Download Statement of purpose

Research Program
Transcriptional control of L-arabinose metabolism in Bacillus subtilis. The AraR (B.
subtilis) protein is a transcription factor (TF) belonging to the GnTR family of regulators.
AraR is responsible for repressing genes that are involved in arabinose metabolism through
binding to seven distinct operators in the promoter region of the L-arabinose operon. In
addition, the TF also binds to a cognate operator in the promoter of its own gene and brings
about self-repression. AraR binds L-arabinose when it is present in the cellular milieu and
this event abrogates the ability of this TF to bind DNA, ultimately leading to the expression of
the metabolic genes. The basic molecular mechanism through which DNA recognition by
AraR is abolished on arabinose binding is still unknown. This project aims to understand the
mechanism of gene repression by AraR and release of this repression at the molecular level.
I have determined crystal structures of AraR (DNA binding domain) in complex with different
operators which reveal the structural basis of recognition of different DNA sequences by
AraR with different affinities. Comparison of the structures of AraR-NTD with two different
operators (ORA1 and ORR3) shows that the relative position of the two monomers on DNA
is different in the two complexes. The structures clearly show that the spacing between the
two sub-sites in the operator sequences is a determinant of the relative positioning of the
two monomers on the DNA. Since the spacing between two sub-sites is variable for
operators of different members of the GnTR family, the differences in spatial position of the
two monomers could be a general feature of this family (Jain and Nair, Manuscript under
review in Nucleic Acids Research). In addition, structures of AraR (DBD) in complex with
two other operators (ORE1 and ORX1) show that AraR exhibits plasticity in the identity of
residues involved in forming sequence specific contacts with DNA. This attribute allows AraR
to to bind to DNA sequences that deviate from the consensus. (Jain and Nair, Manuscript
under preparation).
Efforts are underway to obtain high quality crystals of the FL-AraR:DNA complex.
Overall, the entire study will provide insight into the conformational switch in AraR that
abolishes specific DNA recognition on arabinose binding as well as provide the structural
basis for specific recognition of the operator sequence.
FleQ, master regulator of transcription of flagellar and biofilm genes in Pseudomonas
aeruginosa: Structure and mechanism. Many prokaryotes respond to environment
induced stress by translocating to a more favourable milieu. Bacterial motility is mediated by
the action of flagella that are complex dynamic structures composed of numerous proteins.
The assembly of functional flagella requires coordinated expression of about 40 genes for
synthesis of its structural and regulatory components and the associated chemosensory
apparatus. Synthesis and assembly of flagella is an energetically expensive process as a
result the expression of flagellar genes is tightly controlled to prevent unnecessary and
accidental flagellar assembly and translocation.
The regulation of flagellar genes is brought about primarily at the transcription level through
the action of master regulators. These transcription modulators control the expression of
flagellar genes as well as that of other regulatory proteins in a hierarchical manner.
Pseudomonas aeruginosa (Psa) is a motile gram-negative bacterium that contains a
monotrichous polar flagella. FleQ from Psa is the master regulator that controls the
expression of flagellar genes. Deletion or mutation of FleQ gene makes the bacterium nonmotile. The molecular mechanism by which FleQ activates the target regulatory and flagellar
genes is yet to be elucidated.
In addition, Psa is capable of switching from single, motile cell to multicellular biofilms that
are non-motile, surface associated communities enclosed in an exopolysaccharide matrix
produced by the bacteria. These biofilms are associated with chronic infections in
immunocompromised individuals and can prove to be fatal. Biofilm infections are resistant to
antibiotics and tend to escape the immune system. At the molecular level the
exopolysaccharides genes and other genes responsible for biofilm formation are repressed
by FleQ. The biofilm formation is triggered by increased level of a secondary messenger
called c-di-GMP. It has recently been shown that c-di-GMP binds to FleQ and derepresses
the expression of genes responsible for biofilm formation. The structural basis of release of
FleQ mediated repression by c-di-GMP is not known.
Overall, FleQ appears to be a dual-regulator which can act as an activator of
flagellar genes and repressor of genes critical for biofilm formation. It is seen that bacteria
undergo a transition from a motile state to one wherein they are located within biofilms on
attachment to an anchor in a favourable environment. Through its dual regulatory activity,
FleQ might play an important role in this development of Psa from a flagellated motile stage
to a biofilm resident one.
In order to obtain mechanistic insights regarding the distinct modes of transcription
regulation by FleQ, I propose to study molecular interactions between FleQ and DNA/FleN/cdiGMP using biochemical assays, X-ray crystallography, SAXS and iso-thermal titration
calorimetry. The entire study will provide detailed information regarding (i) interactions
between FleQ and DNA in activation and repression modes, (ii) the protein-protein
interactions and conformational changes that mediate antiactivator activity of FleN towards
FleQ and (iii) the mechanism by which c-di-GMP abrogates DNA binding ability of FleQ to
achieve derepression of biofilm genes.