Murooka Y, Ishibashi K, Yasumoto M, Sasaki M, Sugino H, Azakami H, Yamashita M
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
J Bacteriol. 1990 Apr;172(4):2131-40. doi: 10.1128/jb.172.4.2131-2140.1990.
The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment. In E. coli cells which carried the plasmid, the synthesis of arylsulfatase was repressed by various sources of sulfur; the repression was relieved, in each case, by tyramine. Transfer of the plasmid into atsA or constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsA but not of atsR. The nucleotide sequence of the 3.2-kilobase fragment was determined. Two open reading frames, the atsA gene and an unknown gene (atsB), were found. These are located between a potential promoter and a transcriptional terminator sequence. Deletion analysis suggests that atsB is a potential positive factor for the regulation of arylsulfatase. Analysis of the amino acid sequences of the first 13 amino acids from the N terminus of the purified secreted arysulfatase agrees with that of the nucleotide sequence of atsA. The leader peptide extends over 20 amino acids and has the characteristics of a signal sequence. Primer extension mapping of transcripts generated in vivo suggests that the synthesis of mRNA starts at a site 31 or 32 bases upstream from the ATG initiation codon of the atsB gene. By Northern (RNA) blot analysis of the transcripts induced by tyramine, we found a 2.7-kilobase transcript which is identical in size to the total sequence of the atsB and atsA genes. Thus, the ats operon is composed of two cistrons and is regulated by sulfur and tyramine.
产气克雷伯菌芳基硫酸酯酶(atsA)的结构基因被克隆到大肠杆菌的pKI212载体中。缺失分析表明,带有启动子区域的atsA基因位于一个3.2千碱基的克隆片段内。在携带该质粒的大肠杆菌细胞中,芳基硫酸酯酶的合成受到各种硫源的抑制;在每种情况下,酪胺都能解除这种抑制。将该质粒转移到产气克雷伯菌的atsA或组成型atsR突变株中,可使atsA得到互补,但atsR不能。测定了3.2千碱基片段的核苷酸序列。发现了两个开放阅读框,即atsA基因和一个未知基因(atsB)。它们位于一个潜在的启动子和一个转录终止子序列之间。缺失分析表明,atsB是芳基硫酸酯酶调节的一个潜在正调控因子。对纯化的分泌型芳基硫酸酯酶N端前13个氨基酸的氨基酸序列分析与atsA的核苷酸序列分析结果一致。前导肽延伸超过20个氨基酸,具有信号序列的特征。对体内产生的转录本进行引物延伸图谱分析表明,mRNA的合成起始于atsB基因ATG起始密码子上游31或32个碱基处。通过对酪胺诱导的转录本进行Northern(RNA)印迹分析,我们发现了一个2.7千碱基的转录本,其大小与atsB和atsA基因的总序列相同。因此,ats操纵子由两个顺反子组成,并受硫和酪胺的调控。