Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

Abstract

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons inSinorhizobium meliloti

“…28,29,31,32 Despite of relevant differences in the experimental setups (i.e., biological conditions tested) preclude direct comparison of the mRNAs of the Hfq regulon and those scored as Hfq-associated in our study, an overlap of 322 genes was found between both data sets ( Table S3). Almost half of these genes (158) encode transport proteins (mostly ABC transporter membrane complexes), cytoplasmic metabolic enzymes such as the phosphoenolpyruvate carboxykinase (PckA; Fig.…”

“…Remarkably, the S. meliloti hfq mutants evidenced massive misregulation of transport systems, which anticipates a major role of Hfq in controlling the metabolic versatility of this bacterium. 28,29,31,32 Not unexpectedly, this global influence of Hfq on S. meliloti physiology was also found to compromise an array of symbiotic traits, including competitiveness for Northern analysis of the expression of selected hfq-bound sRNas. Total RNa was obtained from strain sm2B3001 grown in the conditions of coIP experiments.…”

“…Deletion of the chromosomal S. meliloti hfq gene resulted in delayed growth, alterations in the metabolism of certain nitrogen sources, changes in the profiles of quorum sensing (QS) signals, reduced swarming and swimming motilities, and increased vulnerability to oxidative, heat, or pH stresses in free-living bacteria. [27][28][29][30][31] This pleiotropic phenotype is partially explained by the large Hfq-dependent changes in gene expression observed in cultured bacteria. Remarkably, the S. meliloti hfq mutants evidenced massive misregulation of transport systems, which anticipates a major role of Hfq in controlling the metabolic versatility of this bacterium.…”

Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans -Encoded Small RNA MmgR

“…At the moment of the cell harvesting, aliquots of cultures containing 40 OD units each were mixed with their respective pair for proteomic comparison and cooled on ice before centrifuging to collect cell pellets. Proteins were extracted and separated into the cytosolic and membrane subcellular fractions following the protocol described elsewhere (24). The protein concentration in each fraction was determined by the Bradford colorimetric assay (Bradford protein assay Ready-to-Use; Bio-Rad, USA) (65) with a bovine serum albumin solution as a reference, and the quality was verified by SDS-PAGE (62).…”

“…meliloti has been shown to express more than 400 different sRNAs under different growth conditions and lifestyles, i.e., both free-living in the soil and symbiotically associated in a plant (15)(16)(17)(18)(19)(20)(21). In addition, S. meliloti expresses a functional homolog of the Escherichia coli Hfq protein, a chaperone of RNA-RNA interactions and a modulator of RNA activity (22)(23)(24)(25)(26)(27)(28)(29). Nevertheless, the biological implications of the large S. meliloti RNome remain largely unexplored (20), since only four small RNAs have been functionally characterized so far, i.e., the cell cycle regulator sRNA EcpR1 (30), the quorumsensing regulator sRNA RcsR1 (31), and the tandemly encoded orthologs AbcR1 and AbcR2 (32).…”