Abstract
The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.
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References
Bateman A, Bycroft M (2000) The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J Mol Biol 299:1113–1119
Bayan N, Houssin C, Chami M, Leblon G (2003) Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications. J Biotechnol 104:55–67
Benz I, Schmidt A (2002) Never say never again: protein glycosylation in pathogenic bacteria. Mol Microbiol 45:267–276
Clewell DB (1993) Bacterial sex pheromone-induced plasmid transfer. Cell 73:9–12
Cohen-Gonsaud M, Keep NH, Davies AP, Ward J, Henderson B, Labesse G (2004) Resuscitation-promoting factors possess a lysozyme-like domain. Trends Biochem Sci 29:7–10
Dunny GM, Leonhard BAB (1997) Cell–cell communication in gram-positive bacteria. Annu Rev Microbiol 51:527–564
Dusch N, Pühler A, Kalinowski J (1999) Expression of the Corynebacterium glutamicum panD gene encoding l-aspartate-α-decarboxylase leads to panthothenate overproduction in Escherichia coli. Appl Environ Microbiol 65:1530–1539
Greenberg EP, Winans S, Fuqua C (1996) Census and consensus in bacterial ecosystems: the LuxR–LuxI family of quorum-sensing transcriptional regulators. Annu Rev Microbiol 50:727–751
Hartmann M, Tauch A, Eggeling L, Bathe B, Möckel B, Pühler A, Kalinowski J (2003) Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the l-lysine biosynthesis of Corynebacterium glutamicum. J Biotechnol 104:199–211
Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90:5011–5015
Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172
Hermann T, Pfefferle W, Baumann C, Busker E, Schaffer S, Bott M, Sahm H, Dusch N, Kalinowski J, Pühler A, Bendt AK, Krämer R, Burkovski A (2001) Proteome analysis of Corynebacterium glutamicum. Electrophoresis 22:1712–1723
Horton RM (1995) PCR-mediated recombination and mutagenesis—SOEing together tailor-made genes. Mol Biotechnol 3:93–99
Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109
Kaiser D, Losick R (1993) How and why bacteria talk to each other. Cell 73:873–885
Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Hartmann M, Huthmacher K, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefferle W, Pühler A, Rey DA, Rupp O, Rückert C, Sahm H, Wendisch VF, Wiegräbe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25
Kaprelyants AS, Kell DB (1996) Do bacteria need to communicate with each other for growth? Trends Microbiol 4:237–242
Katsumata R, Ozaki A, Oka T, Furuya A (1984) Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J Bacteriol 159:306–311
Kell DB, Young M (2000) Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol 3:238–243
Kell DB, Kaprelyants AS, Grafen A (1995) On pheromones, social behaviour and the function of secondary metabolism in bacteria. Trends Ecol Evol 10:126–129
Kirchner O, Tauch A (2003) Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol 104:287–299
Kleerebezem M, Quadri LEM, Kuipers OP, Vos WM de (1997) Quorum sensing by peptide pheromones and two-component signal-transduction systems in gram-positive bacteria. Mol Microbiol 24:895–904
Lazazzera BA, Grossman AD (1998) The ins and outs of peptide signalling. Trends Microbiol 6:288–294
Lis H, Sharon N (1993) Protein glycosylation—structural and functional aspects. Eur J Biochem 218:1–27
Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB (1998) A bacterial cytokine. Proc Natl Acad Sci USA 95:8916–8921
Mukamolova GV, Kormer SS, Kell DB, Kaprelyants AS (1999) Stimulation of the multiplication of Micrococcus luteus by an autokrine growth factor. Arch Microbiol 172:9–14
Mukamolova GV, Turapov OA, Kazarin K, Telkov M, Kaprelyants AS, Kell DB, Young M (2002a) The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Mol Microbiol 46:611–621
Mukamolova GV, Turapov OA, Young DI, Kaprelyants AS, Kell DB, Young M (2002b) A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol 46:613–635
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Puech V, Chami M, Lemassu A, Lanéelle M-A, Schiffler B, Gounon P, Bayan N, Benz R, Daffé M (2001) Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiology 147:1365–1382
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Schäffer C, Messner P (2001) Glycobiology of surface layer proteins. Biochimie 83:591–599
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
Schäffer C, Graninger M, Messner P (2001) Procaryotic glycosylation. Proteomics 1:248–261
Stephens K (1986) Pheromones among the prokaryontes. Crit Rev Microbiol 13:309–334
Suggs SV, Hirose T, Miyake T, Kawashima EH, Johnson MJ, Itakura K, Wallace RB (1981) Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences. In: Brown DD, Fox CF (eds) Developmental biology using purified genes. Academic, New York, pp 683–693
Swift S, Bainton NJ, Winson MK (1994) Gram-negative bacterial communication by N-acyl homoserine lactones: an universal language? Trends Microbiol 2:193–198
Tauch A, Kirchner O, Wehmeier L, Kalinowski J, Pühler A (1994) Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli. FEMS Microbiol Lett 123:343–348
Tauch A, Homann I, Mormann S, Rüberg S, Billault A, Bathe B, Brand S, Brockmann-Gretza O, Rückert C, Schischka N, Wrenger C, Hoheisel J, Möckel B, Huthmacher K, Pfefferle W, Pühler A, Kalinowski J (2002a) Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library. J Biotechnol 95:25–38
Tauch A, Kirchner O, Löffler B, Götker S, Pühler A, Kalinowski J (2002b) Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr Microbiol 45:362–367
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Tufariello JAM, Jacobs WR Jr, Chan J (2004) Individual Mycobacterium tuberculosis resuscitation-promoting factor homologues are dispensable for growth in vitro and in vivo. Infect Immun 72:515–526
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This work was supported in part by a grant from Hermann Schlosser Stiftung, Frankfurt. The authors thank Degussa AG for providing nucleotide sequence data and financial support.
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Hartmann, M., Barsch, A., Niehaus, K. et al. The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif, is involved in intercellular communication of Corynebacterium glutamicum. Arch Microbiol 182, 299–312 (2004). https://doi.org/10.1007/s00203-004-0713-1
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DOI: https://doi.org/10.1007/s00203-004-0713-1