MUTATION OF THE HTRB LOCUS OF HAEMOPHILUS-INFLUENZAE NONTYPABLE STRAIN-2019 IS ASSOCIATED WITH MODIFICATIONS OF LIPID-A AND PHOSPHORYLATIONOF THE LIPO-OLIGOSACCHARIDE

The HtrB protein was first identified in Escherichia coil as a protein required for cell viability at high temperature, but its expression w as not regulated by temperature. We isolated an htrB homologue from no ntypable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E, coli htrB mutation, The promoter for th e NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two genes are divergently transcribed. The deduced amino acid sequenc e of NTHi 2019 HtrB had 56% homology to E, coil HtrB, In vitro transcr iption-translation analysis confirmed production of a protein with an apparent molecular mass of 32-33 kDa, Primer extension analysis reveal ed that htrB was transcribed from a sigma(70)-dependent consensus prom oter and its expression was not affected by temperature, The expressio n 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 Htr B protein in Haemophilus, we generated two isogenic htrB mutants by sh uttle mutagenesis using a mini-Tn3, The htrB mutants initially showed temperature sensitivity, but they lost the sensitivity after a few pas sages at 30 degrees C and were able to grow at 37 degrees C, They also showed hypersensitivity to deoxycholate and kanamycin, which persiste d on passage, SDS-polyacrylamide gel electrophoresis analysis revealed that the lipo-oligosaccharide (LOS) isolated from these mutants migra ted faster than the wild type LDS and its color changed from black to brown as has been described for E. coil htrB mutants. Immunoblotting a nalysis 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-deac ylated LOS oligosaccharide indicated a modification of the core struct ure characterized in part by a net loss in phosphoethanolamine. Mass s pectrometric analysis of the lipid A of the htrB mutant indicated a lo ss 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.