General Laboratory Interests
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.
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
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
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.