Fig. 1A) leads to a protein consisting of 526 amino acid residues. The tctCBA genes (Fig. 1B) presumably form an operon, since the last nucleotides of the tctC and tctB stop codons correspond to the first nucleotides of the start codons of tctB and tctA, respectively. Downstream of tctA, a Rho-independent transcriptional terminator structure (ΔG° = −128.5 kJ/mol) was identified. TctA (510 amino acid residues) and TctB (188 residues) are integral membrane proteins with 12 and 4 predicted transmembrane helices, respectively. TctC (334 residues) is presumably a periplasmic citrate binding protein that contains a putative transmembrane helix (residues 19 to 39) in the N-terminal region. This transmembrane helix may either tether the protein to the cytoplasmic membrane or be cleaved off by signal peptidase. TctA, TctB, and TctC from C. glutamicum show 38, 29, and 27% sequence identity to the corresponding proteins from S. enterica serovar Typhimurium, the first and most carefully studied members of the TTT transporter family (50-52, 60-62).

FIG. 1.

Physical map of the cit locus of C. glutamicum and DNA sequence of the citA-citH intergenic region (A) and physical map of the tct locus and DNA sequence of the tctC promoter region (B). Transcriptional start sites as determined by primer extension analyses are indicated by asterisks. For citH, three transcriptional start sites were identified. The one resulting in the longest transcript and having the most intense signal was set as +1. The derived −10 and −35 regions of the promoters are indicated by double lines. The proposed start codons of citH, citA, and tctC are indicated in bold, and the putative ribosome binding sites (RBS) are underlined. The proposed N-terminal amino acid residues of CitH, CitA, and TctC are also shown. Downstream of citH and tctA, Rho-independent transcriptional terminator structures are indicated by stem-loop structures.

In order to test whether the C. glutamicum CitH and TctABC proteins are in fact citrate transporters, the corresponding genes were cloned and expressed in the heterologous host E. coli MG1655. This strain does not synthesize a citrate transporter under aerobic conditions and consequently cannot grow on citrate. It is therefore a suitable host to test the functions of putative citrate transporters (42). The recombinant strains were tested for their abilities to utilize citrate as the sole carbon source under aerobic conditions by being streaked onto Simmons citrate agar supplemented with 10 μg/ml chloramphenicol. The formation of colonies and a color change of the pH indicator bromthymol blue from green to blue indicated that MG1655/pXMJ19-citH and MG1655/pXMJ19-tctCBA, but not the control strain, MG1655/pXMJ19, were able to utilize citrate (data not shown). The color change is observed only with cells that are able to metabolize citrate, which leads to the alkalinization of the medium. These results clearly indicate that both CitH and the TctABC complex of C. glutamicum are functional citrate transporters.

Cation dependency of CitH and TctABC tested in the E. coli system.

CitH belongs to the CitMHS family of transporters. Representatives of this family transport citrate in symport with different cations, such as Mg²⁺ (CitM of B. subtilis), Ca²⁺ (CitH of B. subtilis), and Fe³⁺ (CitH of Streptococcus mutans) (4, 29, 30). Since the metal ion specificity cannot be deduced from sequence comparisons, the identity of the metal ion cotransported by C. glutamicum CitH is not yet known. The second citrate transport system of C. glutamicum, TctABC, belongs to the TTT family (63), for which the cation dependency is also not yet clear. Thus, further studies to clarify the cation dependency of the two C. glutamicum citrate transporters were performed.

The growth of the above-mentioned recombinant E. coli MG1655 strains in M9 minimal medium supplemented with 1 g/liter Casamino Acids in order to allow some basic growth and 50 mM sodium citrate as the major carbon source was monitored under aerobic conditions. The medium was supplemented with a 5 mM concentration of either CaCl₂, MgCl₂, NiCl₂, SrCl₂, or ZnCl₂. As shown in Fig. 2, MG1655/pXMJ19-citH can grow on citrate in the presence of Ca²⁺ and Sr²⁺ but not in the presence of Mg²⁺, Ni²⁺, or Zn²⁺. On the other hand, MG1655/pXMJ19-tctCBA can grow on citrate in the presence of Ca²⁺ and Mg²⁺ but not in the presence of Sr²⁺, Ni²⁺, or Zn²⁺. The control strain MG1655/pXMJ19 did not grow on citrate under any tested conditions. These results indicate that both CitH and TctABC of C. glutamicum transport citrate in a complex with Ca²⁺. CitH can also transport an Sr²⁺-citrate complex, and TctABC can also transport an Mg²⁺-citrate complex. It cannot be ruled out that the two transporters are able to take up citrate in a complex with Ni²⁺, as the concentration used also did not allow growth with 50 mM glucose and thus is toxic for the cells (data not shown). In contrast, growth with 50 mM glucose and 5 mM Zn²⁺ was possible, indicating that nongrowth with citrate was due to the situation that neither CitH nor TctABC is active with Zn²⁺.

FIG. 2.

Testing the cation dependency of the citrate transporters CitH and TctABC of C. glutamicum by using the growth of E. coli on citrate as the indicator. E. coli MG1655 transformed with pXMJ19-citH (•), pXMJ19-tctCBA (▪), or pXMJ19 (▴) was grown in a 500-ml Erlenmeyer flask containing 50 ml of M9 minimal medium supplemented with 1 g/liter Casamino Acids, 50 mM sodium citrate, and none or one of the following salts: 5 mM CaCl₂, 5 mM MgCl₂, 5 mM ZnCl₂, 5 mM NiCl₂, and 5 mM SrCl₂. The cultures were incubated at 37°C on a rotary shaker at 120 rpm. The growth curves shown here are representative of those from three independent growth experiments with comparable results.

Cation dependency of the growth of the C. glutamicum wild type on citrate as the sole carbon and energy source.

Based on the fact that C. glutamicum possesses two functional citrate transporters, we tested whether the organism is able to grow on citrate as the sole carbon and energy source. In initial studies, wild-type cells pregrown in standard CGXII minimal medium with 50 mM glucose and 50 mM sodium citrate showed only minimal growth (from an OD₆₀₀ of 0.9 to an OD₆₀₀ of 3) when they were subsequently cultivated in standard CGXII minimal medium containing 50 mM sodium citrate as the sole carbon source (Fig. 3). Standard CGXII minimal medium contains 0.09 mM Ca²⁺ and 1 mM Mg²⁺, indicating that the poor growth may be due to insufficient concentrations of the cations that were found to be required for citrate utilization via CitH and TctABC in E. coli. Therefore, we supplemented the standard CGXII medium with additional CaCl₂, MgCl₂, or SrCl₂. As shown in Fig. 3A, C. glutamicum grew at a rate of 0.31 h⁻¹ to an OD₆₀₀ of about 12 on citrate as the sole carbon source with 2 mM CaCl₂. Increased CaCl₂ concentrations up to 20 mM did not further stimulate either the growth rate or the growth yield. Even higher Ca²⁺ concentrations led to the precipitation of salt, presumably as calcium phosphate. Growth on citrate was accompanied by an increase of the pH of the medium from an initial pH of 7 to 8.5 to 8.7 at the end of growth (data not shown). Growth behavior in the presence of MgCl₂ was different, as the maximal growth rate (0.20 h⁻¹) was obtained only with 100 mM (Fig. 3B) and then decreased with even higher concentrations (data not shown). In the case of SrCl₂, the maximal growth rate (0.14 h⁻¹) and cell yield were obtained with 10 mM (Fig. 3C). Higher concentrations of dissolved SrCl₂ could not be achieved. In the subsequent growth experiments with C. glutamicum on citrate, the medium was usually supplemented with 5 mM CaCl₂.

FIG. 3.

Influence of Ca²⁺ (A), Mg²⁺ (B), or Sr²⁺ (C) on the growth of the C. glutamicum wild type with citrate as the sole carbon and energy source. Cells pregrown in CGXII minimal medium with 50 mM glucose, 50 mM citrate, and 20 mM CaCl₂, 50 mM MgCl₂, or 5 mM SrCl₂, respectively, were used to inoculate 50 ml of CGXII medium (containing 0.09 mM CaCl₂, 1 mM MgSO₄, and 0 mM SrCl₂) containing 50 mM citrate as the sole carbon and energy source. The cultures were incubated at 30°C on a rotary shaker at 120 rpm. The growth curves shown here are representative of those from three independent growth experiments with comparable results.

Testing the roles and cation specificities of CitH and TctABC for the growth of C. glutamicum on citrate.

Since C. glutamicum possesses two functional citrate transporters, the question arises of whether both are required for growth on citrate or if each one alone is sufficient for growth. Moreover, the cation specificities determined for the two transporters in E. coli should also be tested in C. glutamicum. For this purpose, three deletion mutants of C. glutamicum, the ΔcitH, ΔtctCBA, and ΔcitH ΔtctCBA strains, were constructed as described in Materials and Methods. The three strains and, for comparison, the wild type were cultivated in CGXII minimal medium with 50 mM citrate as the sole carbon and energy source, with and without the addition of 5 mM CaCl₂, MgCl₂, or SrCl₂. In the case of MgCl₂ and SrCl₂, the concentration of 5 mM is below the concentration required for maximal growth but still sufficient to allow growth.

As shown in Fig. 4A, the wild type grew best in the presence of Ca²⁺, followed by Mg²⁺ and Sr²⁺. In the presence of Sr²⁺, the wild type reached only about one-third of the OD₆₀₀ obtained with calcium or magnesium ions (a final OD₆₀₀ of 4 compared to a final OD₆₀₀ of 12). The ΔcitH mutant grew well in the presence of magnesium ions, worse with additional calcium ions, and not at all with added strontium ions (Fig. 4B). The ΔtctCBA mutant grew equally well in the presence of calcium and strontium ions but did not grow in the presence of magnesium ions (Fig. 4C). This behavior reflects the cation specificity determined for the TctCBA system (Ca²⁺ and Mg²⁺) and CitH (Ca²⁺ and Sr²⁺) in E. coli. A remarkable result becomes obvious by a comparison of Fig. 4A and C. In the absence of TctABC, Sr²⁺ allows growth on citrate at the same rate and to the same final OD₆₀₀ as Ca²⁺ (Fig. 4C). In the presence of TctABC, however, both the growth rate and the final OD₆₀₀ obtained with Sr²⁺ are much lower than those obtained with Ca²⁺ (Fig. 4A). This result indicates that the simultaneous presence of TctABC and Sr²⁺ ions inhibits growth on citrate in a yet unknown manner. The much better growth of the ΔcitH strain with Mg²⁺ than with Ca²⁺ may indicate that Mg²⁺ is the preferred cation for citrate transport by the TctCBA system.

FIG. 4.

Cation dependency of the growth of the C. glutamicum wild type (A), the ΔcitH strain (B), and the ΔtctCBA strain (C) in CGXII medium with 50 mM citrate as the sole carbon and energy source. The medium contained either no additional salts (⋄) or 5 mM CaCl₂ (•), 5 mM MgCl₂ (▴), or 5 mM SrCl₂ (▪). Cells were pregrown in CGXII minimal medium with 50 mM glucose and 50 mM citrate. The cultures were incubated at 30°C on a rotary shaker at 120 rpm. The growth curves shown here are representative of those from three independent growth experiments with comparable results.

The ΔcitH ΔtctCBA double mutant did not grow on citrate under any tested conditions during the first 10 h of incubation; however, in a few cases, growth up to an OD₆₀₀ of 10 to 12 after 20 to 30 h was observed (data not shown).

The growth of C. glutamicum on citrate is not subject to catabolite repression.

Previous studies (3, 19, 55) had indicated that citrate and glucose are catabolized simultaneously by C. glutamicum. To confirm this hypothesis, we monitored the consumption of the substrates by a culture growing with 50 mM citrate and 50 mM glucose (supplemented with 5 mM CaCl₂). As shown in Fig. 5, the substrates were consumed simultaneously, indicating that the genes required for citrate utilization are not subject to catabolite repression. Interestingly, citrate consumption started before glucose consumption, indicating that citrate initially had some inhibitory effect on glucose utilization. Nevertheless, the two substrates were consumed after the same time period of about 10 h.

FIG. 5.

Simultaneous consumption of carbon sources during the growth (□) of C. glutamicum on citrate and glucose. The wild type was grown in CGXII medium containing 50 mM glucose, 50 mM sodium citrate, and 5 mM CaCl₂. Cells were precultivated in CGXII minimal medium with 50 mM glucose and 50 mM citrate. The consumption of citrate (▴) and glucose (•) was determined via HPLC as described in Materials and Methods. The curves shown here are representative of those from three independent growth experiments with comparable results.

The CitAB two-component system is required for citrate utilization by C. glutamicum.

C. glutamicum possesses genes for 13 TCS (27). One of these TCS, encoded by the citAB genes, belongs to the CitAB/DcuSR subfamily (23). The C. glutamicum sensor kinase CitA (551 amino acid residues) contains two putative transmembrane helices separated by an extracytoplasmic domain extending from residues 48 to 188. It displays 24 and 29% overall sequence identity to the CitA histidine kinases from K. pneumoniae and E. coli, respectively, which were previously shown to function as citrate receptors (22, 23). The C. glutamicum response regulator CitB (218 amino acid residues) shows 32 and 35% sequence identity to the CitB proteins from K. pneumoniae and E. coli, respectively. The affiliation with the CitAB/DcuSR subfamily and the fact that the citAB genes are located immediately upstream of and divergent from the citH gene (Fig. 1A) suggested an involvement of the C. glutamicum CitAB TCS in the regulation of citrate uptake genes. This suggestion was confirmed by the fact that a C. glutamicum mutant lacking the citAB genes (the ΔcitAB strain) was unable to grow with citrate as the sole carbon and energy source within the first 20 h of cultivation but grew like the wild type on glucose (Fig. 6) or pyruvate (data not shown). The growth defect could be abolished by the transformation of the deletion mutant with the citAB expression plasmid pXMJ19-citAB (data not shown). Interestingly, the growth of the ΔcitAB mutant on citrate occurred after prolonged incubation (about 30 h in the experiment with results shown in Fig. 6). When such cultures were used to inoculate fresh citrate minimal medium, cells grew either immediately or with a shortened lag phase, indicating that a suppressor mutation had presumably allowed the expression of either citH or tctCBA.

FIG. 6.

Growth of the C. glutamicum wild type (squares) and the mutant strain ΔcitAB (triangles) in CGXII minimal medium containing either 50 mM glucose (filled symbols) or 50 mM citrate (hollow symbols) as the sole carbon and energy source. Cells were pregrown in CGXII minimal medium with 50 mM glucose and 50 mM citrate. The medium was supplemented with 5 mM CaCl₂. The cultures were incubated at 30°C on a rotary shaker at 120 rpm. The growth curves shown here are representative of those from three independent growth experiments with comparable results.

Transcriptome comparisons reveal that CitAB is responsible for the activation of citH and tctCBA.

To analyze the influence of the citAB deletion on genomewide gene expression in the presence of citrate, the transcriptomes of the ΔcitAB mutant and the wild type were compared using DNA microarrays. For this purpose, the cells were grown in CGXII minimal medium with 50 mM citrate, 50 mM glucose, and 50 mM MgCl₂. Four sets of comparisons, each starting with independent cultures, were made. Sixty genes showed alterations in mRNA levels of more than threefold in at least three of four experiments (P ≤ 0.05). Thirty-six of these genes showed decreases and 29 genes showed increases in mRNA levels in the ΔcitAB mutant compared to those in the wild type. As shown in Table 3, the functions of the encoded proteins are highly diverse, and only those genes showing the most drastic changes in mRNA levels will be dealt with here. The citH and tctCBA mRNA levels in the ΔcitAB mutant were 25- to 100-fold lower than those in the wild type, indicating that the CitAB TCS is responsible for the activation of these genes. Only two other genes, namely, cg2380 and ssuC, showed mRNA levels that were changed to the same extent and in the same manner (Table 3). The former gene encodes a protein of 14,084 Da that contains a single transmembrane helix extending from amino acid residue 65 to 85, with the N-terminal region located extracytoplasmically. Homologs are present in many other corynebacteria, mycobacteria, and other genera of the actinomycetes; however, the function is still unknown. The ssuC gene is part of the ssuD1CBA operon encoding a sulfonatase (ssuD1) and an ABC transporter for aliphatic sulfonates (28). The other genes of this operon, as well as further genes involved in sulfonate metabolism (ssuD2, ssuI, and seuA), also showed altered mRNA levels (Table 3). The reason for the changed expression patterns of these genes in the comparison of the ΔcitAB mutant and the wild type is unclear. In contrast to the genes mentioned above, cg3195, encoding a flavin-containing monooxygenase, showed a 27-fold increase in the mRNA level in the ΔcitAB mutant compared to that in the wild type. Again, an obvious reason for this change is not apparent.

TABLE 3.

Genes with ≥3-fold change in mRNA levels^a

cg designation	NCgl designation	Gene name	Description of (putative) product	Avg mRNA ratio (ΔcitAB strain mRNA/wt mRNA)
cg0089	NCgl0067	citA	Sensor histidine kinase	0.00
cg0090	NCgl0068	citB	Response regulator	0.00
cg3125	NCgl2724	tctA	Tricarboxylate transport membrane protein	0.04
cg3126	NCgl2725	tctB	Tricarboxylate transport membrane protein	0.01
cg3127	NCgl2726	tctC	Tricarboxylate binding protein	0.02
cg2380	NCgl2088		14-kDa membrane protein	0.03
cg0088	NCgl0066	citH	Citrate transporter	0.04
cg1376	NCgl1173	ssuD1	Sulfonatase (alkanesulfonate monooxygenase)	0.10
cg1377	NCgl1174	ssuC	Aliphatic sulfonate transmembrane ABC transporter protein	0.04
cg1379	NCgl1175	ssuB	Aliphatic sulfonate ATP binding ABC transporter protein	0.10
cg1380	NCgl1176	ssuA	Aliphatic sulfonate binding protein	0.20
cg3226	NCgl2816		l-Lactate permease	0.05
cg3227	NCgl2817	lldD	Quinone-dependent l-lactate dehydrogenase	0.15
cg3308	NCgl2881	rpsF	30S ribosomal protein S6	0.09
cg0810	NCgl0676		Hypothetical protein	0.09
cg1628	NCgl1383		Hydrolase of the α/β superfamily	0.12
cg1581	NCgl1341	argJ	Bifunctional ornithine acetyltransferase/N-acetylglutamate synthase protein	0.14
cg1582	NCgl1342	argB	N-Acetylglutamate kinase	0.18
cg1583	NCgl1343	argD	Acetylornithine aminotransferase	0.27
cg3026	NCgl2635	mrpD	Subunit D of multisubunit Na+/H+ antiporter	0.19
cg3027	NCgl2636	mrpE	Subunit E of multisubunit Na+/H+ antiporter	0.15
cg3028	NCgl2637	mrpF	Subunit F of multisubunit Na+/H+ antiporter	0.24
cg3029	NCgl2638	mrpG	Subunit G of multisubunit Na+/H+ antiporter	0.23
cg1156	NCgl0975	ssuD2	Monooxygenase for sulfonate utilization	0.19
cg1451	NCgl1235	serA	Phosphoglycerate dehydrogenase	0.22
cg0576	NCgl0471	rpoB	Subunit β of DNA-directed RNA polymerase	0.23
cg2894	NCgl2523		Drug resistance-related transcriptional repressor	0.24
cg1364	NCgl1161	atpF	Subunit B of ATP synthase	0.26
cg2157	NCgl1892	terC	Tellurium resistance membrane protein	0.30
cg1151	NCgl0972	seuA	Monooxygenase for sulfonate ester utilization	0.30
cg1386	NCgl1182	fixA	Subunit β of putative electron transfer flavoprotein	0.30
cg0653	NCgl0538	rpsK	30S ribosomal protein S11	0.31
cg1147	NCgl0968	ssuI	Flavin mononucleotide binding protein required for sulfonate and sulfonate ester utilization	0.32
cg1349	NCgl1147		Membrane protein containing CBS domain	0.33
cg0825	NCgl0689	fabG	3-Ketoacyl-(acyl carrier protein) reductase	0.35
cg0882	NCgl0738		Hypothetical protein	0.36
cg3420	NCgl2983	sigM	RNA polymerase σ factor of ECF subfamily	2.37
cg2069	NCgl1774	psp1	Putative secreted protein	2.50
cg3351	NCgl2920	nagI	Gentisate 1,2-dioxygenase	2.56
cg2630	NCgl2314	pcaG	Subunit α of protocatechuate dioxygenase	2.57
cg2966	NCgl2588		Phenol 2-monooxygenase	2.71
cg1255	NCgl1060		HNH endonuclease	2.87
cg0638	NCgl0524		HD superfamily hydrolase	3.09
cg3216	NCgl2808	gntP	Gluconate permease	3.10
cg2504	NCgl2201		Hypothetical protein	3.14
cg2181	NCgl1915		Secreted component of ABC-type peptide transport system	3.26
cg2182	NCgl1916		Permease component of ABC-type peptide transport system	3.46
cg2183	NCgl1917		Permease component of ABC-type peptide transport system	3.71
cg2184	NCgl1918		ATPase component of ABC-type peptide transport system; contains duplicated ATPase domains	4.41
cg3151	NCgl2131	tnp2b	Transposase	3.56
cg0759		prpD2	Methylcitrate dehydratase	3.63
cg0760	NCgl0629	prpB2	Methylisocitrate lyase	9.11
cg0762	NCgl0630	prpC2	Methylcitrate synthase	11.05
cg2938	NCgl2563		Permease component of ABC-type dipeptide/oligopeptide/nickel transport system	3.93
cg2940	NCgl2565		ATPase component of ABC-type transport system; contains duplicated ATPase domains	3.69
cg2836	NCgl2476	sucD	Subunit α of succinyl-CoA synthetase	4.92
cg2837	NCgl2477	sucC	Subunit β of succinyl-CoA synthetase	3.82
cg3047	NCgl2656	ackA	Acetate kinase	4.00
cg3048	NCgl2657	pta	Phosphotransacetylase	5.43
cg0961	NCgl0805	metA	Homoserine O-acetyltransferase	4.89
cg2426		tnp2d	Transposase	5.32
cg1612	NCgl1368		Acetyltransferase	5.69
cg2610	NCgl2294		Secreted component of ABC-type dipeptide/oligopeptide/nickel transport system	6.19
cg3107	NCgl2709	adhA	Zn-dependent alcohol dehydrogenase	9.21
cg3195	NCgl2787		Flavin-containing monooxygenase	26.82

^aFor the genes listed, the mRNA level in the ΔcitAB strain was altered ≥3-fold compared to the mRNA level in the wild type (wt) in at least three of four independent experiments after the growth of the strains in CGXII minimal medium containing 50 mM citrate and 50 mM glucose as carbon sources. The table includes those genes for which the mean mRNA ratio showed at least a twofold change (increase or decrease) and had a P value of ≤0.05. The mRNA ratios shown are mean values from three to four independent DNA microarray experiments starting from independent cultures. mRNA was prepared from cells in the exponential growth phase. CBS, cystathionine-β-synthase; ECF, extracytoplasmic function; succinyl-CoA, succinyl coenzyme A.

Requirement of citrate for CitAB-dependent activation of citH and tctCBA.

In order to confirm the microarray data and to test whether the presence of citrate is required for citH and tctCBA induction, primer extension experiments were performed. As shown in Fig. 7, primer extension products were detectable for citH and tctC RNAs of the wild type cultivated on citrate or citrate plus glucose, but not for RNAs of the wild type cultivated on glucose or the ΔcitAB mutant cultivated on glucose or glucose plus citrate. From these results, three conclusions can be drawn: (i) the expression of citH and tctCBA is activated in the presence of citrate, and in the absence of citrate, there is no or very weak expression; (ii) glucose does not inhibit the expression of citH and tctCBA, since the primer extension products for RNAs from cells grown on citrate and those grown on citrate plus glucose were of equal strengths; and (iii) citrate-inducible expression of both citH and tctCBA is strictly dependent on the CitAB TCS.

FIG. 7.

Expression analyses and determination of the transcriptional start sites of the C. glutamicum citH gene (A) and tctCBA operon (B) by primer extension analyses with the oligonucleotides citH-PE-1 and tct-PE-3. Results for genes from the following strains cultivated under the indicated conditions are shown: lanes 1, wild type grown on glucose; lanes 2, wild type grown on citrate; lanes 3, wild type grown on citrate and glucose; lanes 4, ΔcitAB mutant grown on glucose; and lanes 5, ΔcitAB mutant grown on citrate and glucose. RNAs from the strains were isolated after cultivation in CGXII minimal medium with 50 mM MgCl₂ and a 25 mM concentration of the indicated carbon source. In all primer extension experiments for which results are shown, equivalent amounts of total RNA (20 μg) were used. The transcriptional start sites are indicated by asterisks.

In the case of citH, one major and two minor primer extension products were observed. The major transcription start site was located 56 bp upstream of the first putative start codon of citH, and the minor ones were located 55 and 53 bp upstream of the start codon (Fig. 1A and 7). The same start sites were also obtained with a second primer annealing at a different position (data not shown). The −10 region (Fig. 1A) deduced from the transcription start site (5′-TATACT-3′) is very similar to the −10 consensus core hexamer reported previously for C. glutamicum, 5′-TA(C/T)AAT-3′ (38). In the case of tctC, a single primer extension product, which ended 98 bp upstream of the predicted start codon of tctC, was identified (Fig. 1B and 7). Again, the same transcription start site was identified with a second primer annealing at a different position (data not shown), and the sequence of the deduced −10 region (5′-TTTAAT-3′) was similar to the consensus sequence listed above.

Binding of the response regulator CitB to the promoter regions of citH and tctCBA.

To support the assumption that CitB functions as a direct transcriptional activator of citH and tctCBA, EMSAs were performed with purified CitB and the promoter regions of citH and tctC. For this purpose, the CitB protein containing an amino-terminal hexahistidine tag was overproduced in E. coli BL21(DE3) and purified to apparent homogeneity by affinity chromatography (data not shown). DNA fragments covering the promoter regions of citH (bp −179 to +41 with respect to the major citH transcriptional start site, defined as bp +1) and tctC (bp −216 to +120 with respect to the tctC transcriptional start site) were incubated with four different concentrations of CitB and separated by native polyacrylamide gel electrophoresis. As negative controls, a fragment extending from bp +17 to +248 with respect to the major citH transcriptional start site and a promoter fragment of the arbitrarily chosen gene cg2836 (bp −557 to −226 with respect to the translational start site of cg2836) were tested. As shown in Fig. 8, the citH and tct promoter fragments in the presence of only a 10-fold molar excess of CitB were partially shifted, and complete retardation in the presence of a 20- or 30-fold molar excess of CitB was observed. No shift in the two DNA fragments serving as negative controls was observed, even at the highest CitB concentration used. In the case of the citH and tct promoter regions, a second protein-DNA complex was formed with increasing CitB concentrations, indicating either the presence of a second binding site or the binding of further CitB molecules to the initial protein-DNA complex. An inspection of the citH and tctC promoter regions did not reveal a highly conserved sequence motif that might represent the CitB binding site.

FIG. 8.

Binding of CitB to the promoter regions of citH and tctCBA. DNA fragments (219 to 336 bp; final concentrations, 22 to 35 nM) covering the promoter regions of citH and tctCBA and two DNA fragments serving as negative controls (see the text) were incubated for 30 min at room temperature without CitB (far left lanes) or with a 10- to 40-fold molar excess of purified CitB protein, as indicated, before separation by native polyacrylamide (15%) gel electrophoresis and staining with Sybr green I.

DISCUSSION

In this study, we have unraveled the molecular basis for citrate utilization and its regulation in C. glutamicum. As the expression of the C. glutamicum genes citH and tctCBA in E. coli MG1655 enabled growth on citrate, both CitH and TctCBA must be able to catalyze citrate uptake into the cell (Fig. 2). However, the two transporters differ in their cation dependency. CitH is active with Ca²⁺ and Sr²⁺ but not with Mg²⁺, and TctABC is active with Ca²⁺ and Mg²⁺ but not with Sr²⁺. In B. subtilis, the citrate transporters CitM and CitH, which show 42 and 43% sequence identity to C. glutamicum CitH, have complementary metal ion specificities. CitM of B. subtilis transports citrate in complexes with Mg²⁺, Ni²⁺, Mn²⁺, Co²⁺, and Zn²⁺ and CitH in complexes with Ca²⁺, Sr²⁺, and Ba²⁺ (30). As the metal ion specificity of the C. glutamicum homolog clearly resembles that of B. subtilis CitH, the original designation of CitM was changed to CitH. For B. subtilis CitH, apparent K_m values of 33 and 40 μM for the uptake of Ca-citrate and Sr-citrate complexes into E. coli cells were determined previously (30), and similar values can be expected for C. glutamicum CitH. In the case of the TctABC transporter, a number of physiological and biochemical studies were performed with the system of S. enterica serovar Typhimurium (reviewed in reference 63). However, no clear picture of the cation dependency of this system exists. Our results suggest that Ca²⁺ or Mg²⁺ is required for citrate transport catalyzed by C. glutamicum TctABC. Since high concentrations of these cations were required for growth on citrate by using the Tct system, a different cation may be used by this transporter in the natural habitat.

Birnbaum and Demain (3) suggested that citrate transport by C. glutamicum is inducible. The findings from our previous DNA microarray analyses (39) and the primer extension studies in this work clearly show that both citH and tctCBA are expressed only when citrate is present in the medium, and the transcriptome data indicate that the tctCBA operon is activated much more strongly than citH. The reason for this difference and its physiological meaning are not yet clear. The activation of citH and tctCBA in the presence of citrate is strictly dependent on the CitAB TCS, and a ΔcitAB deletion mutant of C. glutamicum was unable to grow on citrate as the sole carbon and energy source within the first ∼30 h of cultivation (see below). These results indicate (i) that the histidine kinase CitA may function as a citrate sensor, as shown previously for the orthologous CitA proteins from K. pneumoniae (23) and E. coli (22), and (ii) that the response regulator CitB presumably functions as a transcriptional activator of citH and the tctCBA operon. The latter hypothesis was supported by results from EMSAs, which showed that purified CitB binds to the promoter regions of citH and tctC. According to these results, the transcriptional control of citrate permease genes by a TCS of the CitAB/DcuSR family occurs not only in gram-negative bacteria and low-GC-content gram-positive bacteria, but also in high-GC-content gram-positive bacteria and thus is distributed in all major lines of the eubacteria. BLAST searches (2) with completed genome sequences of Actinobacteria revealed homologs of CitA/DcuS and CitB/DcuR in many members of this class of bacteria, such as Mycobacterium smegmatis, Nocardia farcinica, and Streptomyces coelicolor, and in nearly all cases, the corresponding genes were localized next to genes for a tricarboxylate or dicarboxylate transporter (Fig. 9).

FIG. 9.

Colocalization of genes encoding two-component systems of the CitAB/DcuSR family with genes for tricarboxylate or dicarboxylate transporters in selected Actinobacteria. Gene identifiers for all sensor kinases are indicated. DAACS, dicarboxylate/amino acid:cation (Na⁺ or H⁺) symporter; SSS, solute:sodium symporter.

Whereas citrate utilization is subject to catabolite repression in many bacteria (see the introduction), C. glutamicum uses citrate and glucose simultaneously (Fig. 5). In accord with this observation, the activation of citH and tctCBA was not influenced by the presence of glucose in the growth medium (39) (Fig. 7). Cometabolism of different carbon sources with glucose is typical for C. glutamicum and was shown previously for, e.g., fructose (13), acetate (58), propionate (12), serine (36), vanillate and protocatechuate (32), and gluconate (17).