Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae.
Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae.
复制标题
流感嗜血杆菌血红素免疫基因的克隆与鉴定。
DOI：
10.1128/jb.179.5.1684-1689.1997
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发表时间：
1997
影响因子：
3.2
通讯作者：
LiPuma,JJ
中科院分区：
文献类型：
--
作者：
Murley,YM;Edlind,TD;Pozsgay,JM;LiPuma,JJ
细菌素血菌素由大多数流感嗜血杆菌的B型菌株（包括不同遗传谱系的菌株）产生，并且对几乎所有不可分型的H.流感病毒株。一个H。携带质粒的流感嗜血杆菌，该质粒具有1.5-kbp的染色体片段，能够赋予对血霉素敏感的H.用部分纯化的血红蛋白筛选流感病毒突变株。缺失和定点诱变定位的血红素免疫基因的3'开放阅读框（ORF）在这个染色体片段。该ORF的亚克隆证明，它足以赋予野生型血霉素敏感的H.流感菌株以及大肠杆菌的溶血素敏感菌株。该ORF命名为hmcl，编码一个105个氨基酸的蛋白质，估计分子量为12.6 kDa。引物延伸分析揭示了一个假定的转录起始位点上游34 bp的起始密码子，并证实了直接上游的hmcI启动子的存在下，通过克隆到一个启动子氯霉素乙酰转移酶载体的基因。为了表征hmcI基因产物，构建了His-HmcI融合蛋白。
The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.