Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021.

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.

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Sequencing the Sinorhizobium meliloti genome.

The Sinorhizobium meliloti genome consists of three replicons. This bacterium forms an intricate symbiotic relationship with the roots of certain legumes and is considered as an agriculturally important nitrogen-fixer. A consortium of 6 European laboratories was organized to sequence its single chromosome (3.7 Mb), whereas the other two elements (pSyma 1.4 Mb and pSymb 1.7 Mb) will be sequenced by other groups.

High-resolution physical map of the pSymb megaplasmid and comparison of the three replicons of Sinorhizobium meliloti strain 1021.

A high-resolution physical map of the larger megaplasmid (pSymb) of Sinorhizobium meliloti strain 1021 has been constructed by using BAC libraries and an original two-step PCR screening method. This method, previously used to map both the chromosome and the smaller megaplasmid (pSyma), allowed us to position over the genome a total of 842 markers with an average density of one marker every 8.3 kb. In addition, we used BLASTX and PRODOM analysis to predict a function for a number of STSs. This work led to the discovery of several interesting loci and to a comparison of the genetic information carried by each replicon. The two main results emerging from this study are (i) a biased distribution of housekeeping genes, mainly detected on chromosome, and (ii) the presence of an unexpected number of transporters, mainly belonging to the ABC superfamily. These are broadly distributed across the whole genome, but particularly found on pSymb.

High-resolution physical map of the Sinorhizobium meliloti 1021 pSyma megaplasmid.

To facilitate sequencing of the Sinorhizobium meliloti 1021 pSyma megaplasmid, a high-resolution map was constructed by ordering 113 overlapping bacterial artificial chromosome clones with 192 markers. The 157 anonymous sequence tagged site markers (81,072 bases) reveal hypothetical functions encoded by the replicon.

A high-density physical map of Sinorhizobium meliloti 1021 chromosome derived from bacterial artificial chromosome library.

As part of the European Sinorhizobium meliloti (strain 1021) chromosome sequencing project, four genomic bacterial artificial chromosome (BAC) libraries have been constructed, one of which was mainly used for chromosome mapping. This library consists of 1,824 clones with an average insert size of 80 kilobases and represents approximately 20-fold total genome coverage [6.8 megabases (Mbs)]. PCR screening of 384 BAC clones with 447 chromosomal markers (PCR primer pairs), consisting of 73 markers representing 118 genes (40 individual genes and 78 genes clustered in 23 operons), two markers from the rrn operon (three loci), four markers from insertion sequences (approximately 16 loci) and 368 sequence-tagged sites allowed the identification of 252 chromosomal BAC clones and the construction of a high-density physical map of the whole 3.7-Mb chromosome of S. meliloti. An average of 5.5 overlapping and colinear BAC clones per marker, correlated with a low rate of deleted or rearranged clones (0.8%) indicate a solid BAC contigation and a correct mapping. Systematic BLASTX analysis of sequence-tagged site marker sequences allowed prediction of a biological function for a number of putative ORFs. Results are available at. This map, whose resolution averages one marker every 9 kilobases, should provide a valuable tool for further sequencing, functional analysis, and positional cloning.

Matrix solid-phase dispersion technique for the determination of moxidectin in bovine tissues.

An HPLC method has been developed for the determination of moxidectin in bovine tissues. The extraction and clean-up procedure is based on the matrix solid-phase dispersion technique. Control and moxidectin-fortified bovine tissue samples (0.25 g) are blended with octadecyl (C18 end-capped) packing material. A column made from the C18-bovine tissue blend is washed with hexane (2 ml); when all the hexane has eluted, an Alumina-B SPE cartridge is attached below the C18-tissue column and, after washing, moxidectin is eluted with methanol (6 ml). Moxidectin is derivatized and determined by HPLC with fluorescence detection. The recovery from fortified samples was greater than 80% in the concentration range 1-100 ng g-1 of tissue. This method permits the determination of moxidectin at levels as low as 1 ng g-1 (1 ppb).