Abstract
An identified pssL gene is yet another one, besides the pssT, pssN and pssP genes, encoding for a protein engaged in polysaccharide polymerization and export in Rhizobium leguminosarum bv. trifolii strain TA1 (RtTA1). Amino acid sequence similarity and hypothetical protein secondary structure placed the PssL protein within Wzx (RfbX) translocases with putative flippase function that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the PssL membrane protein was examined with a series of PssL–PhoA and PssL–LacZ translational fusions. The results support the hypothesis of PssL being a member of PST protein family comprising transporters with 12 membrane spanning segments and amino and carboxyl termini located in the cytoplasm. Results of semi-quantitative RT-PCR showed that the initial abundance of mRNA encoding PssL protein was relatively lower when compared to the quantity of the previously identified PssT membrane protein. PssL might be a good candidate for Wzx-like protein that together with PssT (Wzy protein) could be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation in RtTA1.
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Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Becker A, Kleickmann A, Kuster H, Keller M, Arnold W, Puhler A (1993) Analysis of the Rhizobium meliloti genes exoU, exoV, exoW, exoT, and exoI involved in exopolysaccharide biosynthesis and nodule invasion: exoU and exoW probably encode glucosyltransferases. Mol Plant Microbe Interact 6:735–744
Boyd D, Traxler B, Beckwith J (1993) Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol 175:553–556
Bugert P, Geider K (1995) Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol Microbiol 15:917–933
Burrows LL, Lam JS (1999) Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5. J Bacteriol 181:973–980
Calamia J, Manoil C (1990) lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci USA 87:4937–4941
Chakravorty AK, Żurkowski W, Shine J, Rolfe BGJ (1982) Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation. J Mol Appl Genet 1:585–596
Ciocchini AE, Roset MS, Inon de Iannino N, Ugalde RA (2004) Membrane topology analysis of cyclic glucan synthase, a virulence determinant of Brucella abortus. J Bacteriol 186:7205–7213
Cserzo M, Wallin E. Simon I, von Heijne G, Elofsson A (1997) Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Prot Eng 10:673–676
Daniels C, Vindurampulle C, Morona R (1998) Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol Microbiol 28:1211–1222
Danielsen S, Boyd D, Neuhard J (1995) Membrane topology analysis of the Escherichia coli cytosine permease. Microbiology 141:2905–2913
Ehrle R, Mikhaleva N, Boyd D, Davidson AL, Ehrmann M (2003) Context-dependent effects of charged residues in transmembrane segments of MalF-PhoA fusions. Res Microbiol 154:654–657
Ferguson GP, Datta A, Baumgartner J, Roop II RM, Carlson RW, Walker GC (2004) Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids. Proc Natl Acad Sci USA 101:5012–5017
Gardy JL, Spencer C, Wang K, Ester M, Tusnády GE, Simon I, Hua deFays SK, Lambert C, Nakai K, Brinkman FSL (2003) PSORT-B: improving protein subcellular localization prediction for Gram-negative bacteria. Nucleic Acids Res 31:3613–3617
Garnier J, Gibrat J-F, Robson B (1996) GOR secondary structure prediction method version IV. Meth Enzymol 266:540–553
van Geest M, Lolkema JS (2000) Membrane topology and insertion of membrane proteins: search for topogenic signals. Mol Biol Rev 64:13–33
Goldman E, Rosenberg AH, Zubay G, Studier FW (1995) Consecutive low-usage leucine codons block translation only when near the 5′ end of a message in Escherichia coli. J Mol Biol 245:467–473
Gonzalez JE, Semino CE, Wang LX, Castellano-Torres LE, Walker GC (1998) Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Proc Natl Acad Sci USA 95:13477–13482
Hagting A, van der Velde J, Poolman B, Konings WN (1997) Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis. Biochemistry 36:6777–6785
Hannessey ES, Broome-Smith JK (1993) Gene-fusion techniques for determining membrane-protein topology. Curr Opin Struct Biol 3:524–531
von Heijne G (1992) Membrane protein structure prediction, hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494
Hobohm U, Sander C (1995) A sequence property approach to searching protein databases. J Mol Biol 251:390–399
Hofmann K, Stoffel W (1993) TMbase—a database of membrane spanning proteins segments. Biol Chem Hoppe Seyler 374:166
Jayakumar A, Schulman I, McNeil D, Barnes JR EM (1986) Role of the Escherichia coli glnALG operon in regulation of ammonium transport. J Bacteriol 166:281–284
Jones DT, Taylor WR, Thornton JM (1994) A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33:3038–3049
Król J, Wielbo J, Mazur A, Kopcińska J, Łotocka B, Golinowski W, Skorupska A (1998) Molecular characterization of pssCDE genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD mutant is affected in exopolysaccharide synthesis and endocytosis of bacteria. Mol Plant Microbe Interact 11:1142–1148
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Liu D, Cole RA, Reeves PR (1996) An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178:2102–2107
Macpherson DF, Manning PA, Morona R (1995) Genetic analysis of the rfbX gene of Shigella flexneri. Gene 155:9–17
Manoil C (1991) Analysis of membrane protein topology using alkaline phosphatase and β-galactosidase gene fusion. Methods Cell Biol 34:61–75
Manoil C, Beckwith J (1986) A genetic approach to analyzing membrane protein topology. Science 233:1403–1408
Mazur A, Król J, Skorupska A (2001) Rhizobium leguminosarum bv. trifolii pssNOP genes encoding proteins involved in polymerization and translocation of exopolysaccharide. DNA Seq 12:1–12
Mazur A, Król J, Wielbo J, Urbanik-Sypniewska T, Skorupska A (2002) Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization. Mol Plant Microbe Interact 15:388–397
Mazur A, Król J, Marczak M, Skorupska A (2003) Membrane topology of PssT, the transmembrane protein component of the type I exopolysaccharide transport system in Rhizobium leguminosarum bv. trifolii strain TA1. J Bacteriol 185:2503–2511
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor
Minton NP (1984) Improved vectors for isolation of translational lac gene fusions. Gene 31:269–273
Moller S, Croning MDR, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653
Morona R, Mavris M, Fallarino A, Amanning P (1994) Characterization of the rfc region of Shigella flexneri. J Bacteriol 176:733–747
Morona R, Van Den Bosch L, Daniels C (2000) Evaluation of Wzz/MPA1/MPA2 proteins based on the presence of coiled-coil regions. Microbiology 146:1–4
Paulsen IT, Beness AM, Saier MH (1997) Computer-based analyses of the protein constituents of transport systems catalyzing export of complex carbohydrates in bacteria. Microbiology 143:2685–2699
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence analysis. Proc Natl Acad Sci USA 85:2444–2448
Reuber TL, Long S, Walker GC (1991) Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using Tn phoA fusions. J Bacteriol 173:426–434
Saier Jr MH (2000) A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64:354–411
Sambrook J, Fritsch FE, Maniatis T (1989) Molecular clonning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Schnaitman CA, Klena JD (1993) Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev 57:655–682
Stevenson G, Andrianopoulos K, Hobbs M, Reeves PR (1996) Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid. J Bacteriol 178:4885–4893
Stevenson G, Lan R, Reeves PR (2000) The colanic acid gene cluster of Salmonella enterica has a complex history. FEMS Microbiol Lett 191:11–16
Tao J, Feng L, Guo H, Li Y, Wang L (2004) The O-antigen gene cluster of Shigella boydii O11 and functional identification of its wzy gene. FEMS Microbiol Lett 234:125–132
Traxler B, Boyd D, Beckwith J (1993) The topological analysis of integral cytoplasmic membrane proteins. J Membrane Biol 132:1–11
Tusnády GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J Mol Biol 283:489–506
Vincent JM (1970) A manual for the practical study of root nodule bacteria. Blackwell, Oxford
Whitfield C, Roberts IS (1999) Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol 31:1307–1319
Whitfield C, Paiment A (2003) Biosynthesis and assembly of Group 1 capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria. Carbohydr Res 338:2491–2502
Wielbo J, Mazur A, Król JE, Marczak M, Kutkowska J, Skorupska A (2004) Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants. Arch Microbiol 182:331–336
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This work was supported by a grant from the Polish Committee for Scientific Research no. 2 P04A 0–34 26. A. Mazur was the recipient of a fellowship from The Foundation for Polish Science.
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Mazur, A., Marczak, M., Król, J.E. et al. Topological and transcriptional analysis of pssL gene product: a putative Wzx-like exopolysaccharide translocase in Rhizobium leguminosarum bv. trifolii TA1. Arch Microbiol 184, 1–10 (2005). https://doi.org/10.1007/s00203-005-0018-z
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DOI: https://doi.org/10.1007/s00203-005-0018-z