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|We are studying the genetic elements that control the motility behavior in Bacillus subtilis and Paenibacillus, as well as the role of a newly defined ECF sigma factor, SigY, in B. subtilis. The laboratory is therefore divided into three sub-groups. Members of the B. subtilis motility group are dissecting the molecular nature of the fla/che operon and structural regulation of motility gene expression by the anti-sigma factor, FlgM. Individuals studying swarming in Paenibacillus are using an integrative approach to understand the cell biology, physiology, and genetic regulation of this surface-based motility. Finally, students investigating the role of SigY in B. subtilis are studying its activation in the bacterial cell as well as defining target genes that comprise its regulon.|
The alternate sigma
factor, SigD, initiates transcription of the genes for flagellin protein,
the motility proteins, and several chemotaxis proteins.
Our attention has
turned to identifying an additional promoter(s) in the fla/che operon,
understanding the regulation of its upstream promoters, and studying its
3' end. A new undergraduate in the laboratory, Kelechi Uwaezuoke, is amplifying
large intergenic regions within fla/che so as to introduce them into a
reporter plasmid and thereby localize the additional promoter(s). A senior
undergraduate, Armando Lemus, is studying the importance of anti-termination
regulation in governing fla/che expression. His work promises to provide
evidence of a novel mechanism for controlling the expression of very large
operons in B. subtilis. Heather Werhane, a Master's student that
graduated from the laboratory, utilized RNA protection assay to map the
3' end of the operon. Heather demonstrated that theyxlL gene, which is
immediately downstream of sigD, is the last gene in the fla/che operon
and appears to be involved in the control of SigD activity. Mattew Mendel,
a current Master's student, has completed control experiments for this
study and a manuscript is in preparation.
Further, we are interested
in the environmental signals that control expression of the fla/che operon
and other motility genes. B. subtilis is a developmental organism
that sporulates upon nutrient deprivation.
Finally, we are studying control of flagellin gene expression by the anti-sigma factor, FlgM. The hag gene in B. subtilis is expressed only when a functional hook basal-body (HBB) complex has been formed. This morphogenetic regulation of hag gene expression is controlled by the anti-sigma factor, FlgM that appears to sequester SigD in the cell. A similar regulatory mechanism exists in the enteric bacteria and FlgM appears to be inactivated by its specific export through the HBB. However, the B. subtilis FlgM protein has not been localized outside the cell, and it appears to function as part of a switch between motility functions and the development of natural competence. A new Master's student in the laboratory, Pete Lopez, is studying the molecular mechanism of FlgM control.
part of another study, current Master's student Carla Bonilla identified
a Paenibacillus strain that appeared to either co-exists or arises
from our wild-type B. subtilis. Sylvia Olano, a research associate
in the laboratory, has obtained results that suggests that the organism
co-exists and on-going work is aimed at understanding the cellular basis
for this interaction as
well as the biological relevance of this finding. Peter Ingmire, another research associate in the lab, has written a proposal to sequence the genome of the co-existing Paenibacillus that will facilitate genetic analyses of the organism.
Unlike B. subtilis, the co-existing Paenibacillus displays a sunburst pattern formation on agar plates, consistent with swarming motility. Through live-imaging an undergraduate in the laboratory, Audrey Parangan, has determined that the pattern formation is consistent with a previously described vortex morphotype characterized by cooperative behavior of
individual cells to elicit rotating migration of the entire colony. Further, she has determined that this Paenibacillus is resistant to multiple antibiotics on solid media and is not naturally competent for transformation making it unsuitable for genetic manipulation. Audrey plans to continue her work in the lab as a Master's student this fall and will work towards developing a technique for transforming the Paenibacillus. In this way we hope to better understand the molecular mechanisms that control swarming motility in this organism, and its cellular interaction with B. subtilis.
In Bacillus subtilis,
ten alternative sð factors have been studied and play a role in the
control of sporulation, motility, nutrient uptake, and some stress responses.
The sequence of the B. subtilis genome was recently completed and
genes for seven additional alternative sigma factors identified based
on the homology of their predicted protein products to a new family of
sigma factors. Members of this new family of extracytoplasmic (ECF) sigmas
regulate functions related to the cell membrane, periplasm, or cell wall
such as the heat shock response and antibiotic resistance. One of the
putative sigma factor genes identified, sigY, is the focus of our work.
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Last modified September 2003