The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

ABSTRACT The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS , respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS , but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance

The Sex-Specific VC Neurons Are Mechanically Activated Motor Neurons That Facilitate Serotonin-Induced Egg Laying in C. elegans

Successful execution of behavior requires coordinated activity and communication between multiple cell types. Studies using the relatively simple neural circuits of invertebrates have helped to uncover how conserved molecular and cellular signaling events shape animal behavior. To understand the mechanisms underlying neural circuit activity and behavior, we have been studying a simple circuit that drives egg-laying behavior in the nematode worm Here we show that the sex-specific, ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to serotonin. Ca imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. We also find the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca activity and requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other. Together, our results demonstrate that the VC neurons facilitate efficient execution of egg-laying behavior by coordinating postsynaptic muscle contractility in response to serotonin and mechanosensory feedback. Many animal motor behaviors are modulated by the neurotransmitters, serotonin and ACh. Such motor circuits also respond to mechanosensory feedback, but how neurotransmitters and mechanoreceptors work together to coordinate behavior is not well understood. We address these questions using the egg-laying circuit in where we can manipulate presynaptic neuron and postsynaptic muscle activity in behaving animals while recording circuit responses through Ca imaging. We find that the cholinergic VC motoneurons are important for proper vulval muscle contractility and egg laying in response to serotonin. Muscle contraction also activates the VCs, forming a positive feedback loop that promotes full contraction for egg release. In all, mechanosensory feedback provides a parallel form of modulation that shapes circuit responses to neurotransmitters

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance

Identification of Toxicity Effects of Cu2O Materials on C. elegans as a Function of Environmental Ionic Composition

Previous work has shown that spherical CuO nanomaterials show negative effects on cell and animal physiology. The biological effects of Cu 2 O materials, which posess unique chemical features compared to CuO nanomaterials and can be synthesized in a similarly large variety of shapes and sizes, are comparatively less studied. Here, we synthesized truncated octahedral Cu 2 O particles and characterized their structure, stability, and physiological effects in the nematode worm animal model, Caenorhabditis elegans . Cu 2 O particles were found to be generally stable in aqueous media, although the particles did show signs of oxidation and leaching of Cu 2+ within hours in worm growth media. The particles were found to be especially sensitive to inorganic phosphate (PO 4 3− ) found in standard NGM nematode growth medium. Cu 2 O particles were observed being taken up into the nematode pharynx and detected in the lumen of the gut. Toxicity experiments revealed that treatment with Cu 2 O particles caused a significant reduction in animal size and lifespan. These toxic effects resembled treatment with Cu 2+ , but measurements of Cu leaching, worm size, and long-term behavior experiments show the particles are more toxic than expected from Cu ion leaching alone. These results suggest worm ingestion of intact Cu 2 O particles enhances their toxicity and behavior effects while particle exposure to environmental phosphate precipitates leached Cu 2+ into biounavailable phosphate salts. Interestingly, the worms showed an acute avoidance of bacterial food with Cu 2 O particles, suggesting that animals can detect chemical features of the particles and/or their breakdown products and actively avoid areas with them. These results will help to understand how specific, chemically-defined particles proposed for use in polluted soil and wastewater remediation affect animal toxicity and behaviors in their natural environment. C. elegans worms (grey) encountering Cu 2 O particles (red) can either avoid and survive (left) or they can ingest them (right) and experience toxic effects that resemble effects from Cu 2+ uptake (blue). Phosphate induces Cu 2 O particle oxidation (blue outlines) and Cu ion precipitation, allowing for improved survival even after ingestion (bottom)

Replication origins of <i>C</i>. <i>taeniosporum</i> (Ct), <i>C</i>. <i>botulinum</i> (Cb), and <i>B</i>. <i>subtilis</i> (Bs).

<p>Untranslated, DnaA box-rich segments bracket the <i>dnaA</i> gene (green arrows indicate transcription direction). DnaA boxes which match the low G+C Gram positive bacteria consensus of TTATCCACA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189673#pone.0189673.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189673#pone.0189673.ref033" target="_blank">33</a>] in all 9 (maroon triangles), in 8 (blue triangles) and in 7 (dashed blue triangle) positions. Direct repeats are indicated by yellow bars; a potential DNA Unwinding Element by the red bar. <i>C</i>. <i>botulinum</i> B strain Eklund 17B (NC_010674.1) and <i>B</i>. <i>subtilis</i> strain 168 (NZ_CM000487.1) data were used. The nucleotides are numbered beginning with the first nucleotide of the <i>C</i>. <i>taeniosporum oriC</i> region.</p

Genomics of <i>Clostridium taeniosporum</i>, an organism which forms endospores with ribbon-like appendages

<div><p><i>Clostridium taeniosporum</i>, a non-pathogenic anaerobe closely related to the <i>C</i>. <i>botulinum</i> Group II members, was isolated from Crimean lake silt about 60 years ago. Its endospores are surrounded by an encasement layer which forms a trunk at one spore pole to which about 12–14 large, ribbon-like appendages are attached. The genome consists of one 3,264,813 bp, circular chromosome (with 26.6% GC) and three plasmids. The chromosome contains 2,892 potential protein coding sequences: 2,124 have specific functions, 147 have general functions, 228 are conserved but without known function and 393 are hypothetical based on the fact that no statistically significant orthologs were found. The chromosome also contains 101 genes for stable RNAs, including 7 rRNA clusters. Over 84% of the protein coding sequences and 96% of the stable RNA coding regions are oriented in the same direction as replication. The three known appendage genes are located within a single cluster with five other genes, the protein products of which are closely related, in terms of sequence, to the known appendage proteins. The relatedness of the deduced protein products suggests that all or some of the closely related genes might code for minor appendage proteins or assembly factors. The appendage genes might be unique among the known clostridia; no statistically significant orthologs were found within other clostridial genomes for which sequence data are available. The <i>C</i>. <i>taeniosporum</i> chromosome contains two functional prophages, one <i>Siphoviridae</i> and one <i>Myoviridae</i>, and one defective prophage. Three plasmids of 5.9, 69.7 and 163.1 Kbp are present. These data are expected to contribute to future studies of developmental, structural and evolutionary biology and to potential industrial applications of this organism.</p></div

Functional categories of <i>C</i>. <i>taeniosporum</i> chromosomal protein CDSes.

<p>Functional categories of <i>C</i>. <i>taeniosporum</i> chromosomal protein CDSes.</p

Appendage genes and proteins.

<p>Appendage protein genes P29a, P29b and GP85 and related genes (open arrows) reside on a 9.6 Kbp region of the chromosome and are transcribed in the same direction (arrow points). Extensive homology of the deduced proteins (or regions therein) is indicated by colored bars above the relevant portions of the genes: CRD1 and CRD2 (green), portions of CRD1 and CRD2 with most of P29a and P29b (blue), portions of CRD1 and CRD2 with most of the HYPO2 and HYPO3 proteins (red) and portions of GP85 and CL2 are identical (black) and both have collagen-like regions (orange). Internal repeats and DUF11 sequences within deduced protein sequences are indicated by black arrows and red bars, respectively. Positions of potential mother cell late promoters (red circle) and a potential sigma A-dependent promoter (open red circle) are indicated. The filled circles indicate Kbp positions. Bp 1 corresponds to the complement of chromosomal nucleotide pair 2,172,100.</p

Neighbor-joining phylogenetic tree of a selected cluster of the phages which infect the <i>Firmicute Clostridium</i> and <i>Bacillus</i> genera.

<p>The nucleotide sequences of the 62 phages known to infect the <i>Clostridium</i> or <i>Bacillus</i> genera (and for which complete nucleotides sequences were available) were subjected a MAFFT multi-wise alignment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189673#pone.0189673.ref069" target="_blank">69</a>]. A neighbor-joining tree [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189673#pone.0189673.ref070" target="_blank">70</a>] was constructed (Geneious Pro v.7.1.6) and bootstrap values, expressed in percentage based on 1,000 repetitions, are shown next to each group. The bar represents 0.3 change per nucleotide site.</p

Naturally occurring plasmids of <i>C</i>. <i>taeniosporum</i> and the derivative pCt1-2200.

<p>Locations and transcription directions of genes of pCt1 and pCt1-2200 are indicated by arrows. pCt1 genes include those for mobilization (MOB), relaxase (REL), plasmid replication (REP) and peptidase (PEP) proteins. The replicon of pCt1 (present within a fragment consisting of a portion of the REL gene, the replication gene, the intergenic region and a portion of the PEP gene) was ligated to a chloramphenicol-resistance (CM-R) fragment of pAT4, generating pCt1-2200 which replicates in <i>B</i>. <i>subtilis</i>. pCt2 and pCt3 consist of 62 and 154 potential coding sequences; transcription direction clockwise (green) or counterclockwise (red). The arrow points mark bp 1.</p