Lee N G, Sunshine M G, Engstrom J J, Gibson B W, Apicella M A
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
J Biol Chem. 1995 Nov 10;270(45):27151-9.
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.
HtrB蛋白最初是在大肠杆菌中作为高温下细胞存活所必需的一种蛋白被鉴定出来的，但其表达不受温度调节。我们从不可分型流感嗜血杆菌菌株（NTHi）2019中分离出一个htrB同源物，它能够在功能上互补大肠杆菌的htrB突变。NTHi 2019 htrB基因的启动子与rfaE基因的启动子重叠，且这两个基因是反向转录的。NTHi 2019 HtrB推导的氨基酸序列与大肠杆菌HtrB有56%的同源性。体外转录-翻译分析证实产生了一种表观分子量为32 - 33 kDa的蛋白。引物延伸分析表明htrB是从一个依赖σ70的共有启动子转录而来，其表达不受温度影响。htrB和rfaE在NTHi htrB突变体B29中的表达比亲本菌株高2.5 - 4倍。为了研究HtrB蛋白在嗜血杆菌中的功能，我们使用mini-Tn3通过穿梭诱变产生了两个同源的htrB突变体。htrB突变体最初表现出温度敏感性，但在30℃传代几次后失去了敏感性，并且能够在37℃生长。它们对脱氧胆酸盐和卡那霉素也表现出超敏感性，这种敏感性在传代后持续存在。SDS-聚丙烯酰胺凝胶电泳分析表明，从这些突变体中分离出的脂寡糖（LOS）迁移速度比野生型LOS快，并且其颜色从黑色变为棕色，这与大肠杆菌htrB突变体的情况相同。免疫印迹分析还表明，htrB突变体的LOS对一种与野生型NTHi 2019 LOS结合的单克隆抗体6E4失去了反应性。对O-脱酰化LOS寡糖的电喷雾电离-质谱分析表明核心结构发生了修饰，部分特征是磷酸乙醇胺净损失。对htrB突变体脂质A的质谱分析表明一个或两个肉豆蔻酸取代缺失。这些数据表明HtrB是一种多功能蛋白，可能在调节细胞对各种环境变化的反应中起控制作用。
