ABSTRACT
PrfA,
the master regulator of LIPI-1, is indispensable for the pathogenesis
of the human pathogen Listeria monocytogenes and the animal
pathogen Listeria ivanovii. PrfA is also present in the
apathogenic species Listeria seeligeri, and in this study, we
elucidate the differences between PrfA proteins from the pathogenic and
apathogenic species of the genus Listeria. PrfA proteins of
L. monocytogenes (PrfALm and
PrfA*Lm), L. ivanovii
(PrfALi), and L. seeligeri (PrfALs) were
purified, and their equilibrium constants for binding to the PrfA box
of the hly promoter (Phly
Lm) were
determined by surface plasmon resonance. In addition, the capacities of
these PrfA proteins to bind to the PrfA-dependent promoters
Phly and PactA and to form ternary complexes together
with RNA polymerase were analyzed in electrophoretic mobility shift
assays, and their abilities to initiate transcription in vitro starting
at these promoters were compared. The results show that
PrfALi resembled the constitutively active mutant
PrfA*Lm more than the wild-type PrfALm, whereas
PrfALs showed a drastically reduced capacity to bind to the
PrfA-dependent promoters Phly and PactA. In contrast,
the efficiencies of PrfALm, PrfA*Lm, and
PrfALi forming ternary complexes and initiating
transcription at Phly and PactA were rather similar,
while those of PrfALs were also much lower. The low binding
and transcriptional activation capacities of PrfALs seem to
be in part due to amino acid exchanges in its C-terminal domain
(compared to PrfALm and PrfALi). In contrast to
the significant differences in the biochemical properties of
PrfALm, PrfALi, and PrfALs, the
PrfA-dependent promoters of hly (Phly
Lm,
Phly
L
i, and
Phly
L
s) and actA
(PactA
Lm, PactA
L
i,
and PactA
L
s) of the three
Listeria species did not significantly differ in their binding
affinities to the various PrfA proteins and in their strengths to
promote transcription in vitro. The allelic replacement of
prfA
Lm with prfA
Ls in L.
monocytogenes leads to low expression of PrfA-dependent genes and
to reduced in vivo virulence of L. monocytogenes, suggesting
that the altered properties of PrfALs protein are a major
cause for the low virulence of L.
seeligeri.
E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.