Download Current Members are pictured (clockwise starting with the top row

Download this MSWord file
Current Members are pictured (clockwise starting with the top row, righthand
side):
Past Members: Michael Adedipe, Felix Bergara, Solange Bonilla, Lucia
Catalaa,
Edwin Covarrubias, Boni Cruz, William Estacio, Christian Ibarra, Julia
Iwamasa, Joni Jones, Michelle Lapuz, Jenette Manglicmot, Mary Mathieu, Ivan
Mitchell, Evelyn Monico, Eddie Navarette, Greg Nelson, Elise Olmsted, Juan
Carlos Patarroyo, Charlyn Primose, Julio Ramirez, Kristy Red-Horse, Sonia
Santa Anna-Arriola, Leslie Silva, Toni Piaggio, Heather Werhane, Joyce West,
and Emmy Ypil.
Student Research Projects
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.
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, Armano 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.
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.
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 cotranscribed 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 antisigma, 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
SFSU Home Search What's New Questions Survey Site Map
SFSU, 1600 Holloway Avenue, San Francisco, CA 94132
Last modified May 1, 2001, by the Web Team