Small RNAs in the Genus Clostridium

The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny

Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress

ABSTRACT Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5 0 -UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy

Revisiting autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) mutations in the nicotinic acetylcholine receptor reveal an increase in efficacy regardless of stochiometry

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic form of epilepsy that is caused by mutations in several genes, including genes encoding for the α4 and β2 subunits of the nicotinic acetylcholine (nACh) receptor. Pentameric α4β2 nACh receptors are the most abundant nicotinic receptor in the mammalian brain and form two stoichiometries, the (α4)3(β2)2 and (α4)2(β2)3 receptors that differ in their physiological and pharmacological properties. The purpose of this study was to investigate how ADNFLE mutations β2V287M, β2V287L or α4T293I manifest themselves in different receptor stoichiometries. We expressed wild-type and mutant receptors in Xenopus oocytes and measured the response to ACh and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of ACh at (α4)2(β2)3 receptors was increased. At (α4)3(β2)2 receptors, the efficacy of activation was increased both when two molecules of agonist, either ACh or the siteselective agonist sazetidine-A, were bound at the α4-β2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the mutations increased the current elicited by low concentrations of ACh. Further, the smoking cessation agents, nicotine, varenicline and cytisine increased activation of mutant (α4)3(β2)2 receptors, while only nicotine increased activation of mutant (α4)2(β2)3 receptors. Chronic exposure of all agonists reduced ACh-activation levels at low and high ACh concentrations. From this, we concluded that mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry of the receptor

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4β2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(β2)3 and (α4)3(β2)2 that differ by an ACh-binding site at the α4−α4 interface of (α4)3(β2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4β2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4β2 nACh receptors. Although inhibition by AE succinimide was found to be voltage dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2′−16′ region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13′C)β2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13′ cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed stateThis research was supported by a Project (APP1069417) from the Australian National Health and Medical Research Council (M.D.M. and M.C.) and by a Discovery Project (DP0986469) from the Australian Research Council (M.D.M. and M.C.). T.Q., G.Q., J.I.H. were supported by Australian Postgraduate Award. D.I. was supported by International Postgraduate Research Scholarship, and T.Q., G.X.Q., J.I.H., and D.I. were also supported by the John A. Lamberton scholarshi

Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin, Vc1.1

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca2+ concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)3(α10)2 and (α9)2(α10)3. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9-α9 interface on the hypothesized (α9)3(α10)2 nAChR, in addition to the α10-α9 and or α9-α10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors

Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.

<p>Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.</p

Saz-A and TC-2559 act selectively at the α4-β2 interface.

<p><b>A</b> Schematic showing α4-β2 (black arrow) and α-α (grey arrow) binding sites for ACh at the 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptors. <b>B.</b> Current traces of Saz-A (1μM) and <b>C.</b> TC-2559 (10μM) for 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptor compared to maximum ACh currents on the same oocyte. Peak currents elicited by <b>D</b> Saz-A and <b>E</b> TC-2559 were normalized to the saturating concentration of ACh and fitted to non-linear curve fits. The injection ratios of α4:β2 that correspond to the 3α4:2β2 (blue) and the 2α4:3β2 (red) receptors are 4:1 and 1:4 respectively. Saz-A and TC-2559 do not activate the 3α4:2β2^HQT (green) receptor that has three α4-α4-like interfaces. Dots represent the mean ± s.e.m.</p

Proposed model.

<p>A schematic of proposed receptor model for the activation of WT 3α4:2β2 nAChR. The model includes unbound (R), one agonist bound (AR), two agonist bound (A₂R) and three agonist bound (A₃R) receptor states. Intermediate ‘flip’ state (RF), Open state (RO) and desensitized state (RD) are shown. Blue frame includes receptor activation model for agonists (eg. Saz-A and TC-2559) that selective bind at the α4-β2 interface and red frame shows receptor activation for ligands selective for α4-α4 interface (NS9283). Dashed lines and grey states indicate transitions and states not discretely tested in our experiments but are considered likely to exist.</p

Estimation of open probability.

<p><b><i>A</i></b> Schematic depicting the hypothesized areas for NS206 binding [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161154#pone.0161154.ref034" target="_blank">34</a>]. <b><i>B</i></b> Representative current trace of the response of the 3α4:2β2 receptor to 1 mM ACh (blue) and 1 mM ACh co-applied with 10 μM NS206 to estimate open channel probability. <b><i>C</i></b> The level of NS206 (10μM) positive modulation of ACh currents for the WT. <b><i>D</i></b> Saz-A and <b><i>E</i></b> TC-2559 concentration-response curves in the absence (blue) and presence (green) of 10 μM NS9283 normalized to 1 mM ACh co-applied with 10 μM NS206 (Est <i>P</i>_{<i>o</i>,<i>max</i>}) at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.</p