RESUMEN
Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.
Asunto(s)
Cromosomas Bacterianos/genética , Sinorhizobium meliloti/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , División Celular/genética , Movimiento Celular/genética , Cromosomas Artificiales Bacterianos/genética , Reparación del ADN/genética , Replicación del ADN/genética , ADN Bacteriano/genética , ADN Circular/genética , Metabolismo Energético/genética , Fabaceae/microbiología , Duplicación de Gen , Genes Bacterianos , Datos de Secuencia Molecular , Plantas Medicinales , Replicón/genética , Análisis de Secuencia de ADN , Transducción de Señal/genética , Simbiosis , Transcripción Genética/genética , Virulencia/genéticaRESUMEN
DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.
Asunto(s)
Genes Bacterianos , Rhizobium leguminosarum/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Fabaceae/microbiología , Expresión Génica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Operón , Plantas Medicinales , Homología de Secuencia de Aminoácido , SimbiosisRESUMEN
Whole lupins (Lupinus albus) were roasted with exit temperatures ranging from approximately 130 (moderate heat) to 175 degrees C (high heat). In situ N disappearance after 12 h of incubation in the rumen was 83% for raw lupins, 45% for lupins roasted at moderate temperatures, and 39% for lupins roasted at high temperatures. Lambs fed lupins roasted at moderate temperatures retained more N (P less than .01) than those fed soybean meal (SBM). However, growth rate and feed efficiency were similar among lambs fed diets containing SBM, raw lupins, or roasted lupins. Dehulled lupins commercially roasted at low, moderate, and high temperatures resulted in ruminal in situ N disappearances of 59, 47, and 43% for the respective temperatures. Dehulled lupins (Lupinus albus) were also roasted in a laboratory oven for 2, 4, and 6 h at 120, 140, and 160 degrees C. Simulation of roasting for 2 h had no effect (P greater than .10) on ruminal in situ N disappearance at any of the temperatures. In situ N disappearance was reduced (P less than .05) after roasting for 4 h at 160 degrees C, but acid detergent insoluble N was only moderately increased. Nitrogen retention in lambs fed raw, dehulled lupins was equal (P greater than .10) to that of lambs fed SBM. Whole lupins or dehulled lupins can replace SBM as the sole protein supplement for growing lambs. Although roasting lupins decreased ruminal in situ N disappearance, it had no effect on growth of lambs.