The Efficacy of Sodium Channel Blockers to Prevent Phencyclidine-Induced Cognitive Dysfunction in the Rat: Potential for Novel Treatments for Schizophrenia □ S

ABSTRACT Sodium channel inhibition is a well precedented mechanism used to treat epilepsy and other hyperexcitability disorders. The established sodium channel blocker and broad-spectrum anticonvulsant lamotrigine is also effective in the treatment of bipolar disorder and has been evaluated in patients with schizophrenia. Double-blind placebo-controlled clinical trials found that the drug has potential to reduce cognitive symptoms of the disorder. However, because of compound-related side-effects and the need for dose titration, a conclusive evaluation of the drug's efficacy in patients with schizophrenia has not been possible. In this series of studies in the rat, we compared the efficacy of the two new molecules to prevent a cognitive deficit induced by the N-methyl-D-aspartic acid receptor antagonist phencyclidine (PCP) in the reversal-learning paradigm in the rat. We also explored the effects of the drugs to prevent brain activation and neurochemical effects of PCP. We found that, like lamotrigine, both GSK2 and GSK3 were able to prevent the deficit in reversal learning produced by PCP, thus confirming their potential in the treatment of cognitive symptoms of schizophrenia. However, higher doses than those required for anticonvulsant efficacy of the drugs were needed for activity in the reversal-learning model, suggesting a lower therapeutic window relative to mechanism-dependent central side effects for this indication

A bacterial volatile signal for biofilm formation

Bacteria constantly monitor the environment they reside in and respond to potential changes in the environment through a variety of signal sensing and transduction mechanisms in a timely fashion. Those signaling mechanisms often involve application of small, diffusible chemical molecules. Volatiles are a group of small air-transmittable chemicals that are produced universally by all kingdoms of organisms. Past studies have shown that volatiles can function as cell-cell communication signals not only within species, but also cross-species. However, little is known about how the volatile-mediated signaling mechanism works. In our recent study (Chen, et al. mBio (2015), 6: e00392-15), we demonstrated that the soil bacterium Bacillus subtilis uses acetic acid as a volatile signal to coordinate the timing of biofilm formation within physically separated cells in the community. We also showed that the bacterium possesses an intertwined gene network to produce, secrete, sense, and respond to acetic acid, in stimulating biofilm formation. Interestingly, many of those genes are highly conserved in other bacterial species, raising the possibility that acetic acid may act as a volatile signal for cross-species communication

Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D inCaulobacter crescentus

The Cdz bacteriocin system allows the aquatic oligotrophic bacterium Caulobacter crescentus to kill closely related species in a contact-dependent manner. The toxin, which aggregates on the surfaces of producer cells, is composed of two small hydrophobic proteins, CdzC and CdzD, each bearing an extended glycine-zipper motif, that together induce inner membrane depolarization and kill target cells. To further characterize the mechanism of Cdz delivery and toxicity, we screened for mutations that render a target strain resistant to Cdz-mediated killing. These mutations mapped to four loci, including a TonB-dependent receptor, a three-gene operon (named zerRAB for zipper envelope resistance), and perA (for pentapeptide envelope resistance). Mutations in the zerRAB locus led to its overproduction and to potential changes in cell envelope composition, which may diminish the susceptibility of cells to Cdz toxins. The perA gene is also required to maintain a normal cell envelope, but our screen identified mutations that confer resistance to Cdz toxins without substantially affecting the cell envelope functions of PerA. We demonstrate that PerA, which encodes a pentapeptide repeat protein predicted to form a quadrilateral β-helix, localizes primarily to the outer membrane of cells, where it may serve as a receptor for the Cdz toxins. Collectively, these results provide new insights into the function and mechanisms of an atypical, contact-dependent bacteriocin system. IMPORTANCE Bacteriocins are commonly used by bacteria to kill neighboring cells that compete for resources. Although most bacteriocins are secreted, the aquatic, oligotrophic bacterium Caulobacter crescentus produces a two-peptide bacteriocin, CdzC/D, that remains attached to the outer membranes of cells, enabling contact-dependent killing of cells lacking the immunity protein CdzI. The receptor for CdzC/D has not previously been reported. Here, we describe a genetic screen for mutations that confer resistance to CdzC/D. One locus identified, perA, encodes a pentapeptide repeat protein that resides in the outer membrane of target cells, where it may act as the direct receptor for CdzC/D. Collectively, our results provide new insight into bacteriocin function and diversity.National Institutes of Health (Grant R01GM082899

Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity.

Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity

ssDNA is an allosteric regulator of the C. crescentus SOS-independent DNA damage response transcription activator, DriD

DNA damage repair systems are critical for genomic integrity. However, they must be coordinated with DNA replication and cell division to ensure accurate genomic transmission. In most bacteria, this coordination is mediated by the SOS response through LexA, which triggers a halt in cell division until repair is completed. Recently, an SOS-independent damage response system was revealed in Caulobacter crescentus. This pathway is controlled by the transcription activator, DriD, but how DriD senses and signals DNA damage is unknown. To address this question, we performed biochemical, cellular, and structural studies. We show that DriD binds a specific promoter DNA site via its N-terminal HTH domain to activate transcription of genes, including the cell division inhibitor didA. A structure of the C-terminal portion of DriD revealed a WYL motif domain linked to a WCX dimerization domain. Strikingly, we found that DriD binds ssDNA between the WYL and WCX domains. Comparison of apo and ssDNA-bound DriD structures reveals that ssDNA binding orders and orients the DriD domains, indicating a mechanism for ssDNA-mediated operator DNA binding activation. Biochemical and in vivo studies support the structural model. Our data thus reveal the molecular mechanism underpinning an SOS-independent DNA damage repair pathway.</jats:p

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.</jats:p

A negative feedback regulation on GanA by its catalytic product galactose.

<p><b>(A)</b> The wild type strain (3610) and the <i>ganR</i> mutant (YC222S) were streaked out on LB plates supplemented with 40 μg ml^-1 X-gal, and without (upper panel) or with (lower panel) galactose (0.5%, w/v). Plates were incubated at 37°C overnight before images were taken. <b>(B-C)</b> Assays of ß-galactosidase activities of protein lysates from cells expressing <i>ganA</i> (panel B, YC222S) or <i>lacZ</i> (panel C, YC1074). Assays were done in the presence of 2.5 mM ONPG and a gradient of galactose (from 2.5 to 20 mM). Error bars represent standard deviations from multiple trials. <b>(D)</b> An overview of complex regulations on galactan utilization involving both (1) a positive feedback mechanism on the transcription of the <i>gan</i> operon by ß-1,4-galactobiose and (2) a negative feedback mechanism at the protein level on GanA by its catalytic product galactose.</p

GanR represses the <i>gan</i> operon and the <i>ganR</i> gene.

<p><b>(A)</b> Assays of β-galactosidase activities by the reporter strains bearing either P_<i>ganS</i>-<i>lacZ</i>, or P_<i>ganR</i>-<i>lacZ</i>, or P_<i>ganB</i>-<i>lacZ</i> in the wild type strain (blue bars; YC1073, YC1085, and YC1088) and the <i>ganR</i> mutant (red bars; YC1074, YC1086, and YC1089). A deletion mutation in <i>ganA</i> was also introduced into the above strains. Cells were grown in LB shaking broth to OD₆₀₀ = 1 before harvest and analyses. Assays were done in triplicates and error bars represent standard deviations. <b>(B-C)</b> Assays of ß-galactosidase activities by the wild type reporter strains bearing either P_<i>ganS</i>-<i>lacZ</i>(YC1073, panel B) or P_<i>ganR</i>-<i>lacZ</i>(YC1085, panel C). Cells were grown in LB shaking culture over a period of 5.5 hours after inoculation. Both culture densities (red squares, right-hand y-axis) and ß-galactosidase activities of cells (blue diamonds, left-hand y-axis) were measured. Assays were repeated multiple times and representative data was shown here. <b>(D)</b> Assays of ß-galactosidase activities by the P_<i>ganS</i>-<i>lacZ</i> reporter strains in the wild type background (YC1071), the Δ<i>sinR</i> (YC1091), Δ<i>spo0A</i> (YC1092), Δ<i>degU</i> (YC1248), and Δ<i>ccpA</i> (YC1249) mutants. The <i>ganA</i> deletion mutation was not introduced into the above strains. In some mutants, an <i>epsH</i> deletion mutation was also introduced to prevent cell aggregation during shaking growth [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref045" target="_blank">45</a>]. Cells were grown in LB shaking culture to OD₆₀₀ = 1 before harvest and analyses. Error bars represent standard deviations. <b>(E)</b> Display of the promoter regions of <i>ganS</i> and <i>ganR</i> from <i>B</i>. <i>subtilis</i> NCIB3610 and <i>B</i>. <i>licheniformis</i> ATCC8480. The inverted repeats are highlighted in red, the -35 and -10 motifs of the sigma A-dependent promoter are underlined and shown in italic. ATG or GTG start codons of <i>ganS</i> or <i>ganR</i> are highlighted in blue. The <i>cre</i> box for putative CcpA binding sequences in the <i>ganS</i> promoter regions is highlighted in green. The transcriptional start of the <i>ganS</i> gene in <i>B</i>. <i>subtilis</i> was determined in a very recent study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref029" target="_blank">29</a>] and labeled as +1. <b>(F)</b> The consensus DNA motif logo was generated from a multiple sequence alignment of the putative motifs from the selected promoters using WebLogo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref031" target="_blank">31</a>]. The height of each stack, displayed in bits, is representative of the frequency of the nucleotide in the motif.</p

Mutations in inverted DNA repeats in P_<i>ganS</i> decrease GanR binding.

<p><b>(A-C)</b> Gel mobility shift assays to determine binding of His₆-GanR proteins to the wild type DNA sequence <b>(A)</b>, the Mut1 mutagenic sequence <b>(B)</b>, and Mut2 mutagenic sequence <b>(C)</b> of the <i>ganS</i> promoter. In all lanes, 16 ng (approximately 5 nM) fluorescent DNA probe was added. His₆-GanR proteins were added in a range of concentrations (from 4, 1.3, 0.8, 0.4, to 0.08 μM). In each panel, the right-most lane is the fluorescent probe alone. The left-most lane contains 0.8 μM His₆-GanR proteins, 16 ng of fluorescent probe, and 160 ng of unlabeled cold probe for competitive binding. In the upper section in each gel, shifted DNA bands were indicated by arrows. <b>(D)</b> The ratio of shifted versus total DNA was quantified from panels A-C, and graphed to show percent probe shifted versus protein concentration using WT, Mut1, and Mut2 probes.</p

Strains used in this study.

<p>Strains used in this study.</p