Our lab focuses on understanding how Myxococcus xanthus senses nutrient limitation, and how this event initiates the developmental program. We have previously proposed a model whereby M. xanthus cells use their protein synthetic capacity to measure their nutritional status and (p)ppGpp, a signaling molecule known to couple amino acid availability with a variety of cellular processes in E. coli, acts as a second messenger in this process by activating a variety of starvation responses.
The initial starvation response occurs at the level of the individual cell, allowing each cell to evaluate its own nutritional status. Because M. xanthus is unable to utilize carbohydrates, cells primarily rely on amino acids to serve as carbon and energy sources, as well as substrates for protein synthesis. This relationship between basic metabolic need and the building blocks for protein synthesis presents an intriguing model for the mechanism of the cell to evaluate its nutritional status. Indeed, early physiology studies and our previous studies support our model that M. xanthus senses starvation by monitoring the intracellular level of the nucleotide (p)ppGpp. This model is very attractive as it provides a molecular link between metabolism and development of M. xanthus and remains our starting point to understand this complex sensory pathway.
The Geosmin Project
Project Lead: Eduardo Ruiz
Previous research has shown that M. xanthus secretes attractant molecules which cause prey to move towards the predatory swarm. We propose that one of these attractant molecules is geosmin as it is a volatile compound that is capable of attracting a variety of other organisms. We are currently testing the impact of geosmin in M. xanthus’ hunting strategy by performing knockouts of the geosmin synthase gene in wildtype and non-motile strains of M. xanthus and conducting hunting assays with the resulting mutants.
The Cyclase Project
Project Leads: Elizabeth Moore & Kayleen Lederman
Previous RNA surveys conducted by this lab revealed a significant upregulation in the transcription of 13 Cyclase genes found in Myxococcus xanthus throughout the development cycle. To better understand the effects of these compounds on multicellular fruiting body development, thirteen knockouts are being cloned into M. xanthus for a phenotypic comparison against wild type. We hope this study will prove enlightening to the development of multicellularity as a whole.
The Defensin Project
Project Leads: Bryant Law & Aditya Rao
Myxococcales are unusual amongst prokaryotes for their expression of defensin proteins, which was previously only known to be expressed in Eukaryotes as an antimicrobial. With an incomplete understanding of the role defensins play in humans, our lab studies the use of defensin in Myxococcales’ development and hunting to extrapolate the usage of the protein for both Myxococcales and humans. Finding characteristic defensin cystine pattern in several Myxococcales, our lab attempted to elicit the function and expression of defensins in M. fulvus B02, M. macrosporus HW-1, and M. xanthus DK1622 based on RNA seq. We will assess these genes by performing knockout mutations to assess the bacteria’s ability to hunt via hunting assays with strains based on a paper by Pham et al. and several growth assays. We hope learning and understand the role of defensins in Myxococcales’ physiology will help us to understand the evolution of myxobacteria and multicellularity as well as deepening out understanding of what defensin proteins can do.
Currently, there are four projects in the lab that focus on control of early developmental gene expression and these projects are currently funded by the National Institutes of Health, grant GM54592.
- The nsd gene and its role in nutrient-sensing.
Within the past few years several mutations have been identified that either alter the way cells perceive nutrient availability or alter the timing of development. We are currently focusing on the function of the nsd gene in nutrient sensing and its connection to (p)ppGpp accumulation. The Ω4469 Tn5lac developmental reporter defines a locus designated nsd, for nutrient sensing/utilization. This mutant can initiate development under nutrient-rich conditions, including 0.5% and 0.25% casitone conditions that support the growth of wild type cells. This suggests that nsd may be involved in nutrient sensing/utilization. In addition to determining the role of nsd in nutrient sensing/utilization, we are also interested in understanding how the expression of nsd is controlled by (p)ppGpp levels. We have taken two approaches. First, we have identified the nsd promoter region. Using primer extension and promoter deletions, we have identified a 100-bp region that contains promoter activity. The nsd promoter resembles a σ70-like promoter (sigma-90 [σ90] for M. xanthus) unlike all of the other Class I promoters, which have a σ54-like promoter structure.
- SdeK: its role in controlling developmental gene expression
Previous work from the lab has identified a Histidine Sensor Kinase (HSK), designated SdeK, essential for the control of early developmental gene expression. We found that SdeK acts in concert with the C-signaling pathway to activate developmental gene expression at the 6-hr stage of development. We previously showed that ΔsdeK1 cells were blocked in development approximately 6-hrs post-initiation based on expression studies using a battery of previously described Tn5lac fusions. Three types of fusions were identified: those partially dependent upon sdeK, those totally dependent upon sdeK, and one fusion partially activated in a ΔsdeK1 background. The first two types of fusions showed a similar dependence on C-signal, similar to that of sdeK, which led us to examine the relationship between C-signaling and the SdeK pathway. Preliminary data suggests that the SdeK and the C-signaling pathways independently contribute to the control of expression of these genes at the 6-hr juncture. The identification of SdeK as an HSK predicts the existence of a cognate Response Regulator (RR), which we have designated sdeR. We have been taking several approaches to identify SdeR, including genetic, biochemical and genomic approaches. With the release of the M. xanthus genomic sequence (through special arrangement with TIGR and Monsanto), we initiated a genomic approach to identify sdeR. The simplest hypothesis is that SdeR is a classic response regulator containing the highly conserved receiver domain. With this in mind, we are systematically constructing null alleles of genes encoding response regulators that could identify SdeR. This work is being done in collaboration with Dr. David Hodgson and Dr. David Whitworth from the University of Warwick, in the UK. Though our goal is to identify SdeR, this collaboration will allow us to identify a variety of potentially interesting two component systems. This is a long-term commitment between our labs to systematically, characterize the M. xanthus two-component systems. This approach has identified a response regulator involved in regulating developmental phosphatase activity.
- Identification and characterization of the direct regulators of the Class IA genes
To begin to understand how a rise in (p)ppGpp levels activates Class IA gene expression, we initiated a search for genes that alter their expression. Based on sequence analysis and primer extension experiments, all three of the Class IA genes have a σ54-type promoter. Because σ54-promoters require a highly conserved NtrC-like regulator for activation, we looked for NtrC-like activators that may affect Class IA gene expression. Approximately 53 ntrC-like genes have been identified. Insertion mutations were created in 37 of these and examined for developmental phenotypes (Caberoy et. al., 2003). In collaboration with the Garza lab, we characterizing the nla18 mutant using mRNA slot blots and real time RT-PCR to demonstrate that nla18 is in fact required for Class IA gene expression. However, our recent data suggests that Nla-18 acts upstream and affects (p)ppGpp accumulation. Our current model is that Nla18 is controlling, in part, RelA activation, because relA expression is not altered in the nla18 mutant.
- Chromosome status and M. xanthus development
Several years ago we became interested in examining the possible link between the cell cycle and fruiting body development in M. xanthus. It is evident that cell cycle cues are important in many developmental systems and because little work has been done on this intricate aspect of M. xanthus development in the last 25 years, we initiated a project to examine the role of the cell cycle in development. Because this was a new project it has taken some time to develop the techniques and obtain preliminary data to submit this work for funding from outside agencies. Initially, we investigated the link between the M. xanthus cell cycle and development using fluorescence activated cell sorting (FACS) to determine the DNA content of cells under various physiological states, along with quantitative fluorescence microscopy. Using established FACS protocols for examination of chromosome content in E. coli and Caulobacter crescentus we determined the number and distribution of chromosomes in M. xanthus during vegetative growth and during development. Based on previous studies, our data demonstrates that vegetatively growing M. xanthus cells contain 1-2 chromosome equivalents. This is the expected result of a single time-point taken from an asynchronous population. M. xanthus wild-type cells were allowed to undergo development for 3 days and the DNA content of the resulting spores was determined by FACS. We observed a single population corresponding to two chromosome equivalents.
The M. xanthus Microarray Consortium
The M. xanthus microarray consortium consists of 12 research laboratories dedicated to the construction use of the first M. xanthus DNA microarrays. This project is directly tied to the Monsanto/Cereon and TIGR initiatives to sequence and annotate the M. xanthus genome. We have already designed and printed the first generation arrays consisting of approximately 8,000 M. xanthus ORFs.
Refs: Caberoy NB, RD Welch, JS Jakobsen, SC Slater, and AG Garza. 2003. J Bacteriol 185(20): 6083-6094.