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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.
 
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B. subtilis Motility Group

The alternate sigma factor, SigD, initiates transcription of the genes for flagellin protein, the motility proteins, and several chemotaxis proteins.
The gene for sigma-D, sigD, is encoded in the fla/che operon. The fla/che operon is a >26 kb transcription unit that encodes the majority of the gene products that form the hook basal-body complex (the multi-component structure that tethers the flagellar filament to the bacterial cell), as well as several chemotaxis proteins. Two students that graduated from the laboratory, William Estacio and Michael Adedipe, studied the promoter region of this large operon, and a paper was published (Estacio, et. al.). Subsequently, a post-doctoral fellow, Dr. Joyce West, studied the relative roles of the three known promoters for this operon resulting in a second paper (West, et.al.).

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.
Interestingly, flagellin protein expression increases coincidentally with the initiation of sporulation. We therefore postulated that expression of the gene for flagellin, hag, is controlled by nutrient deprivation and is dependent on some of the same transcriptional regulators and signal-transducing proteins that initiate sporulation. Graduated Master's students Felix Bergara and Elise Olmsted, and past undergraduates Christian
Ibarra, Julia Iwamasa, Mary Mathieu, Juan Carlos Patarroyo, and Julio Ramirez monitored the expression of reporter constructs in different environments and
mutant backgrounds. These studies determined the nature of the nutritional signals and the role of several regulatory proteins in governing expression of the fla/che operon and the hag gene. A paper has been published on this work (Mirel, et. al.) and another is in revision (Bergara, et. al.). Significantly, the second paper demonstrates that fla/che and hag are members
of the CodY regulon.

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.

 
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Paenibacillus Group As 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.
 
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B. subtilis SigY Group

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.

Carla Bonilla, a senior Master's student in the laboratory has demonstrated in collaboration with the Helmann lab at Cornell University that the sigY
gene product, SigY, binds to RNA polymerase and specifically initiates transcription from a template bearing the sigY promoter region. This result provides the first biochemical evidence of SigY activity as a sigma factor.
Further, this result suggests that the sigY gene may be autotranscribed, a feature conserved amongst the structural genes for ECF sigma factors. Again in collaboration with the Helmann lab, she has exploited the biochemical activity of SigY in Run-Off Macroarray Analyses (ROMA) to define genes that may be dependent on the SigY-holoenzyme for expression, and therefore comprise the SigY regulon. Preliminary results suggest that the SigY regulon may consist of genes involved in adaptation to nutrient stress. We have found that a SigY-dependent reporter construct is only expressed in minimal medium, suggesting that nutritional signals govern expression of the SigY regulon that in turn may play a role in adaptation to nutrient limiting conditions.
The current model for ECF sigma function predicts inhibition of SigY activity by an anti-sigma factor encoded within the sigY operon. We postulate that the yxlC gene that is adjacent to sigY, and appears to be co-transcribed with the sigma factor, encodes the cognate anti-sigma factor. A Master's student in the laboratory, Matt Mendel, is studying the mechanism of YxlC function and he is joined in this effort by a new undergraduate in the laboratory, David Bayless. Very little is known about the molecular mechanisms that govern inhibition of ECF sigma function by the cognate anti-sigma, and yet it is clear that this regulatory system is ubiquitous to the bacterial world. Matt and David plan on using genetic, biochemical, and cellular approaches to dissect the putative SigY/YxlC interaction which promises to yield important information about this type of regulation. Finally, a new Master's student in the laboratory, Noe Gomez, is studying the role of other gene products encoded in the sigY operon on SigY function. Significantly, the predicted protein products for the other genes found in this operon appear to con

 

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Márquez-Magaña Lab.
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Last modified September 2003