Molecular characterization of the Sinorhizobium meliloti nlp D gene
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S H O RT C O M M U N I C AT I O N
Wolfgang R. Streit · Kai Hofmann · Wolfgang Liebl
Molecular characterization of the Sinorhizobium meliloti nlp D gene
Received: 11 May 2000 / Revised: 27 July 2000 / Accepted: 31 July 2000 / Published online: 5 September 2000 © Springer-Verlag 2000
Abstract The Sinorhizobium meliloti nlpD gene consists of 1,539 nucleotides and codes for 512 amino acids. Expression of the nlpD gene as a histidine-tagged protein in Escherichia coli resulted in the production of a 57-kDa protein. The deduced polypeptide sequence of NlpD contains one unusual hexamer repeat (KVQRGQ), one tetramer (TVTV) and two direct and inverted trimer repeats (KAA, AAK). The N-terminal amino acid residues displayed similarity with signal peptides of secreted bacterial lipoproteins. Mutations of the S. meliloti nlpD gene caused decreased survival of cells in the stationary phase.
also a unique biotin-regulated gene, bioS, has been identified, located between the pcm and nlpD homologues (Streit and Phillips 1997) (Fig. 1). We have shown that the bioS gene codes for a possible LysR-type regulator and that its expression is linked to the stationary phase of growth in S. meliloti (Heinz et al. 1999; Hofmann et al. 2000). It was therefore of interest to investigate the adjacent nlpD gene and its regulation in this organism.
Keywords Sinorhizobium meliloti · Survival · Stationary growth phase · bioS · Stress · Alfalfa · Root colonization · nlpD
Bacterial strains
Introduction In Escherichia coli and several other gram-negative bacteria, the rpoS gene is located downstream of a group of genes that are expressed in the stationary growth phase and that are organized in a conserved pattern (Lange and Hengge-Aronis 1994; Li et al. 1994). Recently, we have identified a region on the Sinorhizobium meliloti chromosome that encompasses three ORFs whose gene products show significant sequence similarities to proteins known from these prokaryotic “survival operons” (Streit and Phillips 1997) (Fig. 1). Organization of the ORFs surE, pcm, and nlpD in S. meliloti follows the same patterns as described for E. coli and Pseudomonas aeruginosa (Fujita et al. 1994; Li et al. 1994). Interestingly, in S. meliloti,
Dedicated to Prof. Gerhard Gottschalk on the occasion of his 65th birthday. W. R. Streit (✉) · K. Hofmann · W. Liebl Institut für Mikrobiologie und Genetik der Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany e-mail: [email protected], Tel.: +49-551-393775, Fax: +49-551-393793, http://www.gwdg.de/~wstreit/
Material and methods
Escherichia coli was grown at 37 °C on Luria-Bertani (LB) medium (Sambrook et al. 1989) supplemented with appropriate antibiotics. Rhizobia were routinely cultured at 30 °C on TY or LB (Sambrook et al. 1989) medium. For expression studies in the presence or absence of added biotin and seed exudates, cultures were grown on GTS medium (Kiss et al. 1979). β-Galactosidase and β-glucuronidase assays For tests of β-galactosidase and β-glucuronidase, all cultures were grown in flasks containing
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