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Professor;
Ohio Eminent Scholar of Microbiology
Professor, Natural Resources
Professor, Plant Cellular and Molecular
Biology
Director, Plant-Microbe Genomics Facility
Director, Plant Molecular Biology/Biotechnology
Program
Director, Plant Biotechnology
Ph.D., Syracuse University, 1971.
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Microorganisms
are important agents for carbon sequestration
and biofuel/bioenergy production on earth.
My laboratory is concerned with the molecular
regulation, biochemistry, and enzymology
of carbon dioxide assimilation and the mechanisms
by which microbes catalyze the production
of various biofuels. All organisms
require CO2 and many enzyme-catalyzed reactions
employ CO2 as a reactant for processes as
important and varied as carbohydrate metabolism,
lipid biosynthesis, and the production of
important metabolic intermediates for the
cell. With the realization that many
microorganisms require CO2 in order to elicit
pathogenesis, it is not surprising that
CO2 metabolism and its control also has
great health relevance. Most importantly,
CO2 may also be employed as the sole source
of carbon by a large and diverse group of
organisms on this planet. For this
reason, CO2 fixation is a process that is
associated with global issues of agricultural
productivity, carbon cycling, and industrial
productivity. As the chief green house
gas in the atmosphere, CO2 is also recognized
and implicated in the general warming of
the earth's biosphere. For all these
reasons, research on various aspects of
CO2 fixation control, biochemistry, and
ecology have attracted wide interest. In
addition, these studies, and the subsequent
manipulation of various molecular control
mechanisms, have lead to the development
of promising microbial systems for the production
of biofuels and bioenergy. The following
pages summarize our ongoing and future efforts
to probe various aspects of these issues.
MOLECULAR
REGULATION
We are specifically interested in how CO2
fixation structural genes are regulated
in bacteria. The bulk of our work over the
years has concentrated on two or three model
systems, both of which enable organisms
to use CO2 as the sole source of carbon
for growth. One system, the Calvin-Bassham-Benson
(CBB) reductive pentose phosphate pathway,
is undoubtedly the means by which most organic
matter is produced on earth. To understand
how the cbb genes are regulated,
we have concentrated on the metabolically
versatile nonsulfur purple bacteria, especially
Rhodobacter sphaeroides, Rhodobacter
capsulatus, and Rhodopseudomonas
palustris. In these organisms, we have
found that the cbb genes are clustered
in distinct operons, some of which are localized
in different genetic elements, as in Rba.
sphaeroides. In this organism, the
entire cbb regulon is under the control
of a specific transcriptional regulator
gene, cbbR, whose product, CbbR,
positively controls the transcription of
the two major operons required for CO2 fixation.
CbbR must be activated in the cell, after
binding a specific effector molecule, in
order to turn on transcription. We are currently
studying aspects of the biochemistry of
CbbR (Fig. 1) so that we can understand
how the cell is able to convert this constitutively
synthesized protein to the activated state.
Fig.
1. Structural model of Rba. sphaeroides
CbbR monomer. Residues shown to confer constitutive
activity with respect to cbb transcription
are indicated in red. a helices are yellow,
ß sheets are blue, and loops are thin
gray strands. RD-I and RD-II denote coinducer
regulatory domains I and II, respectively.
From Dangel et al. (2005).
Current
studies indicate that several factors, in
addition to CbbR, impinge on control in
these organisms. Of particular note is a
global two-component signal transduction
system (Reg/Prr) that integrates the control
of CO2 assimilation with the nitrogen fixation
(nif) system and other processes
important for energy generation in Rba.
sphaeroides and Rba. capsulatus.
Indeed, molecular signals are apparently
received at the surface of the cell, undoubtedly
reflecting the redox state of some signal
molecule, thus stimulating a membrane-bound
sensor kinase (RegB/PrrB) to become autophosphorylated.
RegB~P then transfers its phosphate to a
response regulator protein, RegA/PrrA; the
phosphorylated form of this molecule (RegB~P)
then binds to DNA, affecting transcription.
This has become a fairly complicated regulatory
system, as good evidence for the presence
of other regulator molecules has also been
obtained. By contrast, a different and rather
unique 2-component regulatory system, consisting
of a novel hybrid sensor kinase and two
response regulator proteins has been found
to influence cbb gene expression
in Rps. palustris. In this instance,
phosphorelay from the sensor kinase to either
of the two response regulator proteins influences
the ability of CbbR to affect transcription
(Romagnoli & Tabita, 2006; 2007).
Integrative
Control of the CO2 Fixation (cbb), Nitrogen
Fixation (nif) and Hydrogen Evolution Systems;
Biofuel/Bioenergy Production
An interesting aspect of our studies on
cbb control was the finding that
the nitrogen fixation (nif) system,
and its control, is intimately involved,
with the Reg/Prr system important for this
interaction. Indeed, knocking out the cbb
system, under conditions where CO2 is normally
used as an electron acceptor and not a carbon
source, causes these organisms to evolve
mutations that allow derepression of nitrogenase
synthesis and expression of the nifHDK
genes, so that reducing equivalents may
now be dissipated as a result of the H+
- reducing hydrogenase activity of nitrogenase.
Thus, the organism exquisitely controls
how it handles environmental signals related
to carbon and nitrogen metabolism. Our current
model for this complex regulatory process
is summarized below for Rba. sphaeroides
(Fig. 1); basically this conceptual model
holds for Rba. capsulatus and Rps.
palustris as well. Note, manipulating
the regulation of this system allows the
organisms to produce copious quantities
of hydrogen gas, a biofuel of enormous significance.
Fig.
2. Conceptual model showing the interplay
of various factors involved in signal transduction
and regulation of cbb gene expression
in Rba. sphaeroides. The link between
the CO2 (cbb) and nitrogen regulatory
system, including the nitrogen fixation
(nif) genes is shown, as is the
means by which high levels of hydrogen gas
may be produced.
These
studies are particularly relevant in Rps.
palustris, as this organism is probably
the most metabolically versatile organism
found on earth; it is able to grow using
virtually any metabolic energy yielding
system, while degrading and removing waste
materials such as lignin monomers. Thus,
we are attempting to couple this metabolic
versatility with the ability to control
the regulation of gene expression so that
genes responsible for copious hydrogen production
are always up-regulated. In this scenario,
where normal regulatory circuits are bypassed,
hydrogen is consistently and always produced
at very high levels (Fig. 3).
Fig.
3. Cartoon illustrating light-energy catalyzed
production of hydrogen gas by Rps. palustris
and other related bacteria. With strains
we have constructed, normal cellular control
mechanisms are altered such that hydrogen
production is always in the “on”
state.
ENZYMOLOGY
RubisCO
We are deeply involved in efforts to determine
how the structure influences the function
of key enzymes and proteins important for
CO2 fixation. How the activity of these
proteins is regulated in the cell is also
of prime importance to our laboratory. Over
the years, we have primarily focused on
ribulose 1,5- bisphosphate carboxylase/oxygenase
(RubisCO), which is the key enzyme of the
CBB pathway. This enzyme is a very poor
catalyst, yet it is the protein that actually
fixes the bulk of CO2 on this planet, and
is thus responsible for the biological removal
of CO2 from the atmosphere. In addition,
RubisCO function directly correlates with
plant and crop productivity as well as the
growth and reproduction of many significant
microbes in the biosphere. These, and other
reasons, probably explains why RubisCO is
the most abundant protein found on earth,
making aspects of its biochemistry and molecular
control (see above) so topical. The major
issue that we study is the basis by which
RubisCO discriminates between CO2 and O2,
two gaseous substrates that compete for
the same active site on the protein. This
is a very important issue as O2 normally
prevents efficient CO2 fixation. Despite
a wealth of structural and mechanistic information,
it is still not clear how closely related
RubisCO molecules possess different specificities
for CO2 and O2. Taking a combined molecular
biological and chemical approach we are
attacking this problem by constructing novel
mutant enzymes, and have developed prokaryotic
genetic selection procedures to facilitate
these efforts (Smith and Tabita, 2003).
An added bonus has been the finding, from
genomic sequencing studies, that anoxic
hyperthermophilic archaea contain RubisCO
genes. We have recently found that at least
some of these putative RubisCO sequences
encode for bona fide RubisCO activity. As
these enzymes are derived from organisms
that never encounter molecular oxygen, such
proteins are proving to be very interesting;
i.e., they serve as model systems to understand
how the active site of RubisCO may have
evolved (Kreel and Tabita, 2007). Moreover,
biological selection, combined with more
traditional molecular and biochemical approaches,
has lead to the identification of several
key amino acid residues involved with influencing
various aspects of catalysis (Fig. 4).
Fig.
4. Residues from adjacent dimers of RubisCO
Interact to influence catalysis
The
RubisCO-Like Protein (RLP)
Some years ago we discovered that some organisms
synthesize a protein that resembles RubisCO,
which we termed the RubisCO-like protein
or RLP (Hanson and Tabita, 2001). Unlike
RubisCO, and depending on the organism,
RLP participates in various aspects of sulfur
metabolism, but does not catalyze CO2 fixation,
yet there are parts of RLP that both resemble
and are distinct from RubisCO. We are interested
in how the active sites of RubisCO and RLP
have evolved for their specific and unique
functions as such knowledge will provide
clues as to how to engineer and improve
aspects of the catalytic properties of these
proteins. Collaborative investigations with
colleagues at UCLA resulted in the first
solved RLP X-ray structure and provided
important insights on the active site of
this protein and how it both resembles and
diverges from RubisCO (Li et al., 2005).
In particular, it was found that the active
site of RLP has been modified and this protein,
along with other alterations, possesses
an interesting structural adaptation which
we termed the CD-loop (Fig. 5).
Fig.
5. The CD-loop of the Chlorobium tepidum
RLP, comprised of amino acids 78 to 91.
Current
studies involve both in vivo and in vitro
approaches to determine both the role and
regulation of RLP in diverse organisms,
as this protein appears to take on different
functions.
RTCA
Cycle Enzymes
We
also study another model CO2 fixation system,
the reductive tricarboxylic acid (RTCA)
pathway, in which several interesting and
unique CO2 fixation catalysts are focal
points. This is a pathway found in many
bacteria and eukaryotic CO2 fixing organisms,
and many of the key reactions are important
in archaea as well. Virtually nothing is
known of the molecular regulation of the
RTCA cycle and we have recently developed
an interesting model system, the green sulfur
bacterium Chlorobium tepidum, for
these studies. This organism, unlike other
organisms that use this pathway, has a fairly
well defined genetic system and the organism
grows rapidly. We have recently isolated
all the relevant and important enzymes of
this pathway, including pyruvate synthase,
alpha-ketoglutarate synthase, ATP-citrate
lyase, and PEP carboxylase, along with several
important electron carriers including rubredoxin,
two ferredoxins, and two cytochromes. The
structural genes for these proteins have
all been isolated and we have begun to study
aspects of the molecular regulation of this
interesting CO2 fixation process. Aspects
of this work was recently published (Yoon
et al.; 2001; Kim and Tabita, 2006).
MOLECULAR ECOLOGY
We have collaborated with marine scientists
at the University of South Florida to understand
how the regulation of key CO2 fixation genes,
like the RubisCO genes, are controlled in
the oceans. This work, a combination of
ship-board and laboratory investigations,
is devoted to a primary problem, namely
the sequestration of CO2 in the environment.
Procedures for the direct examination of
RubisCO transcripts in the open ocean were
developed and applied to the global CO2
fixation problem. As these studies unfolded,
it became possible to identify organisms
which contribute to active CO2 fixation
and sequestration by amplifying and sequencing
specific RubisCO transcripts via RT-PCR
technology. These studies have been performed
in concert with physiological and biochemical
studies with marine cyanobacteria and algae,
such that a coherent picture of how carbon
dioxide assimilation may be controlled in
these organisms, both in the environment
and in the laboratory (Wawrik et al. 2002;
John et al., 2006).
APPLIED STUDIES
Knowledge of the biochemistry and molecular
control of CO2 fixation has stimulated us
to use this knowledge to consider the possibility
that useful compounds of economic and industrial
importance might be synthesized using this
cheap and ubiquitous gas as the starting
material. In one study, we have considered
the possibilty that CO2 might be converted
to ethanol using a genetically engineered
strain with the requisite genes. Other offshoots
of this technology are also being developed
for the production of value-added products.
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Publications on the molecular regulation
of CO2fixation
Dubbs, J. M. and Tabita, F. R.
Interactions of the cbb II promoter-operator
region with CbbR and RegA (PrrA) regulators
indicate distinct mechanisms to control
expression of the two cbb operons of Rhodobacter
sphaeroides. J. Biol. Chem. 278 (2003) 16443
- 16450.
Dangel, A. W., Gibson, J. L., Janssen, A.
P., and Tabita, F. R. Residues
that influence in vivo and in vitro CbbR
function in Rhodobacter sphaeroides
and identification of a specific region
critical for co-inducer recognition. Mol.
Microbiol. 57 (2005) 1397-1414.
Romagnoli, S., and Tabita, F. R.
A novel three-protein two-component system
provides a regulatory twist on an established
circuit to modulate expression of the cbbI
region of Rhodopseudomonas palustris
CGA010. J. Bacteriol. 188 (2006) 2780-2791.
VerBerkmoes, N.C., Shah, M.B., Lankford,
P.K., Pelletier, D.A., Strader, M.B., Tabb,
D.L., McDonald, W.H., Barton, J.W., Hurst,
G.B., Hauser, L., Davison, B.H., Beatty,
J.T., Harwood, C.S., Tabita, F.R.,
Hettich, R.L. and Larimer, F.W. Determination
and comparison of the baseline proteomes
of the versatile microbe Rhodopseudomonas
palustris under its major metabolic
states. J. Proteome Res. 5 (2006) 287-298.
Tabita, F. R. Research
on carbon dioxide fixation in photosynthetic
microorganisms (1971-present) In: Discoveries
in Photosynthesis. Govindjee, Beatty, J.T.,
Gest, H. and Allen, J.F. (eds.). Springer,
pp. 771-788 (2006).
Romagnoli, S., and Tabita, F. R.
Phosphotransfer reactions of the CbbRRS
three-protein two-component system from
Rhodopseudomonas palustris CGA010
appear to be controlled by an internal molecular
switch on the sensor kinase. J. Bacteriol.
189 (2007) 325-335.
Recent publications on the
enzymology and biochemistry of CO2 fixation
Hanson, T.E. and Tabita, F.R.
A RubisCO-like protein from Chlorobium
tepidum that is involved with sulfur
metabolism and the response to oxidative
stress. Proc. Natl. Acad. Sci. USA 98 (2001)
4397-4402.
Yoon,
K.-S., Bobst, C.E.., Hemann, C., Hille,
R., and Tabita, F.R. Spectroscopic
and functional properties of novel 2[4Fe-4S]
clister-containing ferredoxins from the
green sulfur bacterium Chlorobium tepidum.
J. Biol. Chem. 276 (2001) 44027-44036.
Finn,
M. W. and Tabita, F. R.
Synthesis of cataltically active form III
ribulose 1,5-bisphosphate carboxylase/oxygenase
in archaea. J. Bacteriol. 185 (2003) 3049-3059.
Smith, S. A., and Tabita,
F. R. Positive and negative selection
of mutant forms of prokaryotic (cyanobacterial)
ribulose-1, 5-bisphosphate carboxylase/oxygenase.
J. Mol. Biol. 331 (2003) 557-569.
Smith, S. A., and Tabita, F. R.
Glycine 176 affects catalytic properties
and stability of the Synechococcus
sp. strain PCC 6301 ribulose 1,5-bisphosphate
carboxylase/oxygenase. J. Biol. Chem. 279
(2004) 25632-25637.
Finn,
M. W., and Tabita, F. R.
A modified pathway to synthesize ribulose
1,5-bisphosphate in methanogenic archaea.
J. Bacteriol. 186 (2004) 6360-6366.
Li,
H., Sawaya, M. R., Tabita, F. R.,
and Eisenberg, D. Crystal structure of a
novel RuBisCO-like protein from the green
sulfur bacterium Chlorobium tepidum.
Structure 13 (2005) 779-789.
Kim,
W., and Tabita, F. R. Both
subunits of ATP-citrate lyase from Chlorobium
tepidum contribute to catalytic activity.
J. Bacteriol. 188 (2006) 6544-6552.
Kreel,
N. E., and Tabita, F. R.
Substitutions at methionine 295 of the Archaeoglobus
fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase
affects interactions with oxygen binding
and CO2/O2 substrate specificity. J. Biol.
Chem. 282 (2007) 1341-1351.
Recent publications on the
molecular ecology of CO2 fixation
Wawrik, B., Paul, J. H. and Tabita,
F. R. Real-time PCR quantification
of rbcL (ribulose-1,5-bisphosphate
carboxylase/oxygenase) mRNA in diatoms and
pelagophytes. Appl. Environ. Microbiol.
68 (2002) 3771-3779.
John, D.E., Wawrik, B., Tabita,
F. R. and Paul, J.H. Gene diversity
and organization in rbcL-containing genome
fragments from uncultivated Synechococcus
in the Gulf of Mexico. Mar. Ecol. Prog.
Ser. 316 (2006) 23-33.
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