Lemonnier Marc, Lane David
Microbiology (Reading). 1998 Mar;144 ( Pt 3):751-760. doi: 10.1099/00221287-144-3-751.
Certain amino acids are substrates for two decarboxylase enzymes in Escherichia coli, one inducible by anaerobic growth at low pH and the other constitutive. In the case of lysine, an inducible decarboxylase (CadA) has been extensively characterized, but evidence for the existence of a second lysine decarboxylase is fragmentary and uncertain. This paper confirms that a second lysine decarboxylase is encoded by a locus (ldc) previously suggested to be a lysine decarboxylase gene on the basis of sequence comparisons. Overexpression of the cloned gene provided sufficient quantities of enzyme in cell-free extracts for preliminary examination of the properties of the ldc gene product, Ldc. The enzyme is active over a broad range of pH with an optimum at 7.6, much higher than that of CadA, about 5.5. The temperature optimum for both enzymes is similar, at about 52 degrees C, but Ldc is more readily inactivated by heat than CadA. Expression of ldc from its own promoter was very weak for cells growing in a variety of media, although a low level of lysine decarboxylase was present in cells that carried the ldc region on an oligo-copy plasmid when these were grown in minimal-glucose medium. Northern analysis of RNA extracted from such cells revealed a transcript whose length corresponded to that of the ldc gene, suggesting that ldc is normally transcribed from a promoter immediately upstream. However, most of the ldc mRNA was shorter, indicating degradation or premature termination. The ldc upstream sequence promoted transcription of a lacZ gene to which it was fused. Introduction of the upstream sequence as an insert in a multicopy vector increased transcription of the resident lacZ fusion. The low level of expression in single copy, the emergence of expression when the gene is present at moderate copy number, and the derepression by the upstream sequence in trans imply that this second lysine decarboxylase gene may not be constitutive but subject to specific repression by a factor which remains to be identified.
某些氨基酸是大肠杆菌中两种脱羧酶的底物，一种在低pH值下厌氧生长时可诱导产生，另一种则是组成型的。就赖氨酸而言，一种可诱导的脱羧酶（CadA）已得到广泛研究，但关于第二种赖氨酸脱羧酶存在的证据却支离破碎且不确定。本文证实，第二个赖氨酸脱羧酶由一个位点（ldc）编码，该位点先前基于序列比较被认为是一个赖氨酸脱羧酶基因。克隆基因的过表达在无细胞提取物中提供了足够量的酶，用于初步检测ldc基因产物Ldc的特性。该酶在广泛的pH范围内具有活性，最适pH为7.6，远高于CadA的最适pH（约5.5）。两种酶的最适温度相似，约为52℃，但Ldc比CadA更易受热失活。对于在各种培养基中生长的细胞，从其自身启动子表达的ldc非常弱，尽管当携带ldc区域的寡拷贝质粒在最低葡萄糖培养基中生长时，细胞中存在低水平的赖氨酸脱羧酶。对从此类细胞中提取的RNA进行Northern分析，发现了一个转录本，其长度与ldc基因的长度相对应，表明ldc通常从紧邻上游的启动子转录。然而，大多数ldc mRNA较短，表明存在降解或提前终止。ldc上游序列促进了与其融合的lacZ基因的转录。将上游序列作为插入片段导入多拷贝载体中，可增加常驻lacZ融合基因的转录。单拷贝时表达水平低，基因以中等拷贝数存在时出现表达，以及上游序列的反式去阻遏作用，这意味着这第二个赖氨酸脱羧酶基因可能不是组成型的，而是受到一个有待确定的因子的特异性抑制。
