BlcR Protein from Agrobacterium Tumefaciens

CHAPTER 1

A General Introduction to BlcR protein from Agrobacterium tumefaciens

INTRODUCTION

Transcription factors and regulation mechanisms

Transcription factors regulate various biological important processes by binding to specific genes and regulating gene expression. The biophysical and structural studies in the last decades revealed that the regulation mechanisms of transcription factors can be classified into two groups, one as classical allosteric modulation of DNA binding modules, and the other as regulated assembly of oligomeric transcription factor (1). For the classical allosteric modulation, the classical example is Lac repressor, in which sugar functions as the inducing small molecule ligand that bindings to the Lac repressor and modulates the orientation of the DNA binding modules of the Lac repressor, thus decreases the DNA-protein binding affinity and releases the repression effect (2). Alternatively, unlike the well-studied simpler classical allosteric mechanism, the regulated assembly mechanism has greater versatility, as not only the small molecule ligands can regulate the DNA affinity of transcription factors by modulating self-assembly of oligomeric transition factors, but also post-translational modification, DNA architectural interactions with transcription factor, and interactions of transcription factor with other proteins can also influence self-assembly of oligomeric transcription factors and thus influence the regulation effects from transcription factors (1). This regulated assembly mechanism exists widely from prokaryotic homo-oligomeric transcription regulators and eukaryotic hetero-oligomeric transcription complexes. However, so far, there are a limited number of biochemically and biophysically investigated systems on the regulated assembly mechanism.

Isocitrate lyase transcription regulator (IclR) family

Bacteria in the environment are constantly exposed to non-optimal growth conditions, and they often respond to these variations by regulating specific gene expression through the activity of transcription factors. A newly classified transcriptional regulator family, termed the isocitrate lyase transcription regulator (IclR)-type family (3-6), controls a wide range of cellular activities in response to environmental or growth variations. In responding to small effector ligands, which often serve as conditional cues, IclR-type proteins alter their DNA binding capacity, leading to differential gene regulation. So far, roughly 500 different IclR-type proteins have been identified from bacteria and archaea and regulate diverse biological processes, including metabolic pathways (6), multidrug resistance (7), aromatic compound degradation (8, 9), pathogenicity (10), sporulation (11-13), amino acid biosynthesis (14), and quorum-sensing signal degradation (15). Members of the IclR family are usually in the range of 240-280 amino acid residues, and are comprised of two functional domains, the DNA-binding N-terminal domain (NTD) and the ligand-binding C-terminal domain (CTD), which are connected via a linker helix. The NTDs contain a helix-turn-helix (HTH) structural feature which is a prevalent DNA binding motif observed for prokaryotic transcription factors that recognize specific target DNA sequences (16-18). The CTDs include the effector ligand binding sites which are predicted to fold into a GAF (cGMP-regulated cyclic nucleotide phosphodiesterases, certain Adenyl cyclases, the bacterial transcription factor FhlA) structural motif.

The characterized IclR-type proteins

Except for a few systems, the functions of most IclR members are poorly understood. Even in the more intensively studied systems, information on the regulatory DNA sequences, the small effector ligands, and the mode of regulation is rudimentary. The founding member, IclR of Escherichia coli, regulates the aceBAK operon that encodes essential enzymes (isocitrate lyase, malate synthase, and isocitrate dehydroganase kinase/phosphorylase) for E. coli to utilize acetate or fatty acids as a sole carbon source via the glyoxylate pathway (4, 19). The aceBAK operon is repressed by IclR if the preferred carbon source (glucose) is present, and becomes derepressed when acetate or fatty acids are the only carbon source (20). Sequences of the IclR-binding sites in the aceBAK promoter have been identified and are palindromic (21, 22), and different mechanisms have been proposed for IclR to function as a repressor for the aceBAK operon (23). In solution, IclR exists in equilibrium between dimer and tetramer (24, 25), and the tetrameric IclR binds to the DNA promoter (24, 26). However, the cognate effector ligand for IclR is still not clear, although glyoxylate and pyruvate have been shown to bind to IclR: interestingly, these two molecules have the opposite influence on IclR-DNA interaction (24), and the basis for the difference remains undefined.

Structural information on IclR-type proteins is limited. So far, there are two structures of full-length IclR-type proteins, TM-IclR from Thermotoga maritima TM0065 (27) and Pseudomonas putida TtgV (28). As the first characterized crystal structure, TM-IclR crystallizes as a dimer (Fig. 1) (27). The NTDs and the linker helixes participate in the dimerization, whereas the two CTDs are well separated. The two NTDs in the dimeric TM-IclR are symmetric, which is correlated to the inverted-repeat properties of the recognition DNA sequence. However, the two CTDs in the dimeric TM-IclR are asymmetric in respect to their NTDs, and this asymmetric structure may be due to crystal packing (27). The two asymmetric CTDs are involved in the intermolecular interactions with neighboring dimeric TM-IclR in crystal (27). However, for TM-IclR, information on the regulated operon, the target DNA sequence and the cognate effector ligand is not known.

Besides, several structures are available for the isolated ligand-binding CTDs in apo form (AllR (29), YaiJ and KdgR – unpublished) and in the ligand bound form (AllR with glyoxylate (29), E. coli IclR with glyoxylate (24), and E. coli IclR with pyruvate (24)). Details about the interaction between the IclR protein and the target DNA are not clear, although there is a single structure of a full-length IclR-type protein (Pseudomonas putida TtgV) in the DNA bound form (28).

Agrobacterium tumefaciens

Our research interests focus on the BlcR protein of Agrobacterium tumefaciens. A. Tumefaciens is a Gram-negative soil bacterium, and is a plant pathogen causing significant agronomic losses (Fig. 2A) (30). A. Tumefaciens can induce crown gall (Fig. 2B.) on stem and root of many dicotyledonous plants (walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish, rhubarb, and et al.) by transferring T-DNA from its Ti (tumor inducing) plasmid into the nucleus of the plants (Fig. 2C). Because of this DNA transfer activity, A. Tumefaciens is widely utilized in plant genetic engineering (31).

A. Tumefaciens is also an important model for studying host-pathogen and inter-bacterial interactions (32). Ti plasmid is the primary virulence element for A. tumefaciens; in order to be virulent, the bacterium must contain a Ti plasmid (200 kb), which contains the T-DNA and all the genes necessary to transfer it to the plant cell. Many strains and bacteria of A. tumefaciens do not contain a Ti plasmid, and there is a quorum sensing process regulates the conjugal transfer (exchange of plasmids amongst bacteria) of Ti plasmid in A. tumefaciens (33). Quorum sensing is a cell-cell communication of bacteria in adaptation to environmental changes. In quorum sensing mechanism, bacteria synthesize specific small signal molecules, specifically acylhomoserine lactone (AHL) in A. tumefaciens (15). These small molecules are perceived via receptor proteins that in turn regulate expression of specific genes (Fig. 3) (34).

BlcR of Agrobacterium tumefaciens

BlcR (formerly AttJ) of the plant pathogen Agrobacterium tumefaciens is an IclR-type regulator, negatively controlling the blcABC operon that is responsible for the catabolism of γ-butyrolactone (GBL) (34-36) (Fig. 4). The three enzymes (BlcA, a semialdehyde dehydrogenase; BlcB, an alcohol dehydrogenase; and BlcC, a lactonase) convert GBL sequentially to hydroxybutyrate (GHB), succinate semialdehyde (SSA), and succinic acid (SA), the latter being integrated into the TCA cycle. Thus, the GBL pathway allows A. tumefaciens to metabolize GBL, commonly found in plant exudates, as a carbon and energy source. Intriguingly, the lactonase BlcC (formerly AttM), efficiently degrades AHL quorum-sensing signal (Fig. 4) and exerts a profound influence on this intercellular signaling process (15). The observation that BlcR controls the degradation of quorum-sensing signal via BlcC presents an opportunity to regulate T-DNA transfer in A. tumefaciens.

The BlcR-binding site has been mapped by DNA footprinting assay to be a 45-bp DNA fragment that includes two pairs of inverted repeats near the blcABC promoter (Fig. 5) (36), and SSA has been identified as the cognate effector ligand that affects BlcR DNA binding (36). In addition, an in vitro assay of BlcR function via lactonase activity (BlcC) has been established (37). Thus, BlcR represents an experimentally amenable IclR-type protein for biochemical and structural characterizations, to reveal how the IclR-type proteins recognize their inducing ligands and target DNA sequences, how they interact with DNA, and how the effector ligands modulate this activity.

CONCLUSIONS

Transcription factors play an important role in regulating gene expression. The regulation mechanisms of transaction factors can be classified into either classical allosteric modulation or regulated assembly. Compared to the simpler classical allosteric modulation, the regulated assembly has greater versatility and share common features in prokaryotes and eukaryotes. However, the investigation on the regulated assembly mechanisms are limited. In prokaryotes, ligand-responsive transcription regulators are utilized to control biological metabolisms and developments in order to ensure the organisms to be adaptable to environmental or growth changes. The IclR-type family is a newly classified transcriptional regulator family among a broad range of bacteria and archaea, which control a wide range of cellular activities in response to environmental or growth variations (5, 6). However, the biochemical and biophysical understanding of how IclR proteins function to control gene expression and how interaction with the inducing ligands modulates this activity is limited. Compared with other IclR proteins, including those that have been characterized at molecular and biochemical levels, BlcR represents an experimental amenable IclR-type protein for biochemical and biophysical characterizations, to reveal how the IclR-type proteins recognize their inducing ligands and target DNA sequences, how they interact with DNA, and how the effector ligands modulate their DNA binding affinity. Here, we propose to perform detailed in vitro and in vivo analysis of BlcR to serve as a classic model system for the IclR protein family, which will not only reveal conserved mechanisms for different IclR-type regulators in various bacteria and archaea, but also illuminate mechanisms of the regulated assembly for many different transcription regulators in prokaryotes and eukaryotes.

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