title: Mutation of the htrB locus of Haemophilus influenzae nontypable strain 2019 is associated with modifications of lipid A and phosphorylation of the lipo-oligosaccharide.
abstract: 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 non-typable 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 sigma 70-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 degrees C and were able to grow at 37 degrees 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.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City 52242, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Lee
authors.forename: M G
authors.initials: MG
authors.lastname: Sunshine
authors.forename: J J
authors.initials: JJ
authors.lastname: Engstrom
authors.forename: B W
authors.initials: BW
authors.lastname: Gibson
authors.forename: M A
authors.initials: MA
authors.lastname: Apicella
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008050:Lipid A; D008070:Lipopolysaccharides; D012333:RNA, Messenger; C023023:lipid-linked oligosaccharides