Sigma E controls biogenesis of the antisense RNA MicA

Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σ(S). We identified stress conditions that induce high MicA levels even during exponential growth—a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σ(E) is activated. The strict dependence of micA transcription on σ(E) is supported by three observations. Induced overexpression of σ(E) increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σ(E) signature. Thus, MicA is part of the σ(E) regulon and downregulates its target gene, ompA, probably to alleviate membrane stress

Diet change affects intestinal microbiota restoration and improves vertical sleeve gastrectomy outcome in diet-induced obese rats

Purpose: Obesity, a worldwide health problem, is linked to an abnormal gut microbiota and is currently most efectively treated by bariatric surgery. Our aim was to characterize the microbiota of high-fat fed Sprague-Dawley rats when subjected to bariatric surgery (i.e., vertical sleeve gastrectomy) and posterior refeeding with either a high-fat or control diet. We hypothesized that bariatric surgery followed by the control diet was more efective in reverting the microbiota modifcations caused by the high-fat diet when compared to either of the two factors alone. Methods: Using next-generation sequencing of ribosomal RNA amplicons, we analyzed and compared the composition of the cecal microbiota after vertical sleeve gastrectomy with control groups representing non-operated rats, control fed, high-fat fed, and post-operative diet-switched animals. Rats were fed either a high-fat or control low-fat diet and were separated into three comparison groups after eight weeks comprising no surgery, sham surgery, and vertical sleeve gastrectomy. Half of the rats were then moved from the HFD to the control diet. Using next-generation sequencing of ribosomal RNA amplicons, we analyzed the composition of the cecal microbiota of rats allocated to the vertical sleeve gastrectomy group and compared it to that of the non-surgical, control fed, high-fat fed, and post-operative diet-switched groups. Additionally, we correlated diferent biological parameters with the genera exhibiting the highest variation in abundance between the groups. Results: The high-fat diet was the strongest driver of altered taxonomic composition, relative microbial abundance, and diversity in the cecum. These efects were partially reversed in the diet-switched cohort, especially when combined with sleeve gastrectomy, resulting in increased diversity and shifting relative abundances. Several highly-afected genera were correlated with obesity-related parameters. Conclusions: The dysbiotic state caused by high-fat diet was improved by the change to the lower fat, higher fber control diet. Bariatric surgery contributed signifcantly and additively to the diet in restoring microbiome diversity and complexity. These results highlight the importance of dietary intervention following bariatric surgery for improved restoration of cecal diversity, as neither surgery nor change of diet alone had the same efects as when combined

Transcriptional and Post-Transcriptional Regulation of the Escherichia coli luxS mRNA; Involvement of the sRNA MicA

Background: The small RNA (sRNA) MicA has been shown to post-transcriptionally regulate translation of the outer membrane protein A (OmpA) in Escherichia coli. It uses an antisense mechanism to down-regulate OmpA protein synthesis and induce mRNA degradation. MicA is genomically localized between the coding regions of the gshA and luxS genes and is divergently transcribed from its neighbours. Transcription of the luxS gene which originates within or upstream of the MicA sequence would thus be complementary to the sRNA. LuxS regulation is as yet unclear. Methodology/Principal Findings: In this report, I show that the luxS mRNA exists as three long (major) transcripts of sizes that suggest just such interaction. The sRNA MicA’s expression affects the abundance of each of these luxS transcripts. The involvement of the ribonuclease, RNase III in the accumulation of the shortest transcript is demonstrated. When MicA accumulates during growth, or is induced to be over-expressed, the cleaved mRNA species is observed to increase in intensity. Using primer extension and 59-RACE experiments in combination with sRNA overexpression plasmids, I identify the exact origin of two of the three luxS transcripts, one of which is seen to result from a previously unidentified s S dependent promoter. Conclusions/Significance: The presented data provides strong evidence that MicA functions in cis and in trans, targeting both luxS mRNA as well as the previously established ompA and phoP regulation. The proposed luxS regulation by MicA would be in tandem with another sRNA CyaR, shown recently to be involved in inhibiting translation of the luxS mRNA. Regulation of luxS expression is additionally shown to occur on a transcriptional level via s S with variable transcript levels in different growth phases unlike what was previously assumed. This is the first known case of an sRNA in E. coli which targets both in cis (luxS mRNA) and in trans (ompA and phoP mRNAs)

Steady state levels of <i>luxS</i> mRNA transcripts during growth in liquid culture.

<p>Intensity of <i>luxS</i> probing-generated transcripts P1(black) and P2 (open), as well as R3 (shaded), at various stages of growth as assayed by Northern Blot analysis. Signal intensities as derived from band densitometry were normalized to 5S rRNA levels and plotted accordingly.</p

5'- RACE amplified products of first strand synthesized products using the adapter specific B6 primer in combination with the K22 primer which binds to the <i>gshA</i> terminus, or K31 which is complementary to the sequence spanning 31 bp to 50 bp upstream of the <i>luxS</i> ORF.

<p>Resultant PCR products were resolved on 2% agarose gels against pUC <i>MspI</i> marker (Fermentas). TAP  =  tobacco acid pyrophosphatase. The P1-generated and TAP sensitive band was cloned into pTOPO 2.1 plasmids and resultant clones sequenced to give the indicated G (-332) as transcription start site.</p

A. Schematic diagram of the genomic organization of the genes <i>luxS</i>, <i>micA</i>, and <i>gshA</i>.

<p>Arrowed bars indicate direction of <i>luxS</i> and <i>gshA</i> transcription/translation. MicA is transcribed in the opposite direction (in red) from within the intergenic region and numbering is relative to the LuxS translation start site. The relevant regions alone are shown for sake of brevity. Primer binding sites are indicated with arrows above the sequence. Short arrows beneath the sequence signify the transcript ends deduced from primer extension data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013449#pone-0013449-g004" target="_blank">Fig. 4</a>) and are labeled accordingly. The thick arrow labeled P1 indicates the transcript P1 as mapped by primer extension and 5'- RACE. In bold letters upstream of the P1 position are the -10 and -35 boxes. <b>B. Alignment of the </b><b><i>luxS</i></b><b> P1-specific promoter region. </b><i>E. coli</i> sequence (bottom row panel A) was 'BLAST-aligned' against the NCBI database and the highest scoring regions from select bacteria (see text) in the genomic location <i>gshA_micA_luxS</i> were aligned against <i>E. coli's</i>. The strongly σ^S-specific <i>ftsQ</i> P1 promoter is also included below. The RNase III-independent primer extension and 5'-RACE identified mRNA start is labeled as '+1' and indicated with an arrow. The -10 and -35 boxes are indicated by a line above the sequence alignments, the <i>rpoS</i> signatory -13 'C' lies directly outside of the -10 box and is indicated in the figure. Aligned regions correspond to equivalent positions relative to the <i>luxS</i> ORF in each species. Below the diagram are lines descriptive of the regions fused to the <i>luc</i> gene in the transcription assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013449#pone-0013449-g006" target="_blank">Fig 6</a>). (<i>E. coli</i>  =  <i>Escherichia coli</i> K12 (U00096.2); <i>S. typhimurium</i>  =  <i>Salmonella typhimurium</i> LT2 (AE008828.1); <i>S. flexneri</i>  =  <i>Shigella flexneri</i> str.301 (AE005674.1); <i>P. luminescens</i>  =  <i>Photorabdus luminescens</i> subsp. <i>Laumondii</i> (BX571863). The <i>E. coli ftsQ</i> P1 sequence is obtained from (27).</p

Northern blot analysis of <i>luxS</i> mRNA steady state levels in wildtype (lanes 2, 4, 6 & 8) and an isogenic <i>rnc</i>^- mutant strain (lanes 3,5,7 & 9).

<p>The strains carried either no plasmid (lanes 2 & 3); control plasmid (lanes 4 & 5); AntiMicA overexpressing (lanes 6 & 7); MicA overexpressing (lanes 8 & 9). RNA was extracted in stationary phase and 10 μg of total RNA was analyzed on 5% PA gels prior to transfer to charged nylon membranes. Probing was carried out with an in vitro synthesized luxS riboprobe (LuxS RP). The different RNA species are indicated in the figure. Equal loading was ensured by probing and normalizing to 5S RNA.</p

Strains and plasmids.

<p>*Numbering relative to <i>luxS</i> translation start site.</p

Transcriptional fusions to a <i>luc</i> reporter gene of two different lengths of the putative <i>luxS</i> promoter pP1 assayed for activity.

<p>The long fusion p38 (open bars) encompasses 130bp of sequence around the putative promoter whilst the shorter p40 (filled bars) is 38 bp long encompassing only the -35 and -10 boxes. Cells were grown in LB medium until stationary phase, with aliquots taken at various OD₆₀₀ as indicated in the figure. A transcriptionally inactive plasmid was used to normalize for background activity. All values are OD normalized and plotted as fold induction over values at the earliest timepoint.</p

Primer extension assay carried out on total RNA extracted from cells grown to stationary phase.

<p>³²P 5'-end labelled K12 primer was extended with the Superscript II reverse transcriptase and resolved on 7% polyacrylamide gel. Major bands are delineated in the schematic of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013449#pone-0013449-g001" target="_blank">figure 1</a>. P1 represents the longest transcript (> 800 nt); P2 is somewhat shorter (> 650 nt); R3 (∼ 600). An additional band of unexplained origin, X, is also indicated in the figure. Band 2a is seen to be accompanied by an additional unlabelled band in the <i>rnc</i>^- mutant.</p