Biochemistry, molecular biology, molecular ecology, archaea.
My laboratory is concerned with the molecular regulation, biochemistry,and enzymology of carbon dioxide assimilation. 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 CO2metabolism and its control has great health relevance. Carbon dioxide 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. CO2 is also recognized as the chief green house gas and has been 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. The following pages summarize our ongoing and future efforts to probe various aspects of this issue.
We are specifically interested in how CO2 fixation structural genes are regulated in bacteria and various eukaryotic organisms. 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. For these studies, 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 structural ( cbb ) genes are clustered in distinct operons, some of which are localized in different genetic elements, as in R. 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, probably after binding a specific effector molecule, in order to turn on transcription. We are currently studying aspects of the biochemistry of CbbR so that we can understand how the cell is able to convert this constitutively synthesized protein to the activated state. Unlike R. sphaeroides, there are two cbbR genes in R. capsulatus , each of which specifically regulates its cognate cbb operon. 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 that integrates the control of CO2 assimilation with the nitrogen fixation ( nif ) system and other processes important for energy generation. Indeed, molecular signals are apparently received at the surface of the cell, undoubtedly reflecting the redox state of some signal molecule (? in Fig. 1), 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. 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 both organisms to derepress nitrogenase synthesis and 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 R. sphaeroides (Fig. 1); basically this conceptual model holds for R. capsulatus andR. 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. 1. Conceptual model showing the interplay of various factors involved in signal transduction and regulation of cbb gene expression in R. sphaeroides . The link between the CO2 ( cbb ) and nitrogen regulatory system, including the nitrogen fixation ( nif ) genes is shown. Primary signals are received at the cytoplasmic membrane. This is thought to affect the redox potential of some key component (?) influencing RegB/PrrB autophosphorylation and the subsequent formation of RegA~P (PrrA~P). RegA~P (PrrA~P) interacts directly with the cbb and nif operator-promoter regions (Dubbs and Tabita, submitted for publication). Positive regulation is thus conferred both by the CbbR protein and RegA~P(PrrA~P), the phosphorylated response regulator of the Reg (Prr) two-component regulatory system. CbbR is converted to CbbR (the transcriptionally active form of this molecule), presumably by virtue of binding a coinducer molecule produced under CO2 fixation conditions or other growth conditions that favor cbb transcription. The expression of glnB is affected by the cbb system (Qian and Tabita 1998) with glnB influencing nif derepression through the Ntr system and NifA. Blockage of the CBB pathway results in hydrogen evolution by virtue of the hydrogenase activity of the derepressed nitrogenase complex (Joshi and Tabita 1996).The nitrogenase complex and its inherent hydrogenase activity thus serves to remove excess reducing equivalents not dissipated in strains unable to use CO2 as an electron acceptor. p, refers to promoter-operator regions that are activated in a positive manner (+). From Tabita (1999).
The Rhodopseudomomas palustris Microbial Cell Project/Genomes to Life Program
OSU recently initiated a new program to study mechanisms by which a single organism controls the ability to regulate many aspects of metabolism simultaneously ( http://www.osu.edu/researchnews/archive/micromet.htm ). For this purpose, the Department of Energy awarded a large grant to a consortium of investigators from seven institutions to begin the Rhodopseudomonas palustris Microbial Cell Project. Ohio State, with F. R. Tabita, the Principal Investigator, is the lead institute of the consortium.This project involves the genetically tractable and metabolically versatile organism, Rhodopseudomonas palustris , whose entire genomic sequence was recently completed. Postgenomic approaches will be used to determine how the organism regulates the ability to produce useful forms of energy while also removing greenhouse gases (such as carbon dioxide) and degrading toxic compounds. Rps. palustris is metabolically similar, but more versatile than other nonsulfur purple bacteria ( Rhodobacter ) under study in the laboratory (see above).
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 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. This is probably 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 active same active site on the protein. This is a very important issue as O2normally 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, these 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. The structure of the Methanococcus jannaschii and Archaeoglobus fulgidus RubisCO enzymes have been modeled (Fig. 2), providing a ready system for current and future structure-function studies of the archaeal enzyme. More recently, we have shown that these enzymes actually produce physiologically significant RubisCO proteins (Finn and Tabita 2003) and we have elucidated the probable role of this enzyme in archaea.
Fig. 2. Tertiary structure prediction of archaeal RubisCO molecules. The predicted tertiary structure of the M. jannaschii sequence (A) and the A. fulgidus 2 sequence (B) is compared to the known structure of (C), the Synechococcus large subunit. The Synechococcus small subunit is also shown to the lower left of the structure (in amber). Label sizes and shading reflect the distance from the viewer, smaller and darker being further from the viewer. The main features are highlighted as follows: yellow = active site residues within 3.3 � of the bound transition state analogue CABP in the Synechococcus enzyme, and the equivalent residues in the M. jannaschii and A. fulgidus sequences; red = loop-6; cyan = highly divergent ?-helix-6 residues; purple = residues that appear to be absent in the M. jannaschii and A. fulgidussequences (eight residues at the N-terminus of the Synechococcus enzyme were not resolved in the structure determination and therefore are not shown here). Mg 2 + is represented as a green sphere and CABP as a ball and stick model in C. From Watson et al. (1999)
B. Other CBB Enzymes
Other enzymes of the CBB pathway are also under study. RubisCO activase is an enzyme that catlyzes the removal of inhibitory phosphorylated compounds from the active site of RubisCO. Another key enzyme of the pathway that is studied is phosphoribulokinase, the enzyme that catalyses the formation of the substrate for RubisCO. Transketolase and pentose 5-phosphate-3-epimerase are two enzymes whose genes, cbbT and cbbE , respectively, are found in the cbb regulon (Fig.1). These two enzymes catalyze important 5-carbon sugar phosphate transformations and are also studied. Transketolase has particularly been the focus of recent studies, in which the involvement of a specific cysteine residue in cofactor binding was elucidated (Bobst and Tabita submitted)
C. 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. The key CO2 fixation catalysts are redox sensitive proteins which show interesting and novel interactions with electron carriers such as rubredoxin (Fig. 3). This work has recently been published in the Journal of Biological Chemistry (Yoon et al. 1999; 2001) and other manuscripts are in preparation.
Fig. 3. EPR spectrum of C. tepidum rubredoxin before (A) and after (B) reduction with pyruvate ferredoxin oxidoreductase/pyruvate synthase.
An interesting aspect of our work with C. tepidum was the finding of an unusual RubisCO-like protein (RLP) which is involved with sulfur metabolism and the stress response (Hanson and Tabita 2001, 2003).
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.
We also have a collaboratine project to ascertain the role and study the significance of a RubisCO in hydrothermal vent archaeal extremophiles. These studies promise to provide much information as to how CO2 fixation pathways may evolve in potential extraterrestial systems .
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.
In addition to continuing the molecular, biochemical, and ecological efforts described above, we are embarking on new approaches to learn how CO2 fixation control relates to the overall regulation of metabolism in representative organisms. Taking advantage of the many genomes that have been sequenced from CO2 -fixing prokaryotes, including bacteria and archaea which are studied in our laboratory, we have initiated studies employing microarray and proteomics technology to investigate the global expression of CO2 -responsive genes in these organisms. This technology promises to allow us the capability of identifying a large complement of genes important for CO2 fixation that we would not normally identify with standard procedures. Moreover, since we have mutants at our disposal that are known to influence carbon assimilation, it should be feasible to greatly improve our efforts to examine overlapping metabolic schemes. Finally, it is our intention to use this technology to examine genes that are turned on during CO2 -dependent pathogenesis in selected organisms of health significance.