Sanders J D, Cope L D, Hansen E J
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048.
Infect Immun. 1994 Oct;62(10):4515-25. doi: 10.1128/iai.62.10.4515-4525.1994.
Haemophilus influenzae has an absolute requirement for heme for aerobic growth. This organism can satisfy this requirement by synthesizing heme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme. In contrast, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme. A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DL42 the ability to grow on iron and PPIX. Nucleotide sequence analysis revealed that this NTHI DNA insert contained three genes, designated hitA, hitB, and hitC, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmic binding protein-dependent iron transport system in this enteric organism. The NTHI HitA protein also was 69% identical to the ferric-binding protein of Neisseria gonorrhoeae. Inactivation of the cloned NTHI hitC gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth deficiency of Hib DL42. Construction of an isogenic NTHI TN106 mutant lacking a functional hitC gene revealed that this mutation prevented this strain from growing on a medium containing iron and PPIX in place of heme. This NTHI hitC mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the products encoded by the hitABC genes are essential for the utilization of iron by NTHI.
流感嗜血杆菌需血红素才能进行有氧生长。该菌可通过由铁和原卟啉IX（PPIX）合成血红素来满足这一需求。发现b型流感嗜血杆菌（Hib）菌株DL42在含有游离铁和PPIX而非血红素的培养基上生长时无法形成单菌落。相比之下，不可分型流感嗜血杆菌（NTHI）菌株TN106能在同一培养基上轻松生长。用NTHI菌株TN106的基因组文库转化Hib菌株DL42，并在含有铁和PPIX而非血红素的培养基上筛选重组体。一个带有11.5 kb NTHI DNA插入片段的重组质粒显示能赋予Hib菌株DL42在铁和PPIX上生长的能力。核苷酸序列分析表明，该NTHI DNA插入片段包含三个基因，分别命名为hitA、hitB和hitC，它们编码的产物与粘质沙雷氏菌的SfuABC蛋白相似，在这种肠道菌中，SfuABC蛋白已被证明构成一种依赖周质结合蛋白的铁转运系统。NTHI的HitA蛋白与淋病奈瑟菌的铁结合蛋白也有69%的同一性。通过插入抗生素抗性盒使克隆的NTHI hitC基因失活，消除了重组质粒互补Hib DL42生长缺陷的能力。构建一个缺失功能性hitC基因的同基因NTHI TN106突变体表明，该突变阻止了该菌株在含有铁和PPIX而非血红素的培养基上生长。这个NTHI hitC突变体也无法利用与转铁蛋白结合的铁或铁螯合物。这些结果表明，hitABC基因编码的产物对NTHI利用铁至关重要。
