Skibinski David A G, Golby Paul, Chang Yung-Sheng, Sargent Frank, Hoffman Ralf, Harper R, Guest John R, Attwood Margaret M, Berks Ben C, Andrews Simon C
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, United Kingdom.
J Bacteriol. 2002 Dec;184(23):6642-53. doi: 10.1128/JB.184.23.6642-6653.2002.
The hyf locus (hyfABCDEFGHIJ-hyfR-focB) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential sigma(54)-dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was sigma(54) dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located -24 and -12 boxes indicative of a sigma(54)-dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR, suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced ( approximately 1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR-expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ. Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.
大肠杆菌的hyf基因座（hyfABCDEFGHIJ - hyfR - focB）编码一种假定的10亚基氢化酶复合物（氢化酶4 [Hyf]）；一种潜在的依赖于σ⁵⁴的转录激活因子HyfR（与FhlA相关）；以及一种假定的甲酸转运蛋白FocB（与FocA相关）。为了深入了解Hyf系统的生理作用，我们使用hyfA - lacZ转录融合来研究hyf的表达。这项工作表明，hyf在发酵条件下由低pH值的甲酸以FhlA依赖的方式诱导表达。其表达依赖于σ⁵⁴，并且受到甲酸调节子的负转录调节因子HycA的抑制。因此，hyf的表达类似于hyc操纵子的表达。引物延伸分析确定了hyfA结构基因上游30 bp处的一个转录起始位点，具有适当定位的 - 24和 - 12框，表明是一个依赖于σ⁵⁴的启动子。未检测到hyfJ - hyfR的逆转录酶PCR产物，这表明hyfR - focB可能与hyf操纵子的其余部分独立转录。在存在从异源启动子表达hyfR的多拷贝质粒的情况下，hyf的表达被强烈诱导（约1000倍）。这种诱导依赖于低pH值、厌氧和指数后期生长，并且被甲酸微弱增强。表达hyfR的质粒仅使fdhF - lacZ转录增加两倍，但不影响hycB - lacZ的表达。有趣的是，染色体hyfR基因的失活对hyfA - lacZ的表达没有影响。发现纯化的HyfR与hyf启动子/操纵区特异性相互作用。在测试的条件范围内，hyf操纵子的失活对生长没有明显影响。未检测到源自Hyf的氢化酶或甲酸脱氢酶活性，也未观察到与HyfG相对应的含镍蛋白。
