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首页> 美国卫生研究院文献>Journal of Bacteriology >Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.
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Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

机译：用铜绿假单胞菌的几种非弹性蛋白酶生产菌株中的弹性蛋白酶结构基因检测大肠杆菌中的弹性蛋白酶生产。

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The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.
机译：铜绿假单胞菌IFO 3455的弹性蛋白酶结构基因已被克隆并测序。使用此基因作为探针，我们从非产生弹性蛋白酶的菌株（分别为铜绿假单胞菌IFO 3080，N-10和PA103）克隆了弹性蛋白酶基因的DNA片段（pEL3080R，pEL10和pEL103R）。这三种假单胞菌菌株通过蛋白质印迹（免疫印迹）或弹性蛋白酶活性均未显示出可检测水平的弹性蛋白酶抗原性。当将长度约为8 kb的弹性蛋白酶结构基因克隆到大肠杆菌表达载体pUC18中时，我们能够检测到两个细菌克隆（大肠杆菌pEL10和大肠杆菌pEL103R）中的弹性蛋白酶抗原性和弹性。然而，尽管观察到弹性蛋白酶mRNA，但在大肠杆菌pEL3080R克隆中未检测到弹性蛋白酶活性或弹性蛋白酶抗原性。用这三个结构基因的几种限制性酶确定的部分限制性图谱对应于铜绿假单胞菌IFO 3455的图谱。我们对来自非产生弹性蛋白酶的菌株的弹性蛋白酶基因的三个DNA片段进行了测序，并将其与来自弹性蛋白酶的序列进行了比较。生产铜绿假单胞菌菌株IFO 3455和PAO1。在铜绿假单胞菌N-10和PA103中，除了几处核苷酸差异外，其序列与产生弹性蛋白酶的菌株的序列几乎相同。这些微小差异可能反映了弹性蛋白酶基因的微观异质性。这些结果表明，两个不产生弹性蛋白酶的菌株具有正常的弹性蛋白酶结构基因，并且通过调节该基因在铜绿假单胞菌中的表达来抑制弹性蛋白酶的产生。本文提供了这两种菌株中缺乏表达的可能原因。在铜绿假单胞菌IFO 3080中，该序列在编码区具有1个碱基的缺失，这应该引起了氨基酸序列的移码变化。目前，我们还没有解释该菌株的异常后转录行为。

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