Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs.

Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. TCSs and sRNAs mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors to oxygen. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent mechanisms of control. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second requires only the very 5' end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.

Impacts of ocean acidification and warming on post-larval growth and metabolism in two populations of the great scallop (Pecten maximus).

Ocean acidification and warming are key stressors for many marine organisms. Some organisms display physiological acclimatization or plasticity, but this may vary across species ranges, especially if populations are adapted to local climatic conditions. Understanding how acclimatization potential varies among populations is therefore important in predicting species responses to climate change. We carried out a common garden experiment to investigate how different populations of the economically important great scallop (Pecten maximus) from France and Norway responded to variation in temperature and PCO2 concentration. After acclimation, post-larval scallops (spat) were reared for 31 days at one of two temperatures (13°C or 19°C) under either ambient or elevated PCO2 (pH 8.0 and pH 7.7). We combined measures of proteomic, metabolic and phenotypic traits to produce an integrative picture of how physiological plasticity varies between the populations. The proteome of French spat showed significant sensitivity to environmental variation, with 12 metabolic, structural and stress-response proteins responding to temperature and/or PCO2. Principal component analysis revealed seven energy metabolism proteins in French spat that were consistent with countering ROS stress under elevated temperature. Oxygen uptake in French spat did not change under elevated temperature but increased under elevated PCO2. In contrast, Norwegian spat reduced oxygen uptake under both elevated temperature and PCO2. Metabolic plasticity allows French scallops to maintain greater energy availability for growth compared with Norwegian spat. However, increased physiological plasticity and growth in French spat may come at a cost, as they showed reduced survival compared with Norwegian scallops under elevated temperature.

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin.

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment.

Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile.

Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.

A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile.

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development.

In Silico Analysis of Pacific Oyster (Crassostrea gigas) Transcriptome over Developmental Stages Reveals Candidate Genes for Larval Settlement.

Following their planktonic phase, the larvae of benthic marine organisms must locate a suitable habitat to settle and metamorphose. For oysters, larval adhesion occurs at the pediveliger stage with the secretion of a proteinaceous bioadhesive produced by the foot, a specialized and ephemeral organ. Oyster bioadhesive is highly resistant to proteomic extraction and is only produced in very low quantities, which explains why it has been very little examined in larvae to date. In silico analysis of nucleic acid databases could help to identify genes of interest implicated in settlement. In this work, the publicly available transcriptome of Pacific oyster Crassostrea gigas over its developmental stages was mined to select genes highly expressed at the pediveliger stage. Our analysis revealed 59 sequences potentially implicated in adhesion of C. gigas larvae. Some related proteins contain conserved domains already described in other bioadhesives. We propose a hypothetic composition of C. gigas bioadhesive in which the protein constituent is probably composed of collagen and the von Willebrand Factor domain could play a role in adhesive cohesion. Genes coding for enzymes implicated in DOPA chemistry were also detected, indicating that this modification is also potentially present in the adhesive of pediveliger larvae.

Genetic parameters of resistance to Vibrio aestuarianus, and OsHV-1 infections in the Pacific oyster, Crassostrea gigas, at three different life stages.

BACKGROUND: In France, two main diseases threaten Pacific oyster production. Since 2008, Crassostrea gigas spat have suffered massive losses due to the ostreid herpesvirus OsHV-1, and since 2012, significant mortalities in commercial-size adults have been related to infection by the bacterium Vibrio aestuarianus. The genetic basis for resistance to V. aestuarianus and OsHV-1 and the nature of the genetic correlation between these two traits were investigated by using 20 half-sib sire families, each containing two full-sib families. For each disease, controlled infectious challenges were conducted using naïve oysters that were 3 to 26 months old. In addition, siblings were tested under field, pond and raceway conditions to determine whether laboratory trials reflected mortality events that occur in the oyster industry. RESULTS: First, we estimated the genetic basis of resistance to V. aestuarianus in C. gigas. Susceptibility to the infection was low for oysters in spat stage but increased with later life stages. Second, we confirmed a strong genetic basis of resistance to OsHV-1 infection at early stages and demonstrated that it was also strong at later stages. Most families had increased resistance to OsHV-1 infection from the spat to adult stages, while others consistently showed low or high mortality rates related to OsHV-1 infection, regardless of the life stage. Our third main finding was the absence of genetic correlations between resistance to OsHV-1 infection and resistance to V. aestuarianus infection. CONCLUSIONS: Selective breeding to enhance resistance to OsHV-1 infection could be achieved through selective breeding at early stages and would not affect resistance to V. aestuarianus infection. However, our results suggest that the potential to select for improved resistance to V. aestuarianus is lower. Selection for dual resistance to OsHV-1 and V. aestuarianus infection in C. gigas might reduce the impact of these two major diseases by selecting families that have the highest breeding values for resistance to both diseases.

The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.

CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas.

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.