Gail E. Christie
BBSI project: What are the
contributions of prophages to microbial diversity?
Closely related bacterial species, and
even strains within the same species, differ from each other by
the distribution of mobile genetic elements within their genomes.
These elements include insertion sequences, transposons, and bacterial
viruses (integrated as prophages). Prophages often carry additional
genes encoding toxins, virulence factors, or other functions that
give a selective advantage to their bacterial host. We are investigating
the distribution of a family of temperate phages, which appears
to be widespead among the gamma Proteobacteria. What is the distribution
of intact and cryptic P2-related prophages in sequenced bacterial
genomes? What additional genes do they carry, and what phenotypic
properties do they confer upon their bacterial hosts?
Other research interests (see web
page for more details)
1. Determinants for DNA binding by a prokaryotic zinc-finger
Many viruses encode transcription factors that alter the specificity
of the host transcriptional machinery to direct the synthesis of
viral mRNAs. Late gene expression in the P2- related temperate phages
is under the positive control of a family of small, phage-encoded
transcriptional activators exemplified by P2 Ogr. These proteins
constitute a novel class of zinc-binding proteins which bear little
sequence or structural similarity to other known prokaryotic transcription
factors. Genetic analysis and invitro binding studies have identified
an unusual activator binding site upstream of late promoters which
includes an interrupted element of dyad symmetry and is predicted
to span three helical repeats of the DNA major groove. We are presently
investigating the binding of these activators to DNA using NucC,
a member of the P2 Ogr family encoded by a cryptic prophage in Serratia
marcescens. In recent studies, DNA bending by NucC was measured
by a gel mobility shift assay, using fragments derived from a circular
permutation vector carrying a NucC binding site. Specific DNA determinants
important in binding site recognition by NucC have been identified
using a variety of chemical protection and interference studies.
Current studies are directed towards elucidating the stoichiometry
of activator binding and identifying, via genetic and crosslinking
experiments, specific residues in NucC that play a role in DNA recognition.
2. RNA polymerase structure and function
Studies of the interaction of phage-encoded functions with the
host RNA polymerase has led to new insights into the roles of RNA
polymerase subunits. Site-directed mutagenesis and in vitro transcription
studies have defined a surface on the a subunit of E. coli RNA polymerase
that is required for activation of transcription by the P2 Ogr family
of transcription factors. We are currently studying a C-terminal
deletion of the bí subunit of RNA polymerase that affects the action
of proteins modulating the timing of lysis during phage infection.
We have also begun a collaborative effort with the lab of Dr. Dennis
Ohman to develop an in vitro system to study transcription of Pseudomonas
aeruginosa genes under the control of alternate sigma factors.
3. The role of bacteriophages in microbial evolution and pathogenesis
P2-related prophages and cryptic prophages encode a variety of
lysogenic conversion functions. Some of these, like the sopE gene
encoded by a P2-related prophage in Salmonella enterica, have been
implicated in bacterial virulence. Others, like the nucC gene encoded
by a cryptic prophage in Serratia marcescens, have been adapted
for the regulation of genes in the bacterial host. We are characterizing
lysogenic conversion genes encoded by a number of P2-related phages
in order to elucidate the horizontal transfer of these genes and
to understand the contributions of these genes to the physiology
of the bacterial host.