1. A developmental
checkpoint regulated by replication status and DNA damage in Bacillus subtilis.
Bill Burkholder
Cells of Bacillus subtilis can respond to starvation by developing into stress
resistant spores. Spore morphogenesis begins with an asymmetric cell division,
yielding two daughter cells that are both required for the production of a
mature spore. To ensure that both daughter cells receive a complete copy of the
genome, DNA-dependent signals regulate the decision to sporulate. Defects in
replication initiation, replication elongation, or DNA damage inhibit the
initiation of sporulation by preventing activation of a transcription factor
required for sporulation, Spo0A. Spo0A is normally activated during sporulation
by two histidine kinases, KinA and KinB. Using genetic and biochemical
approaches, we found that replication defects and DNA damage inhibit
sporulation by activating transcription of a small open reading frame, sda, that
encodes an inhibitor of KinA and KinB. Transcription of sda is directly
regulated by the replication initiation factor DnaA, which activates
transcription in response to replication defects, and the transcriptional
repressor LexA, which is derepressed in response to DNA damage. Our lab is
currently focusing on three sets of questions: 1) How DnaA coordinates sda
expression with the replication cycle, 2) How Sda specifically recognizes and
inhibits its target kinases, and 3) How the Sda pathway contributes to cell
survival and genomic stability. We will also seek to identify and characterize
other signaling pathways that monitor DNA replication and repair to regulate
growth and development.
2.
Gene expression and comparative genomic studies using a 'pan-Bordetella' DNA
microarray
Craig A. Cummings, Mary Brinig, Simone van de
Pas, Hester Bootsma, Jeff Miller, and David Relman
The closely related bacteria of the genus Bordetella cause respiratory disease in
a wide range of animal hosts. In order
to probe the pathogenesis and phylogeny of these pathogens, we have developed a
DNA microarray for non-redundant representation of three sequenced Bordetella genomes: B. pertussis, B.
bronchiseptica, and B. parapertussis.
Comparative genome hybridization to this microarray
of 42 Bordetella strains confirmed the high degree of sequence conservation
between these three species but also identified numerous regions of difference
(RDs) between them. The RDs are
composed of long, contiguous sequences, and are often species-specific. A few RDs may represent hypervariable
regions within the genome. The
distribution of RDs provides a basis for assessing the evolutionary history of
the genus and suggests mechanisms of differential pathogenicity.
The microarray was also used to study global gene
expression responses to manipulation of the master virulence regulator, BvgAS,
in B. pertussis and B. bronchiseptica. Analysis of bvgS mutant strains and cultures
modulated by the addition of nicotinic acid revealed complex transcriptional
regulation of known virulence factors and identified dozens of previously
unknown BvgAS-regulated genes.
Furthermore, comparison of B.
pertussis and B. bronchiseptica
responses revealed numerous species-specific responses. Because all previously described BvgAS
targets contribute to pathogenesis, we propose that some of these novel,
co-regulated genes may also represent virulence determinants.
3. Molecular aspects of
acclimation of the unicellular alga Chlamydomonas
reinhardtii to sulfur limitation conditions.
Arthur Grossman, Zhaoduo Zhang, Chiung-Wen
Chang and Jeffrey Moseley.
In order to compete for limiting resources,
organisms must cope or acclimate to a continually fluctuating nutrient
environment. These acclimation
processes involve responses ‘specific’ for the limiting nutrient as well as
responses more ‘general’ and that occur when the organism experiences any of a
number of different stress conditions.
The specific responses enable organisms to efficiently scavenge the
limiting nutrient and may involve the induction of high-affinity transport
systems and the synthesis of hydrolytic enzymes that facilitate the release of
nutrients from extracellular organic molecules or from internal reserves. General nutrient limitation responses can
involve changes in the rate of cell division and global alterations in
metabolic activities. In photosynthetic
organisms there must be precise regulation of photosynthetic activity since
when severe nutrient limitation prevents continued cell growth, excitation of
pigment molecules associated with the photosynthetic apparatus could elicit the
formation of reactive oxygen species, which can severely damage structural and
functional features of the cell. In this presentation I will discuss the many
ways in which the unicellular eukaryotic alga Chlamydomonas reinhardtii acclimates to nutrient stress conditions
and describe some of the regulatory elements that enable this organism to
successfully compete as the nutrient concentrations in the environment become
limiting.
4. Fitness cost of resistance to triclosan in Escherichia coli
Clara L. Davis, Ian M. Ehrenreich, Brendan
J.M. Bohannan
Triclosan is an antimicrobial compound that is currently incorporated into a variety of consumer goods. Triclosan resistant mutants have been isolated, raising concerns about resistant strains in the environment, the future efficacy of triclosan, and even the possibility of resistance to clinical drugs. Antibiotic resistance is frequently accompanied by reduced competitive fitness; this trade-off between competitive ability and drug resistance has not been previously examined for triclosan-resistant isolates.
We isolated 45 spontaneous triclosan resistant
mutants of E. coli using gradient
plates of 0-3 ug/ml triclosan. We
quantified their competitive fitness in batch competition assays with the
immediate ancestor and their level of resistance (MIC) to triclosan. To study compensation to the observed
fitness costs, we evolved four of these mutants in batch culture for 24 days
both in media containing sub-MIC levels (0.15 ug/ml) of triclosan, and in media
without triclosan, to determine if the cost could be ameliorated by continued
evolution. Each of the four strains was
replicated three times (12 lineages total per treatment). Fitness levels were variable among the
resistant isolates, ranging from 83% to 100% of wild type. MIC ranged from 0.6-12.2 ug/ml. After
evolution for 24 days without triclosan, a 3% to 32% increase was
observed. After evolution in the
presence of triclosan, 9 of 12 lineages increased in fitness and three
decreased in fitness. The finding of
mutants that carry no cost to resistance implies that such resistant strains
could persist in natural populations, even without the constant presence of
triclosan as a selective agent. The
results of the compensation experiment indicate that amelioration to the cost
of triclosan resistance can occur. There was no apparent relationship between
MIC and fitness level, implying that there is not a direct trade-off between
level of resistance and cost of resistance.
5. The Role of Cellular
Autophagy in Viral Infection
William T. Jackson, Thomas H. Giddings Jr. and
Karla Kirkegaard
Infection by poliovirus, a positive-strand RNA
virus, induces rearrangements of intracellular membranes. In particular, unique 200-400 nm vesicles
are formed, the surfaces of which are used as the site of viral RNA
replication. Ultrastructural and
buoyant-density analyses have demonstrated that the vesicles are
double-membraned and derived from the ER, yet they display cytoplasmic contents
and stain with the lysosomal marker LAMP-1.
These features are similar to those of vacuoles involved in autophagy, a
process in which cellular components are digested in bulk in response to
nutrient deprivation. Whether these
structures are the site of viral RNA replication or part of the host antiviral
response was investigated. Here we
demonstrate that monodansylcadaverine (MDC), a known fluorescent marker for
autophagic vesicles, stains poliovirus- and rhinovirus- infected cells
similarly to cells undergoing autophagy.
MDC co-localizes with the poliovirus protein 3A, part of the viral RNA
replication complex. Both MDC and 3A
also co-localize with the autophagy marker protein LC3 (human Apg8p.) We therefore hypothesize that poliovirus
induces cellular autophagy to create double-membraned vesicles which act as
scaffolds for viral RNA replication.
How the potentially antiviral function of cellular autophagy is
subverted during infection by positive-strand RNA viruses is under
investigation.
6. Phototaxis in the Cyanobacterium Chocystis sp.: The Role
of Type IV Pili and Chemotaxis-like Proteins
Devaki Bhaya
Both filamentous and unicellular species of
cyanobacteria exhibit phototaxis, yet the mechanism and regulation of this
process is not well understood. In an
environment where both light and nutrient levels can fluctuate the ability to
migrate into a favourable niche or move away from a sub-optimal situation may
be critical for the survival of these ubiquitous species. We have examined the mechanism of motility
in the unicellular gliding cyanobacterium Synechocystis
sp. PCC6803 and shown that it requires Type IV pili. Knock-out mutants of
pilA (encoding pilin), pilD (signal peptidase), pilC ( pilus biogenesis/
assembly) and pilT (ATPase with possible motor function) are non-motile. To study signal transduction during
phototaxis we have isolated several mutants with an aberrant phototactic
response (i.e they are non-motile or constitutively exhibit negative
phototaxis). Many of these mutants
mapped to che-like genes. Synechocystis sp has three loci
containing che-like genes; all three of these (tax1, tax2 andtax3 loci) play a
role in phototaxis. Mutants in the tax1
locus are negatively phototactic while mutants in tax3 are non-motile. Our analysis of some of these mutants (using
a variety of techniques that include electron microscopy, time-lapse video
microscopy and protein-tagging technology) will be presented along with our
working model of the signal transduction network that controls phototactic
movement.
7. Stem Cells and Gene Therapy
Microbiological and Immunological Issues
Helen M. Blau
Nuclear transplantation and cell fusion studies
first challenged the dogma that the differentiated state was fixed and
irreversible. Recently, there have been
reports by us and others of additional plasticity of cell fate that may occur
naturally. Like others, we have found
that adult bone marrow-derived cells (BMDC) contribute to diverse adult tissues,
such as brain and brawn at a low frequency.
Nonetheless, the cells derived from adult bone marrow clearly can give
rise to complex mature neurons, Purkinje cells. Studies in our laboratory show that this even occurs in adult
brains of humans. In addition, bone
marrow-derived cells can contribute to muscle fibers at a high frequency in
adult mice. Indeed, we have found that
BMDC contribute to muscles at different frequencies that range from 0.001 to
5%, presumably due to biological differences in these muscles. In recent studies in our laboratory we have
shown that BMDC contribute to muscle tissue in a step-wise biological
progression in response to damage.
Following irradiation-induced damage, transplanted BMDC become satellite
cells: membrane ensheathed mononucleate
muscle-stem-cells. Following a
subsequent exercise-induced damage, multinucleate myofibers are detected. Isolated bone marrow-derived satellite cells
are heritably myogenic and diploid.
These results suggest that two temporally distinct injury related
signals first induce BMDC to occupy the muscle-stem-cell niche, and then to
help regenerate as many as 3.5% of mature muscle fibers, a robust
response. Thus, developmental
plasticity of BMDC is pronounced in response to environmental cues related to
injury. Immunological effectors that
are likely to play a major role remain to be elucidated and are the subject of
major interest to our laboratory. In addition,
vectors that are based on viruses will be used for gene delivery.
8. The yeast casein kinase I homolog Hrr25 phosphorylates and negatively regulates the Crz1 transcription factor
Kim Kafadar, Heng Zhu, Michael Snyder,
Martha Cyert
Calcineurin
is a calcium/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a
variety of environmental stresses. Calcineurin affects cellular responses to
stress by dephosphorylating and thus activating the Zn-finger transcription
factor Crz1p. Upon activation, Crz1p translocates to the nucleus where it
promotes gene transcription. Although much is known regarding the mechanism of
Crz1p dephosphorylation by calcineurin, the kinase(s) responsible for
phosphorylation of Crz1p have not been identified. Using a recently developed
assay that employs protein chips, we have tested 120 yeast kinases for their
ability to phosphorylate Crz1p in vitro. Several kinases were identified and we
have further characterized one of these, the casein kinase I homolog Hrr25p.
Hrr25p is an essential protein that has been shown to be involved in DNA
repair. Here we show that Hrr25p interacts with and phosphorylates Crz1p in
vivo. Interestingly, the interaction between Hrr25p and Crz1p does not depend
on the kinase activity of Hrr25p. Studies are underway to further map this
interaction. In addition, overexpression of HRR25 inhibits Crz1p-dependent
transcription, consistent with its role as a negative regulator of Crz1p. The
effects of its overexpression on Crz1p localization are currently being
investigated. We are also examining the consequences of Hrr25p depletion on
Crz1p activity, phosphorylation, and localization.
9. Genome-wide comparisons
between E. histolytica and E. dispar using microarray analysis.
Preetam Shah, Brendan Loftus, Upinder Singh
We are generating a DNA microarray from Entamoeba histolytica random sheared
genomic library which is currently being sequenced at TIGR and Sanger centers.
The E. histolytica genome has short
intergenic regions and introns making genomic arrays a feasible approach. We
are using the array to identify genetic differences between pathogenic E. histolytica and the closely related
non-pathogenic species E. dispar.
Although several efforts have been made to identify determinants which makes E. histolytica virulent compared to E. dispar, a genetic basis for the
variation in pathogenesis has not been described. Our goal is to identify
genomic clones that have a low genomic abundance or are missing in E. dispar compared to E. histolytica. Our analysis is based on
relative hybridization with equal amounts of flurophore labeled genomic DNA
from E. histolytica and E. dispar. Approximately 6% of the
clones on our array show low E. dispar
/ E. histolytica genomic abundance.
Genes such as Ariel1 and CP1, that have been shown to be absent in E. dispar, were accurately identified as
being absent in E. dispar using the arrays.
The validity of this approach has been tested using Northern blots and confirms
the presence of novel genes that are specific to E. histolytica. This approach represents the first genome-wide
comparison of E. histolytica and E. dispar and promises to unveil novel
aspects of parasite virulence.
Molecular Approaches to
Bioremediation of Hexavalent Chromium
D. Ackerley, C. Gonzalez, C-H. Park, M.
Keyhan, and A. Matin
As a consequence of its toxicity, the accumulation
of chromate [Cr(VI)] in drinking water poses a serious risk to human
health. Conventional approaches to
groundwater remediation and the regeneration of potable water supplies are
extremely expensive and have, to date, been very limited in their effect. From a biological perspective chromium (III)
compounds, the end products of bacterial chromate reduction, are far more
benign; not only are they much less toxic than chromate, they also have very
low solubility, restricting their spread and their biological
availability. Thus, reduction of
chromate by bacteria is an attractive strategy for its remediation. Through biomolecular engineering, it should
be possible to improve the chromate-reducing activities of wild type bacterial
enzymes, and to produce bacteria capable of expressing such activities at a
high level under nutrient-poor and stressful field conditions. With this goal in mind we have identified,
purified, and characterized chromate-reducing enzymes from a variety of
bacteria. The results presented here
indicate not only that these enzymes are efficient reducers of Cr(VI) to
Cr(III) in vitro, but that they also play an important role in chromate
detoxification in vivo. Having
identified such enzymes, the way is now clear for biomolecular engineering
studies aimed at enhancing their chromate-reducing activity and effect. Ongoing studies indicate that these proteins
may also play a role in countering oxidative stress, and this aspect of their
activity is also under investigation.
The relationship among plant
communities, microbial communities, and carbon mineralization in a tropical
soil.
K.M. Carney and P.A. Matson
Soil microbial communities mediate many critical
ecosystem processes. Little is known,
however, about the factors that determine microbial community composition, and
whether community composition influences process rates. Using an experimental
site at La Selva Biological Station, Costa Rica, we are examining whether plant
diversity and community composition and land-use alter soil microbial
communities, and whether microbial community differences can influence
transformations of carbon and nitrogen in soils. Our results from phospholipid
fatty acid analyses indicated that microbial community composition changed
along a plant diversity gradient and between local land-use types. We tested
whether such changes influence the decomposition of labile carbon amendments to
soil. We assessed the catabolic potential of soil communities using a substrate
induced respiration assay that examines differences in responses of soil
microbial communities to 24 labile carbon compounds. Soil catabolic potential changed significantly across plant
diversity and land-use gradients, and, in fact, closely mirrored the observed
shifts in soil microbial community composition. Additionally, soil catabolic evenness was highest in plots with
the highest levels of plant diversity.
We also examined whether microbial community differences could be linked
to litter decomposition rates using a laboratory-based litter transplant
experiment. The experiment demonstrated
a significant relationship between litter decomposition rate and microbial
community composition.
Sophistication in bacterial
gene regulation: Tandem transcriptional and translational sensing of tryptophan
transfer RNA regulates synthesis of the Anti-TRAP protein of B. subtilis
Guang-nan Chen and Charles Yanofsky
Expression of the structural genes of the rtpA-ycbK
operon of Bacillus subtilis is
induced by uncharged tRNATrp acting at tandem segments of the leader transcript
to promote transcription anti-termination and translation initiation. The
leader region of this operon has all the features of operons regulated by an
uncharged tRNA by the T-box transcription attenuation mechanism. In addition,
immediately preceding the rtpA gene there is a coding region, rtpLP, for a ten
residue leader peptide that contain three consecutive tryptophan residues. Here
we show that the presence of rtpLP allows uncharged tRNATrp to regulate
translation of rtpA. Thus, when cells have adequate levels of charged tRNATrp
for protein synthesis, the ribosome translating the rtpLP coding region appears
to block or reduce translation initiation at the rtpA Shine/Dalgarno sequence.
However, when cells are deficient in charged tRNATrp, the ribosome translating
the rtpLP coding region presumably stalls over one of the three tryptophan
codons, freeing the rtpA Shine/Dalgarno region, resulting in efficient
translation of rtpA. The high level of AT produced inactivates the TRAP
protein, leading to transcription of the trp operon, and synthesis of all the
tryptophan biosynthetic enzymes and additional tryptophan.
Genome-wide prokaryotic
codon bias is determined by GC content and organism growth temperature
Swaine L Chen, William Lee, Alison
Hottes, Lucy Shapiro, Harley H McAdams
The concept of nonrandom codon usage, or codon bias,
as a consequence of translation is important for an understanding of molecular
diversity and evolution. The
availability of a large number of sequenced bacterial and archaeal genomes now
provides the opportunity to determine the genome-wide constraints on codon
bias. We have discovered that only two
parameters, genome GC content and organism growth temperature, differentiate
the codon bias of genes from different organisms. Selection for translational accuracy on synonymous codons
contributes to the correlation of growth temperature with codon bias. In vertebrate genomes, evidence for
selection acting on synonymous codons during translation is ambiguous. We have applied our analysis to eukaryotic
codon bias and now show that translational selection on synonymous codons does
occur in higher eukaryotes.
Systemic Screen for Trans-dominant Alleles of Positive-strand RNA
Viruses (Starting with Poliovirus)
Scott Crowder & Karla Kirkegaard
The high polymerase error rates of RNA viruses can create heterogeneous viral populations within infected cells,
even during a single round of replication. The polyploid nature of the
resulting infections may affect the growth of individual genomes, even those
that contain advantageous mutations such as those that confer drug resistance.
We have tested this phenomenon by introducing 22 different lethal mutations
into the poliovirus genome, and assayed each inviable genome for potentially
trans-dominant inhibition of cotransfected wild-type genomes. In this way, we
have found several mutations with pronounced trans-dominant effects on
wild-type growth. Several mutations
within the genomic regions thaencode viral capsids and polymerase inhibit the
growth of wild-type virus more than 10-20-fold. These proteins are known to oligomerize; therefore trans-dominant
alleles may be explained by the oligomerization of mutant with wild-type
proteins to produce non-functional complexes.
Surprisingly, trans-dominant alleles are also observed in the cis-acting
replication element (CRE); perhaps this RNA element titrates limiting factors
in the cell. Other alleles, such as
those of the viral protease 2A,have little effect on the growth of
cotransfected wild-type genomes. We are adapting this screen for other
positive-strand RNA genomes. Differences in the ability of a genome to alter
the viability of sibling genomes should prove useful in the selection of
molecular targets for anti-viral
therapeutics.
Identification of Toxoplasma gondii Proteins Targeted
Beyond the Parasitophorous Vacuole.
J.D. Dunn and John C. Boothroyd
Upon invasion, the obligate intracellular protozoan
parasite Toxoplasma gondii forms a
parasitophorous vacuole (PV), a novel, nonfusogenic compartment in which it
replicates. The parasite targets proteins to the PV and the PV membrane from
specialized organelles called rhoptries and dense granules. Some of these
secreted proteins are thought to have a role in nutrient transport while others
may function in the host-pathogen interaction. Recent evidence from studies on T. gondii cholesterol acquisition, on
attenuation of the host immune response by T.
gondii, and on T. gondii egress
are consistent with the hypothesis that the parasite traffics proteins into the
host cell.
The overarching goal of this study is to understand
the various host cell modifications potentiated by Toxoplasma gondii during its intracellular development. Proteins
secreted by the parasite appear to be the mediators of alterations in host cell
physiology. Thus, our immediate goal is to identify proteins secreted by T. gondii to modify its intracellular
residence. To distinguish between proteins of host and parasite origin, we
metabolically label intracellular parasites subsequently to selectively
inhibiting host protein synthesis with the ricin. Using this technique, we have
identified radiolabeled proteins, putatively of parasite origin, on the surface
of infected host cells. We are also applying this approach to detect parasite
proteins targeted to the host cytosol. Identification of these proteins will
elucidate the nuances of the relationship between Toxoplasma gondii and its host, will potentially reveal new
therapeutic targets, and will provide new insight into the cell biology of both
host and parasite.
Detecting the cytoplasmic
diffusion barrier in C crescentus
predivisional cells
Ellen M. Judd, W. E. Moerner, Lucy
Shapiro, Harley H. McAdams
The global response regulator CtrA controls multiple
cell cycle events in C crescentus. Among these is DNA replication: CtrA binds
to the origin of replication and represses initiation. Replication begins when CtrA is proteolyzed
at the swarmer-to-stalked cell transition, and selectively proteolyzed in the
stalked compartment of the predivisional cell.
This spatially restricted proteolysis suggests that at this time in the
cell cycle a barrier exists that prevents diffusion of cytoplasmic proteins
between the two halves of the cell. We
developed a laser photobleaching technique to detect the presence of such a barrier. C
crescentus cells expressing soluble yellow fluorescent protein (YFP) were
bleached at one end with a focused laser beam.
Diffusion of YFP within the cell is rapid enough for all YFP to be
bleached during the duration of the laser pulse, despite the restriction of the
laser beam to one cell pole. Some cells
had no YFP signal after bleaching; in others, the unbleached half of the cell
remained fluorescent, indicating the existence of a physical barrier. We used this technique to study barrier
formation as a function of time during the C
crescentus cell cycle. Our data
shows that the barrier forms about 15 minutes before the cells divide, for a
150-minute cell cycle. This technique,
in combination with CtrA localization experiments, will reveal if the onset of
CtrA proteolysis in the stalked compartment of the predivisional cell is
coupled to the completion of a cytoplasmic diffusion barrier.
Gene expression during form
transition in Paracoccidioides
brasiliensis.
J.P. Monteiro, K.V. Clemons, L.F. Mirels,
C.R. Lopes, and D.A. Stevens
This project intends to apply high-density DNA
microarray analysis to two isolates of Paracoccidioides
brasiliensis to identify and characterize genes expressed differentially
during the mycelium-to-yeast transition, an event of major importance during
the initiation of infection. Total DNA was extracted from isolate Pb 01 (ATCC
MYA-826), an isolate used for molecular studies by collaborating laboratories,
and randomly sheared into 1.5 kb fragments.
The fragments are being cloned into ë ZAP II phage to generate a random
shear genomic library. We will isolate
approximately 10,000 clones from this library, which will be catalogued and
stored. We will amplify inserts using
PCR, which, after purification to remove PCR reaction components, will be used
for the construction of arrays with around 10,000 elements. Having accomplished this we will isolate
total RNA from the isolates at various time points before, during and after the
thermal dimorphic transformation.
These time points will be close together on the first day after
temperature shift because we expect gene expression changes to occur early
during the dimorphic process.
Polyadenylated RNA will be converted to cDNA using reverse transcriptase
and modified nucleotides. Attaching
fluorescent compounds (Cy3 and Cy5) to the incorporated modified nucleotides
will generate the array probes. These
probes will be hybridized to the arrayed fragments. Arrays will be scanned using confocal laser microscopy thus
generating a dynamic picture of gene expression of the dimorphic process of
each isolate. The spots that reveal
changes or differences in expression will have their corresponding DNA fragment
in the library sequenced and analyzed.
The information gained will be important for, among other issues, understanding
fungal biology, interaction with the host, diagnosis of the disease, and
identification of potential drug targets.
Anaerobic degradation of
vinyl chloride
Jochen Mueller, Galit Meshulam-Simon,
Alfred Spormann
Chlorinated ethylenes, such as perchloroethylene
(PCE) and trichloroethylene (TCE), rank among the most common groundwater
contaminants in the United States. Bioremediation of contaminated sites could
proof as a valuable alternative to current, physical-chemical remediation
strategies. Microbial degradation of PCE and TCE has been observed mostly under
anaerobic conditions. Degradation takes place by stepwise reductive
dehalogenation reactions eventually leading to harmless ethylene. An important
intermediate of those reaction sequences is vinyl chloride (VC), a proven
carcinogen. Anaerobic degradation of VC is comparable slow, which leads to
build-up in concentration of VC at many contaminated sites. Understanding the
underlying molecular mechanisms of VC-degradation could help in assessing and
improving bioremediation of sites contaminated with chlorinated ethylenes.
Anaerobic degradation of VC was studied with a mixed
bacterial culture containing a bacterium closely related to the known
dehalogenating organism Dehalococcoides
ethenogenes. A VC-reductase was partially purified and characterized from
this culture. The corresponding gene was isolated, amplified and sequenced. The
first biochemical and molecular characterization of a VC-reductase identifies
this enzyme as a member of the novel enzyme class of reductive dehalogenases.
Fluorescence in situ hybridization (FISH) was
evaluated as a tool for detecting biological reductive dehalogenation in situ.
Eubacteria- and specific Dehalococcoides 16S
rRNA-targeted probes were applied to the culture. So far, this method was found
inefficient for monitoring the Dehalococcoides-like
microorganism in situ due to low
signal intensity. This is probably a result of low ribosome content and the
cell morphology of these organisms.
A model for measuring
thermal stress in live cells using a Hsp70-luciferase reporter gene
Caitlin E. O'Connell-Rodwell, Dmitri M. Simanovskii,
Cameron McClure, Josh T. Beckman, Yu-an Cao, Weisheng Zhang, Michael H.
Bachmann, Jennifer A. Baran, E. Duco Jansen, Daniel Palanker2, H. Alan
Schwettman2, Christopher H. Contag1
We developed a model for thermal stress using the
stress-inducible heat shock gene (Hsp70A1) and luciferase expression as a
measure of endogenous Hsp70 proteins generated in living cells. A stable line was
created using NIH 3T3 cells transfected with a plasmid containing the
regulatory region from the murine Hsp70A1 gene promoter driving the expression
of the firefly luciferase reporter gene. We applied thermal pulses to cultures
of these cells and assessed luciferase expression every 2 hours (h) for the
first 10 h and at 24 h, using a cooled CCD camera. Fluorescence activated cell sorting (FACS) was used to determine
cell viability. Cells were treated with
different heat regimes for varying times using either heating blocks (ranging
from 42°C for 20min to 64°C for 5 sec), or a CO2 laser (ranging from 50-80°C
for 1-7 msec). Maximum thermotolerance
ranged between 67.5-76°C and evidence of thermally induced cell death ranged
between 76-80°C in the msec treatments.
Our results indicate that the temporal window across thermal gradients
that results in Hsp70 gene expression is narrow and consequently the transition
from thermal resistance to cell death is abrupt. This reporter gene construct
may be useful in medical applications such as predicting collateral thermal
damage created during treatment of biological tissue with lasers and in
defining minimal thresholds needed to express Hsp70 in the targeted delivery of
therapeutics.
Pyrosequencing for microbial
typing
Mostafa Ronaghi, Ronald Davis
Pyrosequencing, is a real-time DNA sequencing
technique generating short reads rapidly and inexpensively. This technology has
the potential advantage of accuracy, ease-of-use, high flexibility and is now
emerging as a popular platform for microbial typing. Here, we review the
methodology and the use of this technique for viral typing, bacterial typing,
and fungal typing. In addition, we describe how to use multiplexing for
accurate and rapid typing.
Communication
Between Plant Pathogen and Host: Screening for Translocated Proteins
Julie Roden and Mary Beth Mudgett
Plants are faced with many pathogens. The bacterial
pathogen Xanthomonas campestris pv.
vesicatoria (Xcv) is the causal agent of black spot, a disease that affects
leaves and fruit of both pepper and tomato plants. During the infection process, the type III secretion system
(TTSS) of Xcv secretes bacterial proteins out of the bacterium and translocates
a subset of these proteins into the plant cell. Resistant plants recognize the presence of the pathogen,
triggering a defense signaling pathway leading to localized cell death of
infected cells and the inability of the bacteria to colonize the tissue. In a susceptible plant, this pathway is not
triggered, the bacteria are able multiply within the tissue, and disease
ensues.
This work is focused on characterizing novel
bacterial proteins translocated into the plant cell during the early stages of
infection. Mutagenesis of Xcv via a Tn5-based
transposon construct will create random gene fusions to a reporter gene that
causes a scoreable resistance response when translocated into plant cells. This reporter gene fragment alone is unable
to be targeted to the plant cell and will only elicit an avrBs2-specific
resistance response if fused to a Xanthomonas protein containing a functional
type III secretion/translocation signal (Xtp).
Further characterization of type III effector proteins identified in
this work and their plant interactors will help to explain how the bacteria is
manipulating its plant host.
Preventing Apoptotic Cell
Death: Cytomegalovirus Immediate-Early Gene UL37x1
Geoffrey B. Smith, A. Louise McCormick,
Edward S. Mocarski
Cytomegalovirus (CMV) asymptomatically infects
40-90% of humans, causing disease in those with weakened immune systems. Transplant recipients, AIDS patients, and
fetuses are all susceptible to CMV disease, manifested as pneumonitis,
hepatitis, renal tubulitis, chorioretinitis, deafness, and microcephaly.
The CMV immediate-early gene UL37x1 encodes a
mitochondrially localized protein that prevents apoptosis. This block in apoptotic signaling occurs at
the same level as the cellular Bcl-2 family member anti-apoptotic proteins,
after caspase-8 activation and Bid cleavage, but prior to cytochrome c
release. Related viruses such as
Epstein-Barr Virus and Kaposi’s
Sarcoma-associated Herpes Virus encode viral anti-apoptotic Bcl-2 homologs, but
UL37x1 lacks homology to Bcl-2 or any other known proteins. UL37 has been shown through
co-immunoprecipitation to interact with human adenine nucleotide translocase
(ANT), a component of the mitochondrial inner membrane. ANT and mitochondrial porin are the
principal components of the permeability transition pore complex (PTPC) thought
to be important for preventing electrochemical de-stabilization of the
mitochondria, which leads to cell death.
By contrast, cellular anti-apoptotic Bcl-2 family members have been
shown to immunoprecipitate with both ANT and porin.
We have determined domains of UL37x1 that are
necessary for mitochondrial localization [aa 5-30] and protection from
apoptosis [aa 115-147]. We have created
point mutants within the anti-apoptotic domain to narrow down the essential
amino acids for its interactions. In
addition, we are conducting a yeast two-hybrid screen for human proteins that
interact with UL37x1. We hope to
confirm the interaction with ANT, as well as determine other cellular
interacting proteins. The
anti-apoptotic effect of UL37x1 may involve as yet uncharacterized cellular
components of apoptosis machinery.
DNA Replication and
Segregation in Caulobacter crescentus
Sherry Wang, Rasmus Jensen, Lucy
Shapiro
TopoIV is a type II topoisomerase that acts to decatenate daughter
chromosomes during DNA replication. In Caulobacter, the parC and parE genes
encode the subunits of TopoIV. A
temperature sensitive mutation in either of the genes causes a cell separation
defect. To investigate the role of TopoIV
in DNA replication and chromosome segregation, we have looked at the
intracellular location of the replisome and the chromosomal origin of
replication in the parC and parE mutants.
We examined the intracellular location of the replisome in the mutants
using YFP fusions to components of the DNA replication machinery. We have previously found that the replisome
assembles on the origin of replication at the pole of the stalked cell,
coincident with the start of DNA replication.
As replication proceeds, the replisome gradually moves closer to the
cell division plane, and finally disassembles upon completion of
replication. It then reassembles only
at the pole of the stalked cell progeny.
At the restrictive temperature, the replisome is mislocalized indicating
a role for TopoIV in the DNA replication process.
We
used FISH to observe the location of the chromosomal origin of replication
sequence in the parC and parE mutants.
In wild type cells, the origin is localized to the pole in swarmer
cells, where DNA replication is inhibited.
When DNA replication initiates in the stalked cells, the duplicated
origin moves rapidly to the opposite pole.
Proteins at that pole might contribute to a “receiver” complex that
traps the origin at the pole. Candidate
proteins include those that localize to the division plane upon completion of
DNA replication at cell division, because the division site becomes the pole of
the newly divided cell. We observe that
the origin is mislocalized in these mutants and that DAPI staining is abnormal
suggesting that TopoIV plays a role not only in chromosome partitioning, but
most importantly, in localization of the origin sequence to the cell pole.
Experimental
setup to measure forces of actin polymerization
Cyrus A. Wilson & Julie A. Theriot
Actin is a ubiquitous and well-conserved protein
among eukaryotic cells. Beyond its classical role in association with myosin,
actin is employed in numerous motility systems which derive their force not
from the conformational changes of a canonical motor protein, but from the
elongation of filaments. The most prevalent application of actin-based motility
is the forward extension of crawling cells; actin polymerization is responsible
for the leading edge protrusions of nearly any cell moving over a solid surface.
Another application of interest is the motility of Listeria monocytogenes and certain other intracellular bacterial
pathogens, which exploit the actin polymerization machinery of the host cell to
move themselves around.
However, biophysical characterization of
polymerization motor systems lags far behind that of motor proteins. We are
developing a setup to physically measure forces generated by actin
polymerization. We envision the eventual experiments to proceed as follows:
Polymerization is nucleated by a parallel bundle of actin filaments isolated
from the acrosomal process of horseshoe crab (Limulus polyphemus) sperm. The bundle is suspended on an artificial
pedestal, such that the barbed ends of the filaments stick out just past the
edge. When monomer (in the form of actin complexed with profilin) is added
above the critical concentration, the elongating filaments press against a bead
suspended in an optical trap. We measure the force exerted on the trapped bead.
We have made progress with direct fluorescent
labeling of the acrosomal actin bundles and preparation of the sequestered
monomer mixture. By fluorescence microscopy we can observe actin polymerization
off the barbed ends of the acrosomal bundles.
Currently
we are focusing our efforts on fabrication of the pedestals that will support
the actin bundles. We are exploring a “soft” lithographic strategy to
efficiently build the structures on glass cover slips. An elastomeric mold is
created by casting against a photoresist master. The mold is then used to cast
polymer replicas on the cover slips.