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Myxococcus xanthus, referred to as a 'social bacterium', demonstrates unique behaviors such as coordinated motility, cooperative feeding, and multicellular structure formation. Its complex social behaviors and developmental processes make M. xanthus a model organism for studying bacterial social behaviors and their underlying mechanisms. Much of the social behavior of M. xanthus hinges on coordination of cell motility among bacteria in close proximity. M. xanthus moves on moist solid surfaces, using its Adventurous (A)-motility and Social (S)-motility systems. A striking feature of M. xanthus motility is the periodic reversal of its direction of movement. The reversal frequency is influenced by chemical and mechanical cues in the surrounding environment. The modulation of the reversal frequency upon physical contact between cells is believed to be a key factor in the bacterium's social behaviors, especially in the formation of complex patterns and structures within the cell population. Here I utilized mathematical modeling to study the motility regulation in M. xanthus, focusing on contact-dependent reversal control, mechanosensing response and impact of motility regulation in solitary (single-cell) predation. My goal is to provide experiment-guiding theories and hypotheses for M. xanthus motility regulation, which is essential to fully understand the social behaviors in this bacterium. In Chapter 2, I developed a single-cell model based on a hypothesis that the motility regulation in M. xanthus is mediated by the interplay between the cell polarity regulation pathway and the A-motility machinery. The aim of this model is to elucidate the cellular mechanism governing contact-dependent motility coordination among cells and to understand how contact-dependent responses at the single-cell level contribute to population-level patterns. This model suggests that the A-motility machinery of M. xanthus potentially serves as a 'mechanosensor' that transduces mechanical cues in the environment into a reversal modulation signal. Chapter 3 addresses a puzzling observation: cells with A-motility alone (A+S−) show a dependence of reversal frequency on substrate stiffness that is opposite to what is observed in wild-type cells that possess both motility systems. Specifically, A+S− cells reverse less frequently on harder substrates, whereas wild-type cells reverse more frequently. To elucidate this perplexing phenomenon, I refined the single-cell model developed in Chapter 2 to study the mechanosensing behaviors with or without S-motility. The base model was sufficient to explain the mechanosensing response in A+S− cells. I then proposed possible interactions between the A-motility and S-motility systems that could explain the contrasting responses to substrate stiffness when S-motility is present or absent. This provides a testable prediction for future experimental investigations. The model suggests that the A-motility system in M. xanthus functions as a central hub of mechanosensing-based reversal control, modulating cell reversal in response to environmental mechanical cues. In Chapter 4, I constructed an agent-based model to investigate the optimal motility strategies for nutrient consumption by M. xanthus during its solitary predation. For different nutrient source types and their uptake latencies, the model identifies 'explore', 'inch', and 'fast explore' as the three most effective motility strategies. Variability in velocity and cell reversal period changes the optimal strategies from 'explore' mode to 'revisit' mode and to 'speed-controlled explore' mode, respectively, for massive remains of prey nutrient sources with moderate uptake latency. The experimental observation that solitary M. xanthus cells combined the 'revisit' and 'inch' mode—as predicted by the model for nutrient acquisition respectively from prey remains and macromolecules—suggests that some of the dead preys may not release its cellular contents immediately and that release of molecular nutrients may require multiple digestion cycles. This model provides insights into the functional role of complex motility regulation in M. xanthus during solitary predation. / Doctor of Philosophy / A fundamental question in biology is how a cell responds to physical, chemical and biological stimuli. Such responses are usually mediated by complex coupling between multiple cellular processes. Bacterial motility and its regulation present many excellent examples of this kind. This dissertation focuses on Myxococcus xanthus, a model organism for bacterial social behavior due to the highly coordinated motility of cells in M. xanthus colonies and their functional cooperation. In this dissertation, I built theoretical models to study the motility regulation in M. xanthus, which is essential for understanding the social behaviors and survival in this bacterium. The specific focuses are to comprehend how environmental mechanical cues regulate M. xanthus's motility, and how the observed motility regulation in M. xanthus facilitates its predatory behavior at the single-cell level. The key aspect of this work is to construct a modeling framework to provide coherent explanations for the experimental observations. It is anticipated that the hypotheses generated through modeling will guide new experiments in the field of myxobacterial biology. The findings offer general insights into how bacterial cells sense, respond, and adapt to the chemical, physical, and biological cues.

Partner-switching regulatory systems (PSRSs) are utilized by many different bacteria to regulate a wide array of cellular responses, from stress response to expression of virulence factors. The filamentous cyanobacterium Nostoc punctiforme can transiently differentiate motile filaments, called hormogonia, in response to various changes in the environment. Hormogonia utilize a Type IV pilus (T4P) complex in conjunction with a secreted polysaccharide for gliding motility along solid surfaces. This study identified three genes, designated hmpU, hmpW, and hmpV, encoding the protein components of a PSRS involved in regulation of hormogonium motility in N. punctiforme. Although mutant strains with in-frame deletions in hmpU, hmpW, and hmpV differentiated morphologically distinct hormogonium-like filaments, further phenotypic analysis demonstrated significant distinctions among the strains. The ∆hmpW strain contained a higher percentage of motile filaments that moved faster than the wild-type strain, while the ∆hmpU and ∆hmpV strains consisted of fewer motile filaments that moved at a slower rate compared to wild type. Immunoblotting and immunofluorescence of PilA, the major component of the pilus in the T4P system, showed that although all mutant strains appeared to express similar levels of PilA protein, the ∆hmpU and ∆hmpV strains displayed reduced extracellular PilA. Lectin blotting and staining with fluorescently-labeled UEA lectin demonstrated a decrease in extracellular hormogonium polysaccharide in the ∆hmpU and ∆hmpV strains, consistent with the current understanding that the polysaccharide is secreted via the T4P system. Epistasis analysis demonstrated that the ∆hmpW, ∆hmpV double-deletion mutant strain displayed reduced spreading in plate motility assays, similar to the ∆hmpV single mutant. Together, these results support a model in which the HmpU phosphatase and HmpW serine kinase control the phosphorylation state of the HmpV protein, modulating its activity on a downstream target to ultimately promote activation of the T4P motor complex and enhance hormogonium motility.

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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.