| Autor: |
Lee, Na-Gyong, Sunshine, Melvin G., Engstrom, Jeffery J., Gibson, Bradford W., Apicella, Michael A. |
| Zdroj: |
Journal of Biological Chemistry; November 1995, Vol. 270 Issue: 45 p27151-27159, 9p |
| Abstrakt: |
The HtrB protein was first identified in Escherichia colias a protein required for cell viability at high temperature, but its expression was not regulated by temperature. We isolated an htrBhomologue from nontypable Haemophilus influenzaestrain (NTHi) 2019, which was able to functionally complement the E. coli htrBmutation. The promoter for the NTHi 2019 htrBgene overlaps the promoter for the rfaEgene, and the two genes are divergently transcribed. The deduced amino acid sequence of NTHi 2019 HtrB had 56% homology to E. coliHtrB. In vitrotranscription-translation analysis confirmed production of a protein with an apparent molecular mass of 32-33 kDa. Primer extension analysis revealed that htrBwas transcribed from a σ70-dependent consensus promoter and its expression was not affected by temperature. The expression of htrBand rfaEwas 2.5-4 times higher in the NTHi htrBmutant B29 than in the parental strain. In order to study the function of the HtrB protein in Haemophilus, we generated two isogenic htrBmutants by shuttle mutagenesis using a mini-Tn3. The htrBmutants 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 htrBmutants. Immunoblotting analysis also showed that the LOS from the htrBmutants 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 htrBmutant 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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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