Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Microbiology, Molecular Biology and Biochemistry
Non Technical Summary
A clear understanding of the mechanism of phase variation in Myxococcus xanthus, a typical soil bacterium, is important because preliminary data from many labs indicate that phase variation plays a critical role in development and gliding motility, two processes that are important for survival of a microorganism in the environment. The motility defect of several of our mutants is manifest only when the cells are in the yellow phase but they can escape the limitations of their defects by going to the tan phase. The bias toward the yellow variants that has been going on for over 30 years, has undermined our ability to appreciate some of the novel attributes of gliding. As we investigate this process with an eye on motility, we expect to uncover the genetic bases for the specific differences between yellow and tan cells and to examine the mechanism of yellow-tan switching. The results we obtain will help us understand the roles that these two cell types play in motility and development. The proposed work promises to expand our understanding the molecular mechanisms underlying phase variation in a non-pathogenic bacterium and may shed new light on the origins of multiple cell-types.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We will test the hypothesis that differences in colonial morphology and gliding motility in Myxococcus xanthus during phase variation are due to a genomic perturbation such as recombinational switching that regulates the production of alternate sets of surface components. Objectives: The objectives for the period of the proposed work are to (1) complete the phenotypic characterization of phase-defective strains with mutations in genes predicted to encode recombinases and (2) determine which genes are differentially transcribed in the yellow and tan phases of Myxococcus vegetative growth. Expected outputs: Myxococcus xanthus cells can vary their phase to produce either yellow (pigmented) or tan (non-pigmented) colonies. In addition to pigment changes, yellow colonies differ greatly from tan colonies in their abilities to swarm and develop. Our study is aimed at identifying genes that contribute to and are regulated during this polymorphism and the processes that maintain an equilibrium between two cell types. Our interest in phase variation stems primarily from the observation that phase variation affects gliding motility. We anticipate this work will reveal links between motility and phase, and possibly explain the wholly unanticipated role of a recombinase in Adventurous motility.
Project Methods
Two recombinases have been identified and mutational analysis shows that they play a role in phase variation. We will determine if either recombinase mediates inversion by acting on nearby inverted repeats. We will map the invertible regions and complete the characterization of these mutants using PCR to look for evidence of inversion and confirm these results by Southern analysis. Standard PCR will be used to determine the orientation of DNA regions in WT tan and yellow strains. We expect to see products from both orientations, albeit at different levels. If our model is correct, the agmG and xerC mutants should give product for only one orientation. We will use semiquantitative RT-PCR to quantify the expression of nearby genes potentially affected by inversion in the agmG and xerC mutants compared with the WT. Some of our recombinase mutants affect motility and pigmentation. To determine if expression of genes for DKxanthine is affected in these strains, we will use oligos for dkxA as a marker for genes involved in yellow pigment production. We will determine the phenotypes of strains engineered to carry target sequences that are immune to the recombinase. We will compare the phenotypes of strains with mutations in genes predicted to encode recombinases. Independent xerC and agmG mutants (50 of each) will be screened for motility and development phenotypes using published methods. Standard assays include: swarm expansion on 0.3 and 1.5% agar, gliding rates and reversal frequency by videomicroscopy, extracellular complementation, biofilm formation, dye-binding (Congo red, calcofluor), growth rate, predataxis, starvation-induced fruiting body formation, and sporulation. We will determine if an alternate set of motility genes is transcribed during phase variation. We will take advantage of published microarray protocols that have been demonstrated to work with M. xanthus and related bacteria. Printed slides have been purchased from Michigan State Univ and will be used to hybridize to labeled cDNA prepared from RNA of yellow and tan phase cells. In order to account for variation due to growth phase, we will harvest RNA from early-log (Klett 50), mid-log, and stationary phase liquid-grown cultures. Each combination will be repeated three times to account for stochastic differences in gene expression resulting in a minimum of 18 analyses. mRNA will be treated with PAP (bacterial polyA polymerase) and ArrayScript (an efficient reverse transcriptase) will be used to make cDNA. Antisense cDNA, labeled with Cy3 and Cy5, will be prepared using RNAyellow and RNAtan and hybridized with slides containing oligonucleotides representing the annotated ORFs from the M. xanthus genome. Hybridization, analysis, interpretation and statistical analysis of data will be done in house. Noteworthy expression changes will be confirmed by real-time PCR. We will focus on studying significantly differentially expressed genes that also show a >3 fold increase or decrease in expression between yellow and tan variants.