Browsing by Author "Wallace, Regina A."

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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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    CRISPR3 Regulates Exopolysaccharide Production in Myxococcus xanthus
    Wallace, Regina A. (Virginia Tech, 2013-10-10)
    Myxococcus xanthus, a model organism for studying development and Type IV pili (T4P), harbors three Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) on its chromosome. CRISPR systems, which function as an adaptive immune system in prokaryotes, are classified into three types based on CRISPR-associated genes. Evidence suggests that these three types mediate immunity slightly differently. M. xanthus CRISPR1 and CRISPR2 are Type I systems while CRISPR3 is a Type III-B system. In a genetic screen, a mariner transposon insertion in the 13th spacer of CRISPR3 (3SP13) was found to restore exopolysaccharide (EPS) production to a pilA mutant. Since the deletion of CRISPR3 failed to suppress a pilA mutation and expression of CRISPR3 from a heterologous promoter led to pilA suppression, it was concluded that the 3SP13 transposon insertion is a gain-of-function mutation. Deletion of the adjacent Repeat Associated Mysterious Proteins (RAMP) genes indicated that they are essential for the 3SP13 transposon insertion to suppress pilA, providing evidence that Type III-B systems may be involved in the regulation of chromosomal genes. We suggest that one of the spacers, once expressed and processed, may inhibit the expression of a negative regulator of EPS production in M. xanthus.
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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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    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.