Expression of the second lysine decarboxylase gene of Escherichia coli.
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.
Lemonnier
Marc
M
Lane
David
D
D001483:Base Sequence; D015152:Blotting, Northern; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
D002262:Carboxy-Lyases; C021520:lysine decarboxylase