A trap transporter for pyruvate and other monocarboxylate 2-oxoacids in the cyanobacterium Anabaena sp. strain PCC 7120

JOURNAL OF BACTERIOLOGY, Nov. 2010, p. 6089–6092 Vol. 192, No. 22 0021-9193/10/$12.00 doi:10.1128/JB.00982-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved. A TRAP Transporter for Pyruvate and Other Monocarboxylate 2-Oxoacids in the Cyanobacterium Anabaena sp. Strain PCC 7120 䌤 Rafael Pernil,† Antonia Herrero, and Enrique Flores* Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain Received 19 August 2010/Accepted 3 September 2010 In the cyanobacterium Anabaena sp. strain PCC 7120, open reading frames (ORFs) alr3026,alr3027, and all3028 encode a tripartite ATP-independent periplasmic transporter (TRAP-T). Wild-type filaments showed significant uptake of [ 14 C]pyruvate, which was impaired in the alr3027 and all3028 mutants and was inhibited by several monocarboxylate 2-oxoacids, identifying this TRAP-T system as a pyruvate/monocarboxylate 2-oxoacid transporter. The tripartite ATP-independent periplasmic transporter (TRAP-T) family of proteins (family 2.A.56 in the transporter classification database [19]) comprises transporters that consist of three components: a small membrane protein usually bearing 4 transmembrane segments (TMSs), a large membrane protein usually bearing 12 TMSs that is the membrane translocator, and a periplasmic substrate binding protein (10). The TRAP transporters use the energy of an electrochemical ion gradient to drive uphill substrate transport (7, 14). TRAP-T family members are widely present in bacteria and archaea, but only a few substrates, including different types of carboxylates, have been identified for them (20). In vitro binding analyses with the periplasmic solute binding proteins RRC01191 from Rhodobacter capsulatus (20) and TakP from Rhodobacter sphaeroides (8) have shown that they bind monocarboxylate 2-oxoacids, including pyruvate. Additionally, pyruvate induces the TRAP-T periplasmic solute binding protein SMb21353 in Sinorhizobium meliloti strain 1021 (13). We are not aware, however, of any study showing a direct role of any of these proteins in pyruvate transport in vivo. Cyanobacteria are a morphologically diverse group of photoautotrophic bacteria that includes unicellular and multicellular (filamentous) organisms (18). Most cyanobacteria can use ammonium or nitrate ions as nitrogen sources, and some can also assimilate urea or fix atmospheric N 2 (5). Some filamentous cyanobacteria fix N 2 in differentiated cells called heterocysts that are formed under combined nitrogen deprivation (6). A TRAP transporter is involved in sodium-dependent glutamate uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (17). It is composed of proteins GtrA and GtrB (small and large membrane subunits, respectively) and GtrC (periplasmic substrate binding protein). A cluster of open reading frames (ORFs), alr3026,alr3027, and all3028, encoding proteins similar to TRAP-T proteins, is found in the genome of the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120 (9). The proteins are Alr3026, with 4 predicted TMSs; Alr3027, with 13 predicted TMSs (however, the N-terminal TMS is a predicted signal peptide that could be removed, producing a mature protein of 12 TMSs); and All3028, a predicted periplasmic solute binding protein. Whereas the two membrane proteins are most similar to proteins of the Synechocystis Gtr glutamate transporter (Alr3026 shares 63% identity with GtrA, and Alr3027, 77% identity with GtrB), the periplasmic solute binding protein, All3028, is more similar to Rhodobacter capsulatus RRC01191 (47% identity) and Rhodobacter sphaeroides TakP (49% identity) than to Synechocystis GtrC (about 18% identity in a 300-amino-acid overlap). It was of interest, therefore, to determine the substrate(s) for this Anabaena transporter, which we approached by mutation and transport analysis. Methods. Anabaena sp. strain PCC 7120 was grown in medium BG11 (nitrate-containing medium [18]) or BG11 0 (BG11 medium lacking NaNO 3 ) at 30°C in 25 ␮E䡠m ⫺2 䡠s ⫺1 of light in a shaker. For the mutants, the medium was supplemented with 10 to 40 ␮gofNm䡠ml ⫺1 or2to5␮gofSp䡠ml ⫺1 and 2 to5␮gofSm䡠ml ⫺1 . For plating, the medium was solidified with 1% separately sterilized agar. Cyanobacterial cell masses were estimated by measuring the concentrations of chlorophyll a(Chl) in the cultures (12). Escherichia coli strain DH5␣was used for plasmid constructions and was grown in LB medium supplemented when appropriate with antibiotics at standard concentrations (1), as were strains HB101 and ED8654, which were used for conjugations with Anabaena sp. To inactivate alr3027, an internal 565-bp fragment was amplified by PCR using primers alr3027-7120-1 and alr3027- 7120-2, which contain BamHI restriction sites in their 5⬘ends (Table 1), and as a template, strain PCC 7120 genomic DNA was used (2). The amplified fragment was cloned into vector pGEM-T and transferred to BamHI-digested pCSV3 (16), producing pCSR11 (Sm r Sp r ), or to BamHI-digested pRL424 (4), producing pCSR18 (Nm r ). To inactivate all3028, an internal 553-bp fragment was amplified by PCR using primers all3028-7120-1 and all3028-7120-2, which contain BamHI restriction sites in their 5⬘ends; strain PCC 7120 genomic DNA was used as a template. The amplified fragment was cloned into vector pGEM-T Easy and transferred to BamHI-digested * Corresponding author. Mailing address: Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Ame´rico Vespucio 49, E-41092, Seville, Spain. Phone: 34 954489523. Fax: 34 954460065. E-mail: [email protected]. † Present address: Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle, NE2 4HH Newcastle upon Tyne, United Kingdom. 䌤 Published ahead of print on 17 September 2010. 6089 on July 20, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from pCSV3, producing pCSR56 (Sm r Sp r ). Conjugation of Anabaena sp. strain PCC 7120 with E. coli HB101 carrying pCSR11, pCSR18, or pCSR56 with helper and methylation plasmid pRL623 was effected by the conjugative plasmid pRL443, carried in E. coli ED8654, and was performed as described previously (3), with selection for resistance to Sm and Sp or Nm. The genetic structures of selected clones were studied by PCR with DNA from the clones and primer pairs alr3027-7120-3/alr3027-7120-4 or all3028-7120-3/all3028- 7120-4 for the alr3027::pCSV3 and all3028::pCSV3 mutants, respectively, or by Southern analysis performed by standard procedures (1), with EcoRV-digested DNA from the wild type and the alr3027::pRL424 mutant and a probe corresponding to the DNA fragment amplified for inactivation of the gene (see above). Anabaena strains homozygous for the mutant chromosomes were obtained and named CSR7a (alr3027::pCSV3), CSR7b (alr3027::pRL424), and CSR13a (all3028::pCSV3), respectively. For uptake assays, filaments of the wild-type and mutant strains were grown in BG11 or BG11 0 medium (supplemented with antibiotics for the mutants), harvested at room temperature, washed, and suspended in 25 mM N-tris(hydroxymethyl)- methylglycine (tricine)-NaOH buffer (pH 8.1) to give a cell density corresponding to 3 to 10 ␮gofChl䡠ml ⫺1 . After a preincubation at 30°C in 200 ␮E䡠m ⫺2 䡠s ⫺1 of light for 5 to 15 min, the assays were started by mixing the suspensions of cells with a solution of [1- 14 C]pyruvate (23 to 28 ␮Ci 䡠␮mol ⫺1 ; GE Healthcare, United Kingdom), D-[1- 14 C]mannitol (59 ␮Ci 䡠␮mol ⫺1 ; Amersham Biosciences, United Kingdom), or 14 C-labeled amino acids as previously described (16). After being incubated for the indicated time periods, 0.3- to 1-ml samples were filtered (0.45-␮m-pore-size Millipore HA filters were used), and the filters carrying the cells were washed with tricine buffer and immediately immersed in a scintillation cocktail to measure their radioactivity. Retention of radioactivity by boiled cells was used as a blank. Each study was done two or three times with different substrate concentrations, and the results of representative experiments are presented in Fig. 2 and 3. Transport kinetics data were analyzed with SigmaPlot (version 11.0, build 11.0.1.80). Impaired transport of pyruvate. Two strains with mutations in alr3027, strains CSR7a and CSR7b, and one with a mutation in all3028, strain CSR13a, were generated as described above (Fig. 1) and used in transport assays that were performed in the presence of 10 mM Na ⫹ ions. Because of the homology of the membrane proteins Alr3026 and Alr3027 to proteins of the Synechocystis Gtr system, the uptake of a set of amino acids, including glutamate (16), was tested in BG11-grown filaments of CSR7b and compared to that of the wild type, but no consistent impairment was observed (data not shown). Because the periplasmic solute binding protein, All3028, is annotated in some databases as an element of a mannitol transporter, we also tested the uptake of [ 14 C]mannitol (88 ␮M), but we found a very low level of uptake activity in wild-type filaments either grown in BG11 medium or incubated for 24 h in BG11 0 medium and no indication of impairment in mutant strain CSR7b. To investigate whether the Anabaena TRAP system could be involved in the transport of monocarboxylate 2-oxoacids, we studied pyruvate uptake. Linear uptake was observed in the wild type for at least 10 min with different concentrations of [1- 14 C]pyruvate (0.44 to 85 ␮M), and the uptake rates were similar for BG11- or BG11 0 -grown filaments. The uptake of [1- 14 C]pyruvate was scarcely affected in the mutants compared to that of the wild type for substrate concentrations below 15 ␮M, but it was affected significantly and to a similar extent for the three mutants at substrate concentrations from 30 to 90 ␮M (Fig. 2). These results suggest that there is a high-affinity mechanism for the uptake of pyruvate that is not inactivated in the mutants. However, the fact that a similar impairment in TABLE 1. Oligodeoxynucleotide primers used in this work a Primer Sequence (5⬘33⬘) all3028-7120-1 ...............GGA TCC CTG TGG AAT GTG GTC ATA C all3028-7120-2 ...............GGA TCC GTT AGC TTC TAC AGT CGC CG all3028-7120-3 ...............GGT GCA GAA ACC GTT GCC all3028-7120-4 ...............CAT TAA CCC GAT TCC AGG CG alr3027-7120-1...............GGA TCC GTG GTG GAC TTG CTT TAG C alr3027-7120-2...............GGA TCC GGT AGT ATC GCA GAC TTG GC alr3027-7120-3...............GAC ACT GGC TTA TGA ATG G alr3027-7120-4...............GCA AAG CCA AAG GGT GG a Introduced restriction enzyme cutting sites are in boldface type. FIG. 1. Schematic of the Anabaena sp. strain PCC 7120 alr3026- alr3027-all3028 genomic region. The approximate locations of plasmid insertions and PCR primers used to clone internal fragments of the genes are shown. FIG. 2. Effect of inactivation of ORFs alr3027 and all3028 on pyruvate uptake. Filaments of strains PCC 7120 (closed circles), CSR13a (all3028::pCSV3; triangles), CSR7a (alr3027::pCSV3; squares), and CSR7b (alr3027::pRL424; open circles) grown in BG11 medium were used in 10-min assays of uptake of [1- 14 C]pyruvate supplied at the indicated concentrations. 6090 NOTES J. BACTERIOL. on July 20, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from pyruvate uptake was observed for the three mutants suggests that Alr3027 and All3028 are proteins involved in the same process and that this system can transport pyruvate, albeit with a relatively low affinity. To deduce the kinetic parameters of pyruvate uptake by the TRAP transporter, the activities obtained with the mutants were subtracted from the activities obtained with the wild type with different substrate concentrations. From the resulting values, we deduced a K m of 66.9 ⫾ 20.5 ␮M and a V max of 21.3 ⫾3.71 nmol (mg Chl) ⫺1 min ⫺1 . Inhibition by monocarboxylate 2-oxoacids. To investigate whether the TRAP transporter could recognize other monocarboxylate 2-oxoacids, the effects of several monocarboxylate 2-oxoacids on the uptake of [1- 14 C]pyruvate were tested. Figure 3 shows that different monocarboxylate 2-oxoacids, tested at 500 ␮M, inhibited the uptake of pyruvate (50 ␮M) more in the wild type than in the mutant strains CSR7b and CSR13a. The different monocarboxylate 2-oxoacids tested showed different degrees of inhibition, in the following order (from more to less inhibitory): 2-oxobutyrate⬎2-oxovalerate⬎2-oxoisovalerate⬎2-oxoisocap- roate⬎2-oxo-3-methylvalerate. These results indicate that the system missing in the mutants is sensitive to the tested monocarboxylate 2-oxoacids and, therefore, that the Anabaena TRAP transporter recognizes monocarboxylate 2-oxoacids other than pyruvate. Concluding remarks. The Anabaena sp. strain PCC 7120 alr3026-alr3027-all3028 gene cluster encodes a TRAP transporter for pyruvate and possibly more generally for monocarboxylate 2-oxoacids. Although this transporter is annotated in some databases as a mannitol transporter, neither our results nor those of other authors studying homologues from Rhodobacter spp. support such an assignment (8, 20). Whereas the Anabaena TRAP transporter shows a relatively low affinity for pyruvate, the dissociation constants of pyruvate binding to the corresponding Rhodobacter binding proteins are very low, suggesting high affinity (8, 20). It is possible that other steps of the transport process, such as translocation through the membrane, affect the apparent affinity of uptake. Our results also show the important role that the solute binding protein has in determining the substrate selectivity of the transporter (20). The Synechocystis and Anabaena TRAP-T membrane proteins are highly similar. However, whereas the Synechocystis system includes a binding protein that, among characterized proteins, is most similar to glutamate binding proteins such as the Campylobacter jejuni PEB1a protein (11) and mediates the uptake of glutamate (17), the Anabaena system includes a binding protein that is most similar to monocarboxylate 2-oxoacid binding proteins such as Rhodobacter sphaeroides TakP (8) and mediates the uptake of pyruvate. In filaments of Anabaena sp. strain PCC 7120 incubated with [1- 14 C]pyruvate, a wide distribution of labeling among metabolic products was observed (15), which indicates that pyruvate is a usable substrate, thus rationalizing the presence of pyruvate transporter(s) in this organism. This work was supported by grant BFU2008-03811 from the Ministerio de Ciencia y Tecnología (Madrid, Spain), cofinanced by FEDER. REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 2010. Current protocols in molecular biology. Greene Publishing and Wiley-Interscience, New York, NY. 2. Cai, Y. P., and C. P. Wolk. 1990. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J. Bacteriol. 172:3138–3145. 3. Elhai, J., A. Vepritskiy, A. M. Muro-Pastor, E. Flores, and C. P. Wolk. 1997. Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J. Bacteriol. 179:1998–2005. 4. Elhai, J., and C. P. Wolk. 1988. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68:119–138. 5. Flores, E., and A. Herrero. 2005. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem. Soc. Trans. 33:164–167. 6. Flores, E., and A. Herrero. 2010. Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat. Rev. Microbiol. 8:39–50. 7. Forward, J. A., M. C. Behrendt, N. R. Wyborn, R. Cross, and D. J. Kelly. 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. 8. Gonin, S., P. Arnoux, B. Pierru, J. Lavergne, B. Alonso, M. Sabaty, and D. Pignol. 2007. Crystal structures of an extracytoplasmic solute receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimmer requiring a cation for ␣-keto acid binding. BMC Struct. Biol. 7:11. 9. Kaneko, T., Y. Nakamura, C. P. Wolk, T. Kuritz, S. Sasamoto, A. Watanabe, M. Iriguchi, A. Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, M. Kohara, M. Matsumoto, A. Matsuno, A. Muraki, N. Nakazaki, S. Shimpo, M. Sugimoto, M. Takazawa, M. Yamada, M. Yasuda, and S. Tabata. 2001. Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 8:205–213. 10. Kelly, D. J., and G. H. Thomas. 2001. The tripartite ATP-independent periplasmic (TRAP) transporters of bacteria and archaea. FEMS Microbiol. Rev. 25:405–424. 11. Leon-Kempis, M. D. R., E. Guccione, F. Mulholland, M. P. Williamson, and D. J. Kelly. 2006. The Campylobacter jejuni PEB1a adhesin is an aspartate/ glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids. Mol. Microbiol. 60:1262–1275. 12. Mackinney, G. 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140:109–112. 13. Mauchline, T. H., J. E. Fowler, A. K. East, A. L. Sarbor, R. Zaheer, A. H. F. Hosie, P. S. Poole, and T. M. Finan. 2006. Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome. Proc. Natl. Acad. Sci. U. S. A. 103:17933–17938. 14. Mulligan, C., E. R. Geertsma, E. Severi, D. J. Kelly, B. Poolman, and G. H. Thomas. 2009. The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter. Proc. Natl. Acad. Sci. U. S. A. 106:1778–1783. FIG. 3. Effects of some monocarboxylate 2-oxoacids on pyruvate uptake in strains PCC 7120, CSR7b, and CSR13a. The uptake of 50 ␮M [1- 14 C]pyruvate was tested in 10-min assays in the absence or presence of 500 ␮M 2-oxobutyrate (2-OB), 2-oxovalerate (2-OV), 2-oxoisovalerate (2-OIV), 2-oxoisocaproate (2-OIC), or 2-oxo-3-meth- ylvalerate (2-OMV). The bars represent the percentages of activity in the presence versus the absence of competitor. Black bars, strain PCC 7120; gray bars, strain CSR7b (alr3027::pRL424); white bars, strain CSR13a (all3028::pCSV3). VOL. 192, 2010 NOTES 6091 on July 20, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from 15. Pernil, R., A. Herrero, and E. Flores. 2010. Catabolic function of compartmentalized alanine dehydrogenase in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 192:5165–5172. 16. Pernil, R., S. Picossi, V. Mariscal, A. Herrero, and E. Flores. 2008. ABC-type amino acid uptake transporters Bgt and N-II of Anabaena sp. strain PCC 7120 share an ATPase subunit and are expressed in vegetative cells and heterocysts. Mol. Microbiol. 67:1067–1080. 17. Quintero, M. J., M. L. Montesinos, A. Herrero, and E. Flores. 2001. Identification of genes encoding amino acid permeases by inactivation of selected ORFs from the Synechocystis genomic sequence. Genome Res. 11:2034–2040. 18. Rippka, R., J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111:1–61. 19. Saier, M. H., Jr., M. R. Yen, K. Noto, D. G. Tamang, and C. Elkan. 2009. The transporter classification database: recent advances. Nucleic Acids Res. 37: D274–D278. 20. Thomas, G. H., T. Southworth, M. R. Leo´n-Kempis, A. Leech, and D. J. Kelly. 2006. Novel ligands for the extracellular solute receptors of two bacterial TRAP transporters. Microbiology 152:187–198. 6092 NOTES J. BACTERIOL. on July 20, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from