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Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.

机译:克隆乳酸乳球菌亚种噬菌体感染所需的染色体基因。乳酸C2。

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摘要

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.
机译:噬菌体抗性突变体,其中乳酸乳球菌亚种的噬菌体感染所需的膜成分存在缺陷。用野生型噬菌体敏感菌株的染色体文库转化乳酸C2。在筛选的4200个噬菌体敏感性转化子中,有3个被阳性鉴定为噬菌体敏感性。基于以下两个标准,确定了克隆的染色体片段与噬菌体敏感性表型之间的因果关系:(i)在没有抗生素选择压力的情况下,克隆片段的丢失频率与噬菌体敏感性丧失的频率; (ii)将噬菌体敏感性转移到用克隆的片段转化的受体的100%噬菌体抗性细胞中。用限制性核酸内切酶对来自三个独立分离株的克隆染色体DNA进行物理定位。克隆的片段的大小为9.6、11.8和9.5 kb。每个片段都包含所有三个片段共有的相同DNA片段,即9.4 kb。赋予噬菌体敏感性的基因通过亚克隆至4.5kb区域而定位。进一步的亚克隆表明,在4.5kb区域内的单个EcoRI位点必须位于基因或其启动子内。对所需的4.5-kb区域进行了测序,发现其编码了一个部分阅读框架和两个完整的开放阅读框架。互补所需的基因在功能上通过Tn5诱变定位,并定位于两个完整的开放阅读框之一,称为pip(噬菌体感染蛋白的缩写)。点的长度是2,703个碱基。潜在的启动子在开放阅读框上游起始206和212个碱基。核糖体结合位点和七碱基间隔子位于GTG(Val)翻译起始密码子之前。从该基因推导的氨基酸序列具有901个残基和99(426)的M(r)。亲水性分析揭示了四到六个潜在的跨膜区域,一个靠近氨基末端,另一个位于极端羧基末端。氨基末端具有信号序列的特征。推定的蛋白质将具有650个残基的中央极性结构域。

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