Jin Hongfan, Ren Zhen, Whitby Paul W, Morton Daniel J, Stull Terrence L
Department of Microbiology/Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Department of Pediatrics, CHO 2308, 940 Northeast 13th Street, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Microbiology (Reading). 1999 Apr;145 ( Pt 4):905-914. doi: 10.1099/13500872-145-4-905.
Haemophilus haemoglobin-haptoglobin complex and utilizes either as a sole source of haem. Previously, a DNA fragment was cloned from H. influenzae that encodes an approximately 120 kDa protein (HgpA) expressing haemoglobin-binding activity in Escherichia coli. Partial sequence analysis revealed significant homology of HgpA with other bacterial haem- and iron-utilization proteins, and a length of CCAA repeating units immediately following the nucleotide sequence encoding the putative leader peptide. In the present study, the complete nucleotide sequence of the cloned DNA fragment was determined and the sequence was analysed. In addition to homology with other haem- and iron-utilization proteins, seven regions typical of TonB-dependent proteins were identified. The transcript of hgpA was determined to be monocistronic by RT-PCR. PCR performed with different colonies of a single H. influenzae strain at one CCAA-repeat-containing locus indicated varying lengths of CCAA repeats, suggesting that haemoglobin and haemoglobin-haptoglobin binding in H. influenzae is regulated by strand slippage across CCAA repeats, as well as by haem repression. E. coli containing cloned hgpA bound both haemoglobin and the haemoglobin-haptoglobin complex. A deletion/insertion mutation of hgpA was constructed in H. influenzae strain H1689. Mutation of hgpA did not affect the ability of H. influenzae either to bind or to utilize haemoglobin or haemoglobin-haptoglobin following growth in haem-deplete media. Affinity purification of haemoglobin-binding proteins from the mutant strain revealed loss of the 120 kDa protein and an increased amount of a 115 kDa protein, suggesting that at least one additional haemoglobin-binding protein exists.
嗜血杆菌血红蛋白 - 触珠蛋白复合物，并将两者中的任何一种用作血红素的唯一来源。此前，从流感嗜血杆菌中克隆出一个DNA片段，该片段编码一种在大肠杆菌中表达血红蛋白结合活性的约120 kDa蛋白质（HgpA）。部分序列分析显示HgpA与其他细菌血红素和铁利用蛋白具有显著同源性，并且在编码推定前导肽的核苷酸序列之后紧接着一段CCAA重复单元。在本研究中，确定了克隆的DNA片段的完整核苷酸序列并进行了序列分析。除了与其他血红素和铁利用蛋白的同源性外，还鉴定出七个典型的依赖TonB的蛋白区域。通过RT-PCR确定hgpA的转录本是单顺反子。在一个含CCAA重复序列的位点对单个流感嗜血杆菌菌株的不同菌落进行PCR，结果表明CCAA重复序列的长度不同，这表明流感嗜血杆菌中血红蛋白和血红蛋白 - 触珠蛋白的结合受CCAA重复序列的链滑动以及血红素抑制的调节。含有克隆hgpA的大肠杆菌能结合血红蛋白和血红蛋白 - 触珠蛋白复合物。在流感嗜血杆菌菌株H1689中构建了hgpA的缺失/插入突变体。在血红素耗尽的培养基中生长后，hgpA的突变并不影响流感嗜血杆菌结合或利用血红蛋白或血红蛋白 - 触珠蛋白的能力。从突变菌株中亲和纯化血红蛋白结合蛋白，结果显示120 kDa蛋白缺失，115 kDa蛋白量增加，这表明至少存在一种额外的血红蛋白结合蛋白。
