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Browsing by Author "Black, Wesley P."

Now showing 1 - 7 of 7
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    A CRISPR with Roles in Myxococcus xanthus Development and Exopolysaccharide Production
    Wallace, Regina A.; Black, Wesley P.; Yang, Xianshuang; Yang, Zhaomin (American Society for Microbiology, 2014-09-08)
    The Gram-negative soil bacterium Myxococcus xanthus utilizes its social (S) gliding motility to move on surfaces during its vegetative and developmental cycles. It is known that S motility requires the type IV pilus (T4P) and the exopolysaccharide (EPS) to function. The T4P is the S motility motor, and it powers cell movement by retraction. As the key regulator of the S motor, EPS is proposed to be the anchor and trigger for T4P retraction. The production of EPS is regulated in turn by the T4P in M. xanthus, and T4P(-) mutants are S- and EPS-. In this study, a Delta pilA strain (T4P(-) and EPS-) was mutagenized by a transposon and screened for EPS- mutants. A pilA suppressor isolated as such harbored an insertion in the 3rd clustered regularly interspaced short palindromic repeat (CRISPR3) in M. xanthus. Evidence indicates that this transposon insertion, designated CRISPR3(star), is a gain-of-function (GOF) mutation. Moreover, CRISPR3(star) eliminated developmental aggregation in both the wild-type and the pilA mutant backgrounds. Upstream of CRISPR3 are genes encoding the repeat-associated mysterious proteins (RAMPs). These RAMP genes are indispensable for CRISPR3(star) to affect development and EPS in M. xanthus. Analysis by reverse transcription (RT)-PCR suggested that CRISPR3(star) led to an increase in the processing of the RNA transcribed from CRISPR3. We propose that certain CRISPR3 transcripts, once expressed and processed, target genes critical for M. xanthus fruiting body development and EPS production in a RAMP-dependent manner.
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    The Hsp70-like StkA functions between T4P and Dif signaling proteins as a negative regulator of exopolysaccharide in Myxococcus xanthus
    Moak, Pamela L.; Black, Wesley P.; Wallace, Regina A.; Li, Zhuo; Yang, Zhaomin (PeerJ, 2015-02-03)
    Myxococcus xanthus displays a form of surface motility known as social (S) gliding. It is mediated by the type IV pilus (T4P) and requires the exopolysaccharide (EPS) to function. It is clear that T4P retraction powers S motility. EPS on a neighboring cell or deposited on a gliding surface is proposed to anchor the distal end of a pilus and trigger T4P retraction at its proximal end. Inversely, T4P has been shown to regulate EPS production upstream of the Dif signaling pathway. Here we describe the isolation of two Tn insertions at the stk locus which had been known to play roles in cellular cohesion and formation of cell groups. An insertion in stkA (MXAN_3474) was identified based on its ability to restore EPS to a pilA deletion mutant. The stkA encodes a DnaK or Hsp70 homolog and it is upstream of stkB (MXAN_3475) and stkC (MXAN_3476). A stkB insertion was identified in a separate genetic screen because it eliminated EPS production of an EPS+ parental strain. Our results with in-frame deletions of these three stk genes indicated that the stkA mutant produced increased level of EPS while stkB and stkC mutants produced less EPS relative to the wild type. S motility and developmental aggregation were affected by deletions of stkA and stkB but only minimally by the deletion of stkC. Genetic epistasis indicated that StkA functions downstream of T4P but upstream of the Dif proteins as a negative regulator of EPS production in M. xanthus.
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    Metagonimoides oregonensis (heterophyidae: digenea) infection in pleurocerid snails and desmognathus quadramaculatus salamander larvae in southern Appalachian streams
    Belden, Lisa K.; Peterman, William E.; Smith, Stephen A.; Brooks, Lauren R.; Benfield, Ernest F.; Black, Wesley P.; Yang, Zhaomin; Wojdak, Jeremy M. (American Society of Parasitology, 2012-08)
    Metagonimoides oregonensis (Heterophyidae) is a little-known digenetic trematode that uses raccoons and possibly mink as definitive hosts, and stream snails and amphibians as intermediate hosts. Some variation in the life cycle and adult morphology in western and eastern populations has been previously noted. In the southern Appalachians, Pleurocera snails and stream salamanders, e.g., Desmognathus spp., are used as intermediate hosts in the life cycle. We completed a series of studies in this system examining some aspects of larval trematode morphology and first and second intermediate host use. Molecular sequencing of the 28S rDNA of cercariae in our survey placed them clearly within the heterophyid family. However, light and scanning electron microscopy revealed both lateral and dorso-ventral finfolds on the cercariae in our region, whereas original descriptions of M. oregonensis cercariae from the west coast indicate only a dorso-ventral finfold, so further work on the systematics of this group may be warranted. A survey of first intermediate host, Pleurocera proxima, from 7 streams in the region identified only M. oregonensis, virgulate-type cercariae, and cotylomicrocercous-type cercariae in the streams, with M. oregonensis having the highest prevalence, and the only type present that use amphibians as second intermediate hosts. Based on clearing and staining of 6 Desmognathus quadramaculatus salamander larvae, we found that individual salamanders could have over 600 metacercariae, which form between muscle fibers throughout the body. Histological observations suggest that the metacercariae do not cause excessive tissue damage or inflammation, and likely persist through metamorphosis, thereby transmitting potentially large numbers of worms to definitive host raccoons foraging along streams.
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    The orphan response regulator EpsW is a substrate of the DifE kinase and it regulates exopolysaccharide in Myxococcus Xanthus
    Black, Wesley P.; Wang, Linglin; Davis, Manli Y.; Yang, Zhaomin (Nature, 2015-12-07)
    Here we attempted to identify the downstream target of the DifE histidine kinase in the regulation of exopolysaccharide (EPS) production in the Gram-negative bacterium Myxococcus xanthus. This bacterium is an important model system for the studies of Type IV pilus (T4P) because it is motile by social (S) motility which is powered by T4P retraction. EPS is critical for S motility because it is the preferred anchor for T4P retraction in this bacterium. Previous studies identified the Dif chemosensory pathway as crucial for the regulation of EPS production. However, the downstream target of the DifE kinase in this pathway was unknown. In this study, EpsW, an orphan and single-domain response regulator (RR), was identified as a potential DifE target first by bioinformatics. Subsequent experiments demonstrated that epsW is essential for EPS biosynthesis in vivo and that EpsW is directly phosphorylated by DifE in vitro. Targted mutagenesis of epsW suggests that EpsW is unlikely the terminal RR of the Dif pathway. We propose instead that EpsW is an intermediary in a multistep phosphorelay that regulates EPS in M. xanthus.
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    Regulation of Exopolysaccharide Production in Myxococcus Xanthus
    Black, Wesley P. (Virginia Tech, 2005-12-15)
    The surface gliding motility of Myxococcus xanthus is required for a multicellular developmental process initiated by unfavorable growth conditions. One form of the M. xanthus surface motility, social (S) gliding, is mediated by the extension and retraction of polarly localized type IV pili (Tfp). Besides Tfp, exopolysaccharides (EPS), another cell surface associated component, are also required for M. xanthus S motility. Previous studies demonstrated that the Dif chemotaxis-like signal transduction pathway is central to the regulation of EPS production in M. xanthus. Specifically, difA, difC and difE mutants were found to be defective in EPS production and S motility. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA, respectively, are therefore positive regulators of EPS. This study, undertaken to better understand the regulation of EPS production, led to a few major findings. First, DifD and DifG, homologous to CheY and CheC, respectively, were found to be negative regulators of EPS production. Both DifD and DifG likely function upstream of the DifE kinase in EPS regulation. DifB, which has no homology to known chemotaxis proteins, was found not to be involved in EPS production. Secondly, this study led to the recognition that Tfp likely function upstream of the Dif pathway in the regulation of EPS production. Extracellular complementation experiments suggest that Tfp may act as sensors instead of signals for the Dif chemotaxis-like pathway. We propose a regulatory feedback loop that couples EPS production with Tfp function through the Dif signaling proteins. Lastly, we sought to identify additional genes involved in EPS production. Our efforts identified a mutation in a separate chemotaxis gene cluster as a suppressor of difA mutations, suggesting potential cross-talks among the multiple chemotaxis-like pathways in M. xanthus. In addition, we identified twenty-five previously uncharacterized genes that are predicted to be involved in M. xanthus EPS production. These genes appear to encode additional EPS regulators and proteins with biosynthetic function.
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    The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus
    Black, Wesley P.; Wang, Lingling; Jing, Xing; Saldaña, Rafael Castañeda; Li, Feng; Scharf, Birgit E.; Schubot, Florian D.; Yang, Zhaomin (Nature Publishing Group, 2017-08-04)
    Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppressor screen isolated a pilB mutation that restored EPS production to a T4P− mutant. An additional PilB mutant variant, which is deficient in ATP hydrolysis and T4P assembly, supports EPS production without the T4P, indicating PilB can regulate EPS production independently of its function in T4P assembly. Further analysis confirms that PilB functions downstream of the T4P filament but upstream of the Dif proteins. In vitro studies suggest that the nucleotide-free form of PilB assumes the active signaling conformation in EPS regulation. Since M. xanthus PilB possesses conserved motifs with high affinity for c-di-GMP binding, the findings here suggest that c-di-GMP can regulate both motility and biofilm formation through a single effector in this surface-motile bacterium.
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    Type IV Pilus Proteins Form an Integrated Structure Extending from the Cytoplasm to the Outer Membrane
    Li, Chengyun; Wallace, Regina A.; Black, Wesley P.; Li, Yue-zhong; Yang, Zhaomin (PLoS, 2013-07-26)
    The bacterial type IV pilus (T4P) is the strongest biological motor known to date as its retraction can generate forces well over 100 pN. Myxococcus xanthus, a 𝞭-proteobacterium, provides a good model for T4P investigations because its social (S) gliding motility is powered by T4P. In this study, the interactions among M. xanthus T4P proteins were investigated using genetics and the yeast two-hybrid (Y2H) system. Our genetic analysis suggests that there is an integrated T4P structure that crosses the inner membrane (IM), periplasm and the outer membrane (OM). Moreover, this structure exists in the absence of the pilus filament. A systematic Y2H survey provided evidence for direct interactions among IM and OM proteins exposed to the periplasm. For example, the IM lipoprotein PilP interacted with its cognate OM protein PilQ. In addition, interactions among T4P proteins from the thermophile Thermus thermophilus were investigated by Y2H. The results indicated similar protein-protein interactions in the T4P system of this non-proteobacterium despite significant sequence divergence between T4P proteins in T. thermophilus and M. xanthus. The observations here support the model of an integrated T4P structure in the absence of a pilus in diverse bacterial species.