XylE as a marker gene for microorganisms

Abstract

The xylE gene encodes catechol 2,3 dioxygenase (C23O) (E.C. 1.13.1.2), a critical enzyme in the dissimilation of toluenes, benzoates and their methyl derivatives via the meta pathway [12]. The enzyme breaks down catechol to the yellow-coloured product 2-hydroxymuconic semialdehyde. The presence of C23O in bacteria or protein preparations is thus evident from the formation of the resulting yellow product. The xylE system does not require specific or selective media in order to detect positive colonies [15]. Bacteria can be grown on any appropriate growth substrate or media, if necessary replicated on to separate plates and the original sprayed with catechol to detect C23O+ colonies [6]. C23O is inactivated in the presence of oxygen and is therefore rapidly destroyed outside the bacterial cell unless protected by acetone or ethanol [7]. The detection of active C23O should therefore be indicative of intact and therefore probably viable host cells [6]. As with other indicator genes there may be problems with the presence of indigenous bacteria bearing xylE or related genes. However, these have been estimated to constitute a very small fraction of the microbial population in most environments and are completely absent from others [1]. Any problem with background can be overcome by the use of additional genetic markers and/or the characteristic induction of xylE when coupled to inducible promoters (see below). The presence of yellow-pigmented bacteria in test systems does not seem to be a problem since yellow haloes around colonies and changes in intensity of yellow-colouration on induction of xylE distinguish positive colonies from other bacteria.