Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions.

Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Transcriptome analysis of Sinorhizobium meliloti during symbiosis.

BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis. RESULTS: The expression of 214 S. meliloti genes was monitored under ten environmental conditions, including free-living aerobic and microaerobic conditions, addition of the plant symbiotic elicitor luteolin, and a variety of symbiotic conditions. Five new genes induced by luteolin have been identified as well as nine new genes induced in mature nitrogen-fixing bacteroids. A bacterial and a plant symbiotic mutant affected in nodule development have been found of particular interest to decipher gene expression at the intermediate stage of the symbiotic interaction. S. meliloti gene expression in the cultivated legume Medicago sativa (alfalfa) and the model plant M. truncatula were compared and a small number of differences was found. CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome analysis of the model rhizobium S. meliloti, the present work has highlighted the differential expression of several classes of genes during symbiosis. These genes are related to invasion, oxidative stress protection, iron mobilization, and signaling, thus emphasizing possible common mechanisms between symbiosis and pathogenesis.

Development of Sinorhizobium meliloti pilot macroarrays for transcriptome analysis.

In order to prepare for whole-genome expression analysis in Sinorhizobium meliloti, pilot DNA macroarrays were designed for 34 genes of known regulation. The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5' end of the primers, and the method of RNA labeling. Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method. The sizes of PCR products were also found to be important, whereas the influence of 5' tags was minimal. The variability between replicated spots on a membrane was found to be low. These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression.

Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods.

The use of denaturing gradient gel electrophoresis (DGGE) and traditional culture-depending methods for examining the bacterial community of traditional cassava starch fermentation were investigated. It appeared that DGGE profiles of total DNA of cassava dough exhibited 10 distinguishable bands. In contrast, DGGE fingerprints of bacteria recovered from enrichment cultures of fermented dough gave variable profiles containing fewer bands. Bands corresponding to five bacterial species detected by direct PCR-DGGE of total DNA from of cassava dough were also observed in DGGE patterns of enrichment cultures. Eighteen strains were isolated from cultures selected on the basis of their DGGE banding patterns. Assessment of bacterial identification by 16S rDNA sequence similarity revealed that band comigration implied sequence identity. Comparison of 16S rDNA sequences of excised DGGE bands and recovered pure culture isolates with those in GENBANK and the RDP databases revealed that representative bacteria of fermented cassava dough were Lactobacillus and Pediococcus species as well as species of Clostridium, Propionibacterium and Bacillus. Some Lactobacillus species detected in dough samples by sequence analysis of DGGE bands were not recovered in any of the five culture media and conditions used. On the other hand, some species recovered as pure cultures from enrichments were not detected by direct DGGE analysis of total bacterial DNA from cassava dough. Our results provide evidence of the necessity to combine both culture-dependent and culture-independent methods for better description of microbial communities in indigenous cassava starch fermentations.