Quantitative Proteomic Analysis of the Hfq-Regulon in <em>Sinorhizobium meliloti</em> 2011

<div><p>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 <em>Sinorhizobium meliloti</em> 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 <em>hfq</em> mutant and the wild type <em>S. meliloti</em> 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 <em>S. meliloti</em>, 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 <em>S. meliloti.</em> The comparative analysis of the <em>hfq</em> mutant proteome allowed identification of novel strongly Hfq-regulated genes in <em>S. meliloti</em>.</p> </div

Summary of the quantitative LC/MS proteomic analysis of ¹⁵N labelled proteins.

<p>A) Representative Venn diagram of identified and quantified unique proteins in each of the subcellular fractions. B) Functional distribution of positively Hfq-regulated proteins. C) Functional distribution of negatively Hfq-regulated proteins.</p

The absence of Hfq results in deficient growth in MDM defined medium.

<p>(•), wild type strain 2011; (○), Δ<i>hfq</i> mutant strain 20PS01. Each curve represents the average from three different cultures ± SD. The experiment was repeated twice, with essentially the same results. Arrows point to the growth stage in which cells were harvested for comparative quantitative analysis of ¹⁵N-labelled proteins.</p

Hfq is required for normal siderophore production.

<p>The results are presented as siderophore relative units measured by the CAS interference assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048494#pone.0048494-Schwyn1" target="_blank">[35]</a>. Values represent the average of three independent cultures ± SD. Different letters indicate statistically significant differences among strains based on pairwise comparisons (Student’s <i>t</i>-test, <i>P</i><0.05.).</p

Bialaphos and sodium glufosinate sensitivity assay.

<p>In both cases, bacterial growth was estimated by OD₆₀₀ measurements after 72 h in the presence of different concentrations of the toxic tripeptide Bialaphos (A) or sodium glufosinate (B). (•), wild type strain 2011; (○), Δ<i>hfq</i> mutant strain 20PS01. Data represent the growth measured for each <i>S. meliloti</i> strain in the presence of a given concentration of the toxic reagent relative to the growth in the absence of the chemical. Each bar shows average values for n = 3 replicate cultures ± SD. The experiment was repeated twice with similar results. The chemical structure of both chemicals is shown within each panel.</p

5-fluorouracil sensitivity assay.

<p>A) Schematic representation of the chromosomal <i>S. meliloti</i> 1021 locus encompassing the annotation SMc01827. The arrows indicate possible transcription start sites (J.-P. Schlüter & A. Becker, personal communication). B) Bacterial growth was estimated by OD₆₀₀ measurements after 96 h in the presence of different concentrations of the uracil analogue. (•), wild type strain 2011; (○), Δ<i>hfq</i> mutant strain 20PS01. Values represent the relative growth of <i>S. meliloti</i> 2011 and 20PS01 in presence and absence of the toxic analogue. Each bar shows average values for n = 3 replicate cultures ± SD. The experiment was repeated twice with similar results. The chemical structure of the uracil analogue is shown.</p

Hfq contributes to tolerance to oxidative stress.

<p>Bacterial growth in MDM was estimated by OD₆₀₀ measurements after 72 h in the presence of increasing concentrations of H₂O₂. (•), wild type strain 2011; (○), Δ<i>hfq</i> mutant strain 20PS01. Values represent the relative growth of <i>S. meliloti</i> 2011 and 20PS01 in presence and absence of the oxidative stress agent. Each bar shows average values for n = 3 replicate cultures ± SD. The experiment was repeated twice with similar results.</p

Comparison of experimentally determined and predicted SugR binding sites located upstream of the coding regions of , , , and and construction of a consensus sequence

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> A) Predicted binding motifs were determined by sequence comparisons of proven motifA and motifB to the DNA fragments showing positive SugR binding in the EMSA studies. Proven and predicted motifs are separated by a horizontal, dashed line. Boxed letters in the experimentally proven motifs A and B and the putative SugR binding sequences located upstream the coding regions of , , and denote identical nucleotides in all sequences. Distances to the according translation starts (TL) are indicated. B) A frequency plot of the deduced consensus sequence of all motifs is constructed by means of the WebLogo tool. The overall height of each stack of letters indicates the sequence conservation at each position of the 21-bp motif, whereas the height of each symbol within the stack reflects the relative frequency of the corresponding nucleotide at that position

Transcriptional regulation of the extended PTS cluster genes of cultures grown in liquid media containing glucose, fructose, or acetate

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The strain RES167 was grown in liquid media containing glucose, fructose, or acetate. By RT-PCR the mRNA levels of the genes , , , , , , and of cultures grown in glucose (white bars) or fructose (grey bars) were compared to those of cultures grown in acetate containing media. Results are means of four measurements from two biological replicates. Standard deviations are indicated by error bars

Electrophoretic mobility shift assays (EMSA) with selected upstream DNA fragments of PTS coding regions using the purified SugR protein

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The physical maps of the extended fructose-PTS gene cluster as well as of the genes and are shown. Beneath the maps the fluorescently labelled PCR fragments are indicated which were used for EMSA studies. These studies were carried out with 15 pmol of purified SugR protein and 0.05 pmol of labeled PCR fragments. The results obtained for each PCR fragment are presented by agarose gel photos. In each picture, the left lane shows the shift in presence of the SugR protein, whereas the right lane shows the negative control without added SugR protein. Transcriptional terminators are denoted as stem loop structures