Post-Transcriptional Control of the Escherichia coli PhoQ-PhoP Two-Component System by Multiple sRNAs Involves a Novel Pairing Region of GcvB

Coornaert, Audrey; Chiaruttini, Claude; Springer, Mathias; Guillier, Maude

doi:10.1371/journal.pgen.1003156

Cited by 74 publications

(78 citation statements)

References 45 publications

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“…The presence of these amino acids is required for the survival of the bacterium and their lack is responsible for the expression of both genes for their own biosynthesis, and virulence-related genes, as demonstrated in pigs (Wagner and Mulks 2006;Subashchandrabose et al 2009). GcvB is also known to regulate the PhoQ-PhoP two-component system, which is involved in magnesium homeostasis, pathogenicity, cell envelope composition, and acid resistance in several bacterial species (Coornaert et al 2013). Also involved in stress response, the sRNAs of the RtT family were first discovered as oligonucleotides released from the primary transcript of the tyrT and many other tRNA operons in E. coli during the tRNA processing.…”

Section: Discussionmentioning

confidence: 99%

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

Rossi

Bossé

et al. 2016

RNA

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Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.

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Section: Discussionmentioning

confidence: 99%

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

Rossi

Bossé

et al. 2016

RNA

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“…Naturally occurring antisense small noncoding RNAs (sRNA) have been reported to repress genes downstream of the targeted ORF. Two examples are sRNAs of the phoPQ operon in E. coli (37) and the YPK_1206-1205 operon in Yersinia pseudotuberculosis (38), where sRNAs silence the target and downstream genes. It will be interesting to see if upstream silencing exists in nature as more sRNAs are identified and characterized.…”

Section: Discussionmentioning

confidence: 99%

Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

Goh

Hohmeier

Stone

et al. 2015

Appl Environ Microbiol

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Essential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, the Escherichia coli ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL 50 ). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL 50 values (means and standard deviations) of ribF, ileS, and lspA were 0.65 ؎ 0.18, 0.64 ؎ 0.06, and 0.76 ؎ 0.10, respectively. However, they were not significantly different (P ‫؍‬ 0.71 by weighted one-way analysis of variance). The gene requirement for ispH could not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

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“…Two hundred and fifty micromolars dNTPs and 2.5 µM of initiator tRNA fMet were added with 0.5 µM 30S subunits, and the mixtures were incubated at 37°C for 10 min. One unit AMV RT (Finnzymes) was then added and cDNA synthesis was performed at 37°C for 20 min (Coornaert et al 2013). A sequencing ladder was produced by asymmetric PCR with the pnp PCR fragment (15 fmol/µL) amplified in the presence of 5 ′ -Cy5-end-labeled primer RNX1 and one of the 4 ddNTP mixes (at a ratio ddNTP:dNTP of 25:1 except for ddGTP which was at a ratio of 2.5:1).…”

Section: Toe-print Analysismentioning

confidence: 99%

The small RNA SraG participates in PNPase homeostasis

Fontaine

Gasiorowski

Gracia

et al. 2016

RNA

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The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3 ′ -5 ′ exoribonuclease involved in mRNA decay in Escherichia coli. The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp. Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.

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Post-Transcriptional Control of the Escherichia coli PhoQ-PhoP Two-Component System by Multiple sRNAs Involves a Novel Pairing Region of GcvB

Cited by 74 publications

References 45 publications

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

The small RNA SraG participates in PNPase homeostasis

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