Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives ofSinorhizobium meliloti strain 2011

Abstract

Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.

sinI- andexpR-Dependent Quorum Sensing inSinorhizobium meliloti

“…Proteins were extracted from freeze-dried cells as previously described (8) and quantified based on a modified Bradford protein assay (23). Protein concentrations were normalized, and the samples were subjected to two-dimensional (2D) gel separation as previously described (9,48). Preparative gels were stained with Coomassie brilliant blue in a stepwise colloidal staining procedure (49).…”

Proteomic Analysis of Wild-Type Sinorhizobium meliloti Responses to N -Acyl Homoserine Lactone Quorum-Sensing Signals and the Transition to Stationary Phase

“…S. meliloti 1021 was grown in TA (YTB) medium (7) for proteome analysis so that results could be compared to earlier proteomic studies of this bacterium (7,8,21,22,32,48) and in a defined NM medium (39) supplemented with 3g of glucose/liter, 0.505 g of KNO 3 /liter, and Gotz vitamins for AHL extraction and purification in order to minimize contamination of AHL preparations with organic components from rich media. The AHL reporter strains, Escherichia coli pSB401 (LuxRIЈ:: luxCDABE) and pSB1075 (LasRIЈ::luxCDABE) (53) were grown in Luria-Bertani (LB) medium (18).…”

“…Proteins were extracted from freeze-dried cells and the samples were subjected to proteomic analysis as described previously (7,8). Isoelectric focusing was performed with linear immobilized pH 4 to 7 IPG strips (Amersham Pharmacia Biotechnology, Uppsala, Sweden).…”

A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote–host interaction

“…Determining the complete genome sequence of S. meliloti set the stage for exploring this wealth of information by transcriptional and proteomic studies (15)(16)(17)(18)(19)(20)(21)(22). Here we describe the construction of a dual-genome Symbiosis Chip and how we have used this unique tool, which allows us to probe global gene expression in both partners simultaneously.…”