Skip to main content
SpringerLink
Log in

Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: identification of the exsA gene encoding an ABC transporter protein, and the exsB gene which probably codes for a regulator of succinoglycan biosynthesis

  • Original Paper
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

Two new genes, designated exsA and exsB, were identified adjacent to the 24 kb exo gene cluster of Rhizobium meliloti, which is involved in succinoglycan (EPS I) biosynthesis. The derived amino acid sequence of ExsA displayed significant homologies to ATP binding cassette (ABC) transporter proteins. R. meliloti strains mutated in exsA were characterized by a decreased ratio of HMW to LMW EPS I, indicating a function for ExsA in EPS I biosynthesis. The R. meliloti NdvA protein, which is involved in the transport of cyclic β-(1,2)-glucans, was identified as the closest homologue of ExsA. R. meliloti exsB mutants produced a three-fold increased amount of EPS I in comparison to the wild-type strain. In contrast, high copy number of exsB resulted in a decrease in the EPS I level to 20% of wild type, indicating that the exsB gene product can negatively influence EPS I biosynthesis. It was demonstrated that this influence is not due to transcriptional regulation of the exo genes by the exsB gene product. By plasmid integration it was shown that exsA and exsB represent monocistronic transcription units.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Altschul SF, Gish W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Google Scholar 

  • Aman P, McNeil M, Franzen L, Darvill AG, Albersheim P (1981) Structural elucidation using HPLC-MS and GLC-MS of the acidic polysaccharide secreted by Rhizobium meliloti strain 1021. Carbohydr Res 95:263–282

    Google Scholar 

  • Battisti L, Lara JC, Leigh JA (1992) Specific oligosaccharide form of the Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa. Proc Natl Acad Sci USA 89:5625–5629

    Google Scholar 

  • Becker A, Kleickmann A, Arnold W, Pühler A (1993a) Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted β-1,3-1,4-glucanases and ExoH resembles membrane proteins. Mol Gen Genet 238:145–154

    Google Scholar 

  • Becker A, Kleickmann A, Arnold W, Keller M, Pühler A (1993b) Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment. Mol Gen Genet 241:367–379

    Google Scholar 

  • Becker A, Kleickmann A, Küster H, Keller M, Arnold W, Pühler A (1993c). 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

    Google Scholar 

  • Becker A, Niehaus K, Pühler A (1995a) Low molecular weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal and a missing C-terminal domain. Mol Microbiol 16:191–203

    Google Scholar 

  • Becker A, Schmidt M, Jäger W, Pühler A (1995b) New gentamicin resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162:37–39

    Google Scholar 

  • Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198

    Google Scholar 

  • Bronner D, Clarke BR, Whitfield C (1994) Identification of an ATP-binding cassette transport system required for the translocation of lipopoysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. Mol Microbiol 14:505–519

    Google Scholar 

  • Buendia AM, Enenkel B, Köplin R, Niehaus K, Arnold W, Pühler A (1991) The Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose-4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol Microbiol 5:1519–1530

    Google Scholar 

  • Bullock WC, Fernandez JM, Short JM (1987) XL1-Blue: a high effiecient plasmid transforming recA − Escherichia coli strain with beta-galactosidase selection. BioTechniques 5:376–379

    Google Scholar 

  • Casse F, Boucher C, Julliot JS, Michel M, Dénarié J (1979) Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J Bacteriol 113:229–242

    Google Scholar 

  • Coplin DL, Cook D (1990) Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol Plant-Microbe Interact 3:271–279

    Google Scholar 

  • Doherty D, Leigh J, Glazebrook J, Walker GC (1988) Rhizobium meliloti mutants that overproduce the R. meliloti acidic Calcofluor-binding exopolysaccharide. J Bacteriol 170:4249–4256

    Google Scholar 

  • Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179:125–142

    Google Scholar 

  • Fath MJ, Kolter, R (1993) ABC transporter: bacterial exporters. Microbiol Rev 57:995–1017

    Google Scholar 

  • Finke A, Bronner D, Nikolaev AV, Jann B, Jann K (1991) Biosynthesis of the Escherichia coli K-5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product. J Bacteriol 173:4088–4094

    Google Scholar 

  • Frosch M, Edwards U, Bousset K, Kraube B, Weisgerber C (1991) Evidence for a common molecular origin of the capsule gene loci in gram-negative bacteria expressing group II capsular polysaccharides. Mol Micribiol 5:1251–1263

    Google Scholar 

  • Glucksmann MA, Reuber TL, Walker GC (1993a) Family of glycosyltransferases needed for the synthesis of succinoglycan by Rhizobium meliloti. J Bacteriol 175:7033–7044

    Google Scholar 

  • Glucksmann MA, Reuber TL, Walker GC (1993b) Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis. J Bacteriol 175:7045–7055

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestions with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

    Google Scholar 

  • Hynes MF, Simon R, Müller P, Niehaus K, Labes M, Pühler A (1986) The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa. Mol Gen Genet 202:356–362

    Google Scholar 

  • Keller M, Roxlau A, Weng WM, Schmidt M, Quandt J, Niehaus K, Jording D, Arnold W, Pühler A (1995) Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharide succinoglycan and galactoglucan. Mol Plant Microbe Interact 2:267–277

    Google Scholar 

  • Kessler C (1992) Nonradioactive labeling and detection of biomolecules. Springer Verlag, Berlin

    Google Scholar 

  • Köplin R, Wang G, Hötte B, Priefer U, Pühler A (1993) A 3.9 kb DNA region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose. J Bacteriol 175:7786–7792

    Google Scholar 

  • Kroll JS, Loynds B, Brophy LM, Moxon ER (1990) The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol Microbiol 4:1853–1862

    Google Scholar 

  • Labes M, Pühler A, Simon R (1990) A new family of RSF1010-derived expression and lac fusion broad-host-range vectors for Gram-negative bacteria. Gene 89:37–46

    Google Scholar 

  • Leigh JA, Lee CC (1988) Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules. J Bacteriol 170:3327–3332

    Google Scholar 

  • Leigh JA, Walker GC (1994) Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function. Trends Genet 10:63–67

    Google Scholar 

  • Leigh JA, Reed JW, Hanks JF, Hirsch AM, Walker GC (1987) Rhizobium meliloti mutants that fail to succinylate their Calcofluor-binding exopolysaccharide are deficient in nodule invasion. Cell 51:579–587

    Google Scholar 

  • Long S, Reed JW, Himawan J, Walker GC (1988) Genetic analysis of a cluster of genes required for synthesis of the Calcofluor-binding exopolysaccharide of Rhizobium meliloti. J Bacteriol 170:4239–4248

    Google Scholar 

  • Masepohl B, Klipp W, Pühler A (1988) Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet 212: 27–37

    Google Scholar 

  • Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Müller P, Hynes M, Kapp D, Niehaus K, Pühler A (1988) Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with modulation mutants. Mol Gen Genet 211:17–26

    Google Scholar 

  • Müller P, Keller M, Weng WM, Quandt J, Arnold W, Pühler A (1993) Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein. Mol Plant Microbe Interact 6:55–65

    Google Scholar 

  • Pridmore RD (1987) New and versatile cloning vectors with kanamycin-resistance markers. Gene 56:309–312

    Google Scholar 

  • Priefer UB, Simon R, Pühler A (1985) Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions. J Bacteriol 163:324–330

    Google Scholar 

  • Reed JW, Glazebrook J, Walker GC (1991) The exoR gene of Rhizobium melioti affects RNA levels of other exo genes but lacks homology to known transcriptional regulators. J Bacteriol 173:3789–3794

    Google Scholar 

  • Reinhold BB, Chan SY, Reuber TL, Marra A, Walker GC, Reinhold VN (1994) Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021. J Bacteriol 176:1997–2002

    Google Scholar 

  • Reuber TL, Walker GC (1993a) The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021. J Bacteriol 175:3653–3655

    Google Scholar 

  • Reuber TL, Walker GC (1993b) Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74:269–280

    Google Scholar 

  • Rolfe BG, Gresshoff PM, Shine J (1980) Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci Lett 19:277–284

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Google Scholar 

  • Simon R (1984) High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet 196:413–420

    Google Scholar 

  • Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784–791

    Google Scholar 

  • Smith AN, Boulnois GJ, Roberts IS (1990) Molecular analysis of Escherichia coli K5 kps locus: identification and characterization of an inner-membrane capsular polysaccharide transport system. Mol Microbiol 4:1863–1869

    Google Scholar 

  • Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–232

    Google Scholar 

  • Staden R, McLachlan AD (1982) Codon preference and its use in identifying protein coding regions in large DNA sequences. Nucleic Acids Res 10:141–156

    Google Scholar 

  • Stanfield SW, Ielpi L, O'Brochta D, Helinski DR, Ditta GS (1988) The ndvA gene product of Rhizobium meliloti is required for β-(1 → 2)glucan production and has homology to the ATP-binding export protein HlyB. J Bacteriol 170:3523–3530

    Google Scholar 

  • Steenbergen SM, Vimr ER (1990) Mechanism of polysiatic acid chain elongation in Escherichia coli K1. Mol Microbiol 4:603–611

    Google Scholar 

  • Tolmasky ME, Staneloni RJ, Ugalde RA, Leloi LF (1980) Lipid-bound sugars in Rhizobium meliloti. Arch Biochem Biophys 203:358–364

    Google Scholar 

  • Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194

    Google Scholar 

  • Vincent JM (1970) A manual for the practical study of root nodule bacteria. IBP Handbook No. 15, Oxford

  • Walker JE, Sarste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the α and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951

    Google Scholar 

  • Zhan H, Leigh JA (1990) Two genes that regulate exopolysaccharide production in Rhizobium meliloti. J Bacteriol 172:5254–5259

    Google Scholar 

  • Zimmermann J, Voss H, Schwager C, Stegemann J, Erfle H, Stucky K, Kristensen T, Ansorge W (1990) A simplified protocol for fast plasmid DNA sequencing. Nucleic Acids Res 18:1067

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, 33501, Bielefeld, Germany

    Anke Becker, Helge Küster, Karsten Niehaus & Alfred Pühler

Authors
  1. Anke Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helge Küster
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karsten Niehaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alfred Pühler
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by A. Kondorosi

Rights and permissions

Reprints and permissions

About this article

Cite this article

Becker, A., Küster, H., Niehaus, K. et al. Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: identification of the exsA gene encoding an ABC transporter protein, and the exsB gene which probably codes for a regulator of succinoglycan biosynthesis. Molec. Gen. Genet. 249, 487–497 (1995). https://doi.org/10.1007/BF00290574

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00290574

Key words

Search

Navigation