Department

Molecular Biology Department

First Advisor

Dr. Daniel Wall

Description

M. xanthus possesses two motility systems that control gliding motility, namely S- and A-motility. Cells use these motility systems to socially interact with their siblings. Interestingly, we discovered when motile are mixed with isogenic nonmotile cells, e.g. strain DK8601, motile cells are inhibited from moving. This process is called swarm inhibition. In prior work we identified two key players – TraA and TraB involved in swarm inhibition [1]. To better understand how this cell-cell interaction works, our objective here was to identify additional mutants to test if other proteins are involved in this process. Both spontaneous and U.V. induced mutants were isolated in motile cells that showed a relief of swarm inhibition. Here, our experimental design was to spot motile cells with nonmotile cells in an equal ratio and put on an agar plate to see if any mutants, either spontaneous or U.V. induced, showed relief of inhibition. Motile cells or flares that “brokeout” from the spots were carefully streaked out to isolate individual colonies and those cells were retested for relief of swarm inhibition. Once we confirmed their phenotype, an extra copy of the traA and traB genes were transformed into the mutant strains to test for complementation if the new mutations were in the traA or traB or different genes. The resulting transformed colonies were verified for their kanamycin resistance and retested for relief of swarm inhibition. From these experiments four positive mutants (swarm relief) were found out and sent for whole genome sequencing to identify the new gene(s) involved in this process. In contrast, 12 mutants were complemented by the traA plasmid. The identification of new players or genes will help give us a better mechanistic understanding of how cell-cell signaling regulates swarming behavior in M. xanthus. (1. Pathak, D.T. Wei, X. Bucuvalas, A. Haft, D.H. Gerloff, D.L. Wall, D. 2012.Cell contact dependent outer membrane exchange in myxobacteria: genetic determinants and mechanism. PLoS Genetics. 2012 Apr; 8(4): e1002626)

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Relief of Swarm Inhibition in Myxococcus xanthus

M. xanthus possesses two motility systems that control gliding motility, namely S- and A-motility. Cells use these motility systems to socially interact with their siblings. Interestingly, we discovered when motile are mixed with isogenic nonmotile cells, e.g. strain DK8601, motile cells are inhibited from moving. This process is called swarm inhibition. In prior work we identified two key players – TraA and TraB involved in swarm inhibition [1]. To better understand how this cell-cell interaction works, our objective here was to identify additional mutants to test if other proteins are involved in this process. Both spontaneous and U.V. induced mutants were isolated in motile cells that showed a relief of swarm inhibition. Here, our experimental design was to spot motile cells with nonmotile cells in an equal ratio and put on an agar plate to see if any mutants, either spontaneous or U.V. induced, showed relief of inhibition. Motile cells or flares that “brokeout” from the spots were carefully streaked out to isolate individual colonies and those cells were retested for relief of swarm inhibition. Once we confirmed their phenotype, an extra copy of the traA and traB genes were transformed into the mutant strains to test for complementation if the new mutations were in the traA or traB or different genes. The resulting transformed colonies were verified for their kanamycin resistance and retested for relief of swarm inhibition. From these experiments four positive mutants (swarm relief) were found out and sent for whole genome sequencing to identify the new gene(s) involved in this process. In contrast, 12 mutants were complemented by the traA plasmid. The identification of new players or genes will help give us a better mechanistic understanding of how cell-cell signaling regulates swarming behavior in M. xanthus. (1. Pathak, D.T. Wei, X. Bucuvalas, A. Haft, D.H. Gerloff, D.L. Wall, D. 2012.Cell contact dependent outer membrane exchange in myxobacteria: genetic determinants and mechanism. PLoS Genetics. 2012 Apr; 8(4): e1002626)