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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China
2 Graduate University of Chinese Academy of Sciences, Beijing, 100049, China
3 Lehrstuhl für Biochemie der Pflanzen, Ruhr Universität, Bochum, Germany
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-ketoadipate, gentisate and resorcinol pathways in Corynebacterium glutamicum. The gentisate (GenK/NCgl2922) and vanillate (VanK/NCgl2302) transporters have been identified previously. In this study, physiological functions of the remaining four putative transporters as well as the vanillate transporter (VanK/NCgl2302) were examined by genetic disruption/complementation and uptake assays. Results indicated that ncgl1031 encodes PcaK for 4-hydroxybenzoate and protocatechuate transport, and ncgl2302 encodes VanK for vanillate transport. Genetic studies and uptake assays indicated that both ncgl2325/benK and ncgl2326/benE are involved in benzoate transport in C. glutamicum. When growth rates were compared for two benzoate transporter mutants, benK and benE, a high growth rate was observed for the benE mutant. Sequence alignments revealed that PcaK, VanK, BenK and GenK belong to the major facilitator superfamily (MFS). Modelling of secondary structures based on previously characterized MFS members revealed that NCgl1031, NCgl2302, NCgl2325 and NCgl2922 are typical 12 helix transmembrane proteins but NCgl2326 contains only 11 
-helices. Thus the functionally identified NCgl2326 belongs to a novel type of benzoate transporters. Attempts to identify the phenotype of a hydK/ncgl2953 mutant failed, so the function of ncgl2953 remains unclear.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Membrane proteins are the main players in this transport. Sequencing data from several prokaryotic and eukaryotic genomes have revealed that about 30 % of all the integral membrane proteins are involved in transport across biological membranes (Paulsen et al., 2000).
Among the various categories of transporters classified (Ren et al., 2004), the major facilitator superfamily (MFS; Pao et al., 1998; Saier et al., 1999) is the largest (54 families), best known and functionally the most diverse superfamily of the electrochemical potential-driven transporter subclass. Recently, the structures and functions of three MFS transporters have been studied in detail (Hirai et al., 2002; Abramson et al., 2003; Huang et al., 2003). The aromatic acid : H+ symporter (AAHS) family of the MFS presently contains six members, namely PcaK of Pseudomonas putida (Nichols & Harwood, 1997), TfdK of Ralstonia eutropha (Leveau et al., 1998), BenK, VanK and MucK of Acinetobacter sp. ADP1 (Collier et al., 1997; Williams & Shaw, 1997; D'Argenio et al., 1999) and MhpT of Escherichia coli, which are all from Gram-negative bacteria.
Corynebacterium glutamicum is a Gram-positive, non-pathogenic, non-motile, aerobic, coryneform bacterium with a high G+C content that belongs to the actinomycetes subphylum (Stackebrandt et al., 1997). Since its isolation (Kinoshita et al., 1957), C. glutamicum has been extensively employed for industrial scale production of amino acids (L-glutamate, L-lysine, etc.), vitamins (D-pantothenic acid, etc.) and nucleotides, and is among the most important microorganisms for industrial biotechnology. The accessibility of genome data for C. glutamicum (Ikeda & Nakagawa, 2003; Kalinowski et al., 2003) has greatly stimulated studies that use C. glutamicum as a model microorganism in genetic and physiological research. Recent studies have demonstrated that C. glutamicum can degrade various aromatic compounds (Shen et al., 2004, 2005a; Shen & Liu, 2005), and a novel mycothiol-dependent gentisate pathway has been identified (Feng et al., 2006). Although transport systems for amino acids (Simic et al., 2001; Kennerknecht et al., 2002; Eggeling & Sahm, 2003; Ren et al., 2004) and sugars (Dominguez & Lindley, 1996; Dominguez et al., 1998; Gourdon et al., 2003) have been well characterized in C. glutamicum, knowledge of aromatic compound transport is very limited. In this study, the entire genome of C. glutamicum ATCC 13032 was searched for aromatic acid transporter genes and six putative genes encoding transporters were identified through genetic disruption/complementation and uptake assays for various aromatic compounds.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli strains were grown in Luria–Bertani (LB) broth on a rotary shaker (150 r.p.m.) or on LB plates containing 1.5 % (w/v) agar, at 37 °C. C. glutamicum strains were routinely cultivated in LB broth or BHIS (brain heart infusion broth supplemented with 50 mM sorbitol). To test their ability to grow on various aromatic compounds, wild-type C. glutamicum and mutants were inoculated into minimal medium (MM, pH 9.0; Konopka, 1993). Yeast extract (0.1 g per litre) was added to meet the vitamin requirements of bacterial strains. Benzoate, protocatechuate and vanillate were autoclaved in MM broth. Stock solutions of gentisate (100 mM), resorcinol (200 mM), 3-hydroxybenzoate (100 mM) and 4-hydroxybenzoate (100 mM) were sterilized by filtration through 0.2 µm pore size filters and were added to MM at final concentrations of 2 mM. All the C. glutamicum strains were grown with rotary shaking at 150 r.p.m. at 30 °C. Growth was monitored by measuring turbidity at 600 nm (OD600). For selection of mutants, in accordance with the vector used, antibiotics were added at the following concentrations: ampicillin, 100 µg ml–1 for E. coli; chloramphenicol, 20 µg ml–1 for E. coli and 10 µg ml–1 for C. glutamicum; kanamycin, 50 µg ml–1 for E. coli and 25 µg ml–1 for C. glutamicum; nalidixic acid, 50 µg ml–1 for C. glutamicum.
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. DNA manipulation, plasmid isolation and agarose gel electrophoresis were routinely carried out using standard methods (Sambrook et al., 1989). Restriction endonucleases, ligase and DNA polymerase were used according to the manufacturer's instructions. Vectors were electroporated into E. coli and C. glutamicum by the methods of Tauch et al. (2002). Primers used in this work for amplification of intact and disrupted genes are listed in Table 2. To facilitate cloning, both forward and reverse primers were flanked with cleavage sites for restriction endonucleases. PCR consisted of 30 cycles of denaturation at 95 °C for 1 min, annealing at 42 °C for 1 min and extension at 72 °C for 1.5 min followed by a final extension at 72 °C for 10 min. Amplified DNA was analysed by electrophoresis using 1 % agarose, purified by extraction from the gel, restricted with the corresponding endonucleases to obtain the disrupted gene, and finally ligated with pGEM-T Easy vector (Promega).
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ncgl1031, pK18mobsacBncgl2302, pK18mobsacBncgl2325, pK18mobsacBncgl2326 and pK18mobsacBncgl2953 were constructed. All pK18mobsacB derivatives were constructed by ligation of the appropriate disrupted gene (restricted from pGEM-T Easy) with pK18mobsacB. Plasmids were electroporated into C. glutamicum RES167 competent cells. Integration of introduced plasmids into the chromosome by first crossover was selected on BHIS plates containing kanamycin (25 mg ml–1) and nalidixic acid (50 mg ml–1). These antibiotic-resistant cells were grown overnight in LB medium and spread on LB plates containing 10 % (w/v) sucrose. The second crossover of chromosomal DNA led to kanamycin-sensitive (KmS) cells that were tested for their ability to grow in MM supplemented with aromatic compounds (benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, protocatechuate, resorcinol and vanillate) as sole carbon and energy sources. Mutant strains that were unable to grow on a particular aromatic acid were tested for gene deletion by PCR amplification using the same primers as used for amplification of intact genes. The knockout mutants were designated RES167ncgl1031, RES167ncgl2302, RES167ncgl2325, RES167ncgl2326 and RES167ncgl2953. A double knockout mutant, RES167ncgl(2325-2326), was also constructed by disrupting both ncgl2325 and ncgl2326. The deletion of target genes in pK18mobsacB derivatives and in C. glutamicum mutants was also confirmed by DNA sequencing.
Genetic complementation.
For genetic complementation, ncgl2031, ncgl2302, ncgl2325, ncgl2922 and ncgl2953 were amplified by PCR. The native ribosome-binding site (RBS) was replaced with the consensus RBS sequence identified by Amador et al. (1999). The endonuclease-digested PCR product was ligated into E. coli–C. glutamicum shuttle expression vector pXMJ19 (Jakoby et al., 1999) restricted with the appropriate endonucleases. These complementation plasmids were introduced into C. glutamicum RES167 mutants by electroporation to produce complemented strains.
Determination of growth rates of two mutants for benzoate transport at different benzoate concentrations.
In order to differentiate the function of the two benzoate transporters, mutants RES167ncgl2325 and RES167ncgl2326 were inoculated in MM (pH 9.0) containing different benzoate concentrations (1–5 mM). Triplicate cultures were incubated using a rotary shaker (150 r.p.m.) at 30 °C. Growth was measured by the increase in OD600 and growth rates for the two mutants at different benzoate concentrations were determined.
Assay for uptake of aromatic acids by resting cells.
C. glutamicum RES167 and the mutant strains were grown in 100 ml MM (pH 9.0) supplemented with 40 mM sodium acetate to an OD600 of 2.0. Cultures were centrifuged at 8000 r.p.m. for 5 min at 4 °C and washed twice with 50 mM phosphate buffer (pH 8.0). Cells were resuspended in the same buffer and incubated at 30 °C for 30 min to exhaust all endocellular carbon reserves. Cells were again centrifuged, washed and resuspended (at OD600 2.0) in 50 ml phosphate buffer (pH 8.0) containing 2 mM aromatic acid as substrate. Uptake of aromatic acids, as indicated by the decrease in their concentrations in the supernatant, was analysed by HPLC in a 1050 Hewlett Packard chromatograph equipped with a reverse-phase RP-18 column (4.6 mmx240 mmx0.5 mm) and a photodiode array detector. Standards were prepared in phosphate buffer (pH 8.0). Aqueous solvent contained 20 % (v/v) methanol in 100 mM ammonium acetate buffer (pH 4.2). Benzoate, 4-hydroxybenzoate and vanillate were typically eluted and monitored with this system at 7.9 min and 225 nm, at 6.0 min and 250 nm, and at 7.1 min and 253 nm, respectively.
Sequence data analysis.
The nucleotide sequence of C. glutamicum ATCC 13032 genome was obtained from GenBank (accession no. NC003450; Ikeda & Nakagawa, 2003). Sequence comparisons and protein sequence similarity searches were performed using BLAST programs at the BLAST server of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Pairwise and multiple sequence alignments were made with the CLUSTAL W program (Thompson et al., 1994).
Secondary structure modelling and search for conserved domains and residues.
Five topology prediction methods, MEMSAT (Jones et al., 1994), HMMTOP (Tusnády & Simon, 2001), TMHMM (Krogh et al., 2001; Kyte–Doolittle (Kyte & Doolittle, 1982; Fariselli et al., 2005) and HTMR (Fariselli et al., 2005) were employed to predict topology of aromatic acid transporters in C. glutamicum and the three well characterized MFS transporters. The domains conserved in MFS transporters (Jessen-Marshall et al., 1995; Pao et al., 1998) and charged amino acid residues conserved in the AAHS family (Ditty & Harwood, 1999) were located on the basis of predicted secondary structures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kalinowski et al., 2003) of the total coding sequences. Using BLAST searches based on sequence similarities as listed in Table 3, six genes (ncgl1031, ncgl2302, ncgl2325, ncgl2326, ncgl2922 and ncgl2953) were identified as encoding putative transport proteins for aromatic acids. The physical map and genetic organization are shown in Fig. 1. Three genes, ncgl2302, ncgl2325 and ncgl2326, are located in the -ketoadipate metabolic island, which constitutes almost 1 % of the C. glutamicum genome and contains all the genes necessary for catabolism of aromatic compounds via the -ketoadipate pathway. In contrast to the location of all necessary genes for the protocatechuate branch of this pathway in the -ketoadipate metabolic island, the putative transporter gene, ncgl1031, is located elsewhere in the genome. The ncgl2922 gene is located in another genetic cluster, and this gene has been previously shown to be necessary for 3-hydroxybenzoate and gentisate assimilation (Shen et al., 2005b). The ncgl2953 gene is located in one of the two genetic clusters (ncgl1110–ncgl1113 and ncgl2950–ncgl2953) that have recently been determined to be involved in resorcinol and hydroxyquinol degradation (Huang et al., 2006).
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ncgl1031, had lost the ability to grow with 4-hydroxybenzoate and protocatechuate (Fig. 2a) but retained the ability to grow with benzoate and other substituted benzoates. The prevention of mutant growth in the absence of the transporter may possibly be related to the high pH to which the cells were cultivated. When this mutant was complemented with pXMJ19-ncgl1031, the ability to grow on 4-hydroxybenzoate and protocatechuate was restored (Fig. 2a). This clearly indicated that ncgl1031 is essential for 4-hydroxybenzoate and protocatechuate assimilation in C. glutamicum. Furthermore, assays with resting cells indicated that uptake of 4-hydroxybenzoate was active in wild-type RES167 but not in mutant RES167ncgl1031. Within the first 10 min of culture the concentration of 4-hydroxybenzoate was halved with wild-type RES167, whereas no significant change in 4-hydroxybenzoate was observed with RES167ncgl1031 (Fig. 3a). Thus, it is concluded that NCgl1031 is a transporter for 4-hydroxybenzoate and protocatechuate in C. glutamicum.
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ncgl2302 had lost the ability to grow with vanillate (Fig. 2b
) without effects on growth on benzoate, 3- and 4-hydroxybenzoate, protocatechuate, gentisate, or resorcinol. After complementation with pXMJ19-ncgl2302, the ability to grow on vanillate was restored (Fig. 1b). Uptake of vanillate by resting cells of wild-type strain RES167 was active, but was inactive in mutant RES167ncgl2302 (Fig. 3b). Combining these results, we concluded that ncgl2302 encoded vanillate transporter in C. glutamicum.
Both ncgl2325 (benK) and ncgl2326 (benE) are involved in transport of benzoate
NCgl2325 and NCgl2326 are active in uptake of benzoate.
Both ncgl2325 and ncgl2326 are putative benzoate transporters. NCgl2325 shows 58 % and 55 % similarity with putative benzoate transporters identified from the genomic annotations of Rhodococcus sp. strain 19070 (AAK58907) and Rhodococcus sp. RHA1 (YP702351), respectively. Homologues of NCgl2326 were identified from genomes of Rhodococcus sp. RHA1 (YP705462) and Moorella thermoacetica ATCC 39073 (ZP00575807), but the function of these homologues had not been experimentally characterized. We found that individual disruption of ncgl2325 and ncgl2326 did not result in any phenotypic variation from wild-type in assimilation of aromatic compounds, including benzoate. However, the double-knockout mutant RES167ncgl(2325-2326) lost the ability to grow on benzoate (Fig. 1c, d) but there was no effect on growth on 3- or 4-hydroxybenzoate, protocatechuate, gentisate, resorcinol or vanillate. These results indicated that both ncgl2325 and ncgl2326 are involved in benzoate assimilation and in the absence of either, the other is functional. The involvement of ncgl2325 and ncgl2326 in benzoate assimilation was further confirmed by complementation experiments. When either one of these genes was introduced into the double-knockout mutant using pXMJ19 the ability of the mutants to grow on benzoate was restored (Fig. 1c, d). Results for uptake of benzoate from resting cell assays indicated that there was no uptake by the double knockout mutant RES167ncgl(2325–2326); the single knockout mutants RES167ncgl2325 and RES167ncgl2326 retained the ability to take up benzoate, and the uptake rates for benzoate in RES167ncgl2326 were higher than in RES167ncgl2325 (Fig. 3c). It was also observed that the growth rate of RES167ncgl2326 (0.123 h–1) was higher than that of RES167ncgl2325 (0.100 h–1).
NCgl2326 (BenE) represents a novel type of benzoate transporter.
Sequence alignments and secondary structure prediction revealed that NCgl2325 (BenK), NCgl1031 (PcaK) and NCgl2032 (VanK) all had the typical 12 transmembrane -helices, and had conserved domains at loops 2–3 and 8–9 and conserved charged amino acid residues, characteristic of MFS transporters (Jessen-Marshall et al., 1995; Pao et al., 1998; Ditty & Harwood, 1999). Strikingly, the transporter encoded by ncgl2326 was predicted to have 11 transmembrane -helices, a structure entirely different from transporters of the MFS, and did not contain any of the above-mentioned conserved features. Since both BenE and BenK are benzoate transporters, alignment of the currently known BenE and BenK sequences (including putative and functionally identified) was performed and results indicated that they formed two clearly separate clusters (Fig. 4). NCgl2326 (BenE) is the first functionally characterized benzoate transporter of its type and was predicted to have 11 helices.
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), it was assumed to be involved in transport of related aromatic compounds. Moreover, it shows 41 % similarity with the permease of the MFS in Brevibacterium linens BL2. However, disruption of this gene did not result in any observable phenotypic change regarding assimilation of aromatic compounds as tested in this study. It is thus concluded that ncgl2953 is not involved in aromatic acid transport in the three degradation pathways mentioned earlier. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, b), yet how these aromatic compounds enter cells had not been determined. In this study, out of 660 predicted membrane-protein-encoding genes in C. glutamicum (Kalinowski et al., 2003), five genes have been confirmed to code for aromatic acid transporters. As demonstrated in this study, vanillate is transported via VanK, and VanK in C. glutamicum (encoded by ncgl2302) was found to be exclusively responsible for vanillate transport, and was devoid of any involvement in transport of protocatechuate or 4-hydroxybenzoate. This is different from Acinetobacter sp. strain ADP1, in which VanK was reported to be also involved in 4-hydroxybenzoate and protocatechuate transport (D'Argenio et al., 1999). While working on nitrogen metabolism, Merkens et al. (2005) reported that vanK mutation in C. glutamicum led to decreased growth on protocatechuate and thus proposed the occurrence of an additional protocatechuate transporter. We indeed found that NCgl1301 (PcaK) was responsible for protocatechuate and 4-hydroxybenzoate transport in C. glutamicum. However, our results were significantly different from those of Merkens et al. (2005). A similar transporter was identified in P. putida (Nichols & Harwood, 1997); expression of pcaK was repressed by benzoate and preferential degradation of benzoate over 4-hydroxybenzoate was observed (Nichols & Harwood, 1995).
Two modes have been reported for benzoate transport in bacteria. The first is by simple or facilitated diffusion (Harwood & Gibson, 1986), and the second by specific membrane transporters (Miguez et al., 1995; Collier et al., 1997). In C. glutamicum this transport is through membrane transporters. In the present research two benzoate transporters, BenK and BenE, have been identified in C. glutamicum. It is of most interest that the predicted 11 helix transmembrane protein, NCgl2526 (BenE), is active in benzoate transport. Homologues of BenE were also identified in Agrobacterium tumefaciens, Acinetobacter sp. strain ADP1, Rhodococcus sp. RHA1 and P. putida. In all of these cases, sequence similarities between BenK and BenE are very low, i.e. 15.0 % identity for C. glutamicum, 15.5 % for Ag. tumefaciens strain C58 (accession numbers NP355471 and NP532938), 15.0 % for Acinetobacter sp. strain ADP1 (YP046120 and YP046126), 14.4 % for Rhodococcus sp. RHA1 (YP702351 and YP705462) and 14.0 % for P. putida KT2440 (NP745309 and NP745311). Further work is needed on characterization of the structure–function relationship of this novel type of benzoate transporter (BenE) and on comparison of benzoate uptake kinetics between BenK and BenE.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 September 2006;
revised 14 November 2006;
accepted 16 November 2006.
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, Parent strain RES167; 
, mutant strain; 
, complemented strain.
, parent strain RES167; 
, RES167