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A Global Analysis of Protein Expression Profiles in Sinorhizobium meliloti: Discovery of New Genes for Nodule Occupancy and Stress Adaptation

¹Genomic Interactions Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, A.C.T. 2601 Australia; ²Laboratoire de Génétique Cellulaire-INRA, Chemin de Borde Rouge-Auzeville BP27, 31326 Castanet Tolosan, France; ³Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Apdo. Postal 565-A Cuernavaca, Morelos, CP62210, México

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrientstress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodulespecific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Additional keywords: iron sequestering , MALDI-TOF mass spectrometry , molecular networks , Nex18 , reactive oxygen species , TspO .