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(Received for publication, July 17, 1995; and in revised form, September 8, 1995)
The HtrB protein was first identified in Escherichia coli as a protein required for cell viability at high temperature, but
its expression was not regulated by temperature. We isolated an htrB homologue from nontypable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E. coli htrB mutation. The promoter for the NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two
genes are divergently transcribed. The deduced amino acid sequence of
NTHi 2019 HtrB had 56% homology to E. coli HtrB. In vitro transcription-translation analysis confirmed production of a
protein with an apparent molecular mass of 32-33 kDa. Primer
extension analysis revealed that htrB was transcribed from a
-dependent consensus promoter and its expression was
not affected by temperature. The expression of htrB and rfaE was 2.5-4 times higher in the NTHi htrB mutant B29 than in the parental strain. In order to study the
function of the HtrB protein in Haemophilus, we generated two
isogenic htrB mutants by shuttle mutagenesis using a mini-Tn3.
The htrB mutants initially showed temperature sensitivity, but
they lost the sensitivity after a few passages at 30 °C and were
able to grow at 37 °C. They also showed hypersensitivity to
deoxycholate and kanamycin, which persisted on passage.
SDS-polyacrylamide gel electrophoresis analysis revealed that the
lipo-oligosaccharide (LOS) isolated from these mutants migrated faster
than the wild type LOS and its color changed from black to brown as has
been described for E. coli htrB mutants. Immunoblotting
analysis also showed that the LOS from the htrB mutants lost
reactivity to a monoclonal antibody, 6E4, which binds to the wild type
NTHi 2019 LOS. Electrospray ionization-mass spectrometry analysis of
the O-deacylated LOS oligosaccharide indicated a modification
of the core structure characterized in part by a net loss in
phosphoethanolamine. Mass spectrometric analysis of the lipid A of the htrB mutant indicated a loss of one or both myristic acid
substitutions. These data suggest that HtrB is a multifunctional
protein and may play a controlling role in regulating cell responses to
various environmental changes.
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