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. / Master of Science

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science

Myxococcus xanthus, a Gram-negative bacterium with a developmental cycle, displays a type IV pili (TFP) mediated surface motility known as social (S) gliding. Beside the polarly localized TFP, the fibril or extracellular polysaccharide (EPS) is also required for S-motility to function. It is proposed that S-motility, along with the related bacterial twitching motility in other species, is powered by TFP retraction. EPS is proposed to anchor and trigger such retractions in M. xanthus. EPS production is known to be regulated by TFP and the Dif signal transduction pathway. Two genetic screens were performed previously to identify additional genes important for EPS production. The first was for the isolation of pilA suppressors, the second for the identification of mutants underproducing EPS in a difA suppressor background. Both screens identified transposon insertions at the stk locus. In particular, StkA, a DnaK homolog, was identified as a possible negative regulator of EPS production by a stkA transposon insertion that suppressed a pilA mutation. A stkB transposon insertion was found to have diminished EPS production in a difA suppressor background. In this study, in-frame deletion mutants of the five genes at the stk locus, stkY, stkZ, stkA, stkB and stkC, were constructed and examined. In addition, mutations of rbp and bskL, two genes downstream of the stk locus, were constructed. Like transposon insertions, the stkA in-frame deletion resulted in overproduction of EPS. The stkB and to a less extent the stkC mutants underproduced EPS. Mutations in the other genes had no obvious effects on EPS production. Genetic epistasis suggests that StkA functions downstream of TFP and upstream of the Dif sensory proteins in EPS regulation in M. xanthus. Epistasis analysis involving stkB was inconclusive. It is unresolved whether StkB plays a role in the biosynthesis or the regulation of EPS production in M. xanthus. / Master of Science

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. / Ph. D.

The Myxococcus xanthus Dif and Frz chemosensory pathways play important roles in the regulation of gliding motility. The Dif system regulates the production of exopolysaccheride (EPS), which is essential for social motility and fruiting body formation. The Frz pathway controls reversal frequency, which is fundamental for directed movement by this surface-gliding bacterium. In addition, both pathways are involved in the chemotactic response towards several phosphatidylethanolamine (PE) species such that the Dif pathway is required for excitation while the Frz pathway is essential for adaptation. In this study we addressed three crucial questions regarding the signal processing of these two chemosensory pathways by focusing on DifA and FrzCD, the MCP homologs from their respective pathways. First, the receptor protein in the Dif pathway, DifA, lacks a perisplasmic domain, the typical signal-sensing structure. To examine whether DifA shares similar transmembrane signaling mechanism with typical transmembrane sensor proteins (MCPs and sensor kinases), we constructed a chimeric protein that is composed of the N-terminus of NarX (nitrate sensor kinase) and the C-terminus of DifA. This NarX-DifA chimera restores the DifA functionality (EPS production, agglutination, S-motility and development) to a "difA mutant in a nitrate-dependent manner, suggesting DifA shares a similar transmembrane signaling mechanism with typical MCPs and sensor kinases despite its unorthodox structure. Second, the M. xanthus chemotaxis is still controversial. It has been argued that the taxis-like response in this slowly gliding bacterium could result from physiological effects of certain chemicals. To study motility regulation by the Frz pathway, we constructed two chimeras between the N-terminus of NarX and C-terminus of FrzCD, which is the receptor protein of the Frz pathway. The two chimeras, NazDF and NazDR, are identical except that NazDR contains a G51R mutation in the otherwise wild-type NarX sensory module. This G51R mutation was shown to reverse the signaling output of a NarX-Tar chimera to nitrate. We discovered that nitrate specifically decreased the reversal frequency of NazDF-expressing cells and increased that of NazDR-expressing cells. These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazDR strain failed to adapt to nitrate in temporal assays, as did the wild type to known repellents. Therefore, the lack of temporal adaptation to negative stimuli is an intrinsic property in M. xanthus motility regulation. Third, the Dif and Frz pathways are both involved in the chemotactic response towards certain PE molecules such that the Dif pathway is required for excitation and while the Frz system is essential for adaptation. In addition, 12:0 PE, known to be sensed by DifA, results in increased FrzCD methylation. These findings suggested that in the regulation of PE response, two pathways communicate with each other to mediate adaptation. Here we provided evidence to indicate that DifA does not undergo methylation during EPS regulation and PE chemotaxis. On the other hand, using mutants expressing the NarX-DifA chimera, it was found that signal transduction through DifA, DifC (CheW-like) and DifE (CheA-like) modulates FrzCD methylation. Surprisingly, the attractant 12:0 PE can modulate FrzCD methylation in two ways distinguishable by the dependency on DifA, DifC and DifE. The DifACE-independent mechanism, which may result from specific sensing of 12:0 PE by FrzCD, increases FrzCD methylation as expected. Unexpectedly, 12:0 PE decreases FrzCD methylation with the DifACE-dependent mechanism. This "opposite" FrzCD methylation by DifACE-dependent signaling was supported by results from NafA-expressing mutants because nitrate, which acts as a repellent, increases FrzCD methylation. Based on these findings, we proposed a model for chemotaxis toward 12:0 PE (and 16:1 PE). In this model, DifA and FrzCD both sense the same signal and activate the pathways of excitation (Dif) and adaptation (Frz) independently. The two pathways communicate with each other via methylation crosstalk between DifACE and FrzCD in such a way that processes of excitation and adaptation can be coordinated. / Ph. D.