Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium

Abstract

The phenomenon of phase variation in the insect-pathogenic bacterium Xenorhabdus luminescens was investigated. Differential activity of the lipase enzyme (EC 3.1.1.3) was observed between the two phases of the bacteria. The enzyme was found to be secreted into the culture medium, and about five to six times greater specific activity was secreted by the primary phase than by the secondary form. The lipase gene (lip-1) was cloned and sequenced. The data imply that there is only a single Tween 80-utilizing lipase gene in X. luminescens K122. The sequence revealed a translation product of 645 amino acids, from which a hydrophobic leader sequence of 24 amino acids is removed during processing. The structure of the gene was shown to be the same in the primary and secondary forms of X. luminescens. In addition, transcription was found to start at the same position, 169 bp upstream of the translation initiation codon, in the two forms of the bacteria. Equal amounts of lipase RNA accumulated in the two forms, and at least as much lipase protein was secreted by the secondary form as by the primary. This suggests that the difference in specific activity between the enzymes secreted by the two phases probably arises from a posttranslational type of regulation.

A Phosphopantetheinyl Transferase Homolog Is Essential for Photorhabdus luminescens To Support Growth and Reproduction of the Entomopathogenic Nematode Heterorhabditis bacteriophora

“…The secondary-phase variants appear at high frequency during prolonged culturing, while more rare is the generation of primary-phase cells from secondary phase (6). The secondaryphase cells differ from the primary-phase cells in colony morphology, cell size, and dye uptake characteristics (6,7,9,52). Also, typical primary-phase characteristics such as bioluminescence, pigment synthesis, phospholipase and siderophore activities, and production of intracellular crystalline inclusion proteins are depressed or absent in secondary-phase cells.…”

“…Genetic studies of P. luminescens have been limited, probably because of a low frequency of transformation (7) and the current inability to introduce recombinant DNA into P. luminescens by conjugation (52). Success has been achieved in using allelic exchange to construct disruptions in the genes encoding intracellular inclusion proteins CipA and CipB (7) and insecticidal toxin genes tca, tcb, tcc, and tcd (10) of P. luminescens.…”

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

“…Previous studies have suggested that primary-phase variants of Photorhabdus temperata produce more active lipase enzyme than the secondary variants, whilst levels of lipase gene transcription remain the same (Wang & Dowds, 1993). This led to the hypothesis that the difference in the specific activity of the lipase between the two phenotypic variants may be associated with post-translational regulation (Wang & Dowds, 1993). Whilst we did not examine levels of PrtA gene transcription in the two variants, we note that differences in the association of PrtA with its candidate inhibitor represent a potential mechanism for generating the apparent differences in expression we observed in this study.…”

“…This may be due to an activation of inherently inhibited phase II secreted protease following treatment with SDS, reducing agent and heating. Previous studies have suggested that primary-phase variants of Photorhabdus temperata produce more active lipase enzyme than the secondary variants, whilst levels of lipase gene transcription remain the same (Wang & Dowds, 1993). This led to the hypothesis that the difference in the specific activity of the lipase between the two phenotypic variants may be associated with post-translational regulation (Wang & Dowds, 1993).…”

A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize

“…proteins in this group are known to possess lipolytic activity. These include a lipase/acyltransferase from Aeromonas hydrophila [10], a hemolysin from Vibrio parahaemolyticus [11] and a lipase from Xenorhabdus luminescens [12]. In addition, we have already used our motif scheme to identify the catalytic triad of the Aeromonas hydrophila enzyme [13].…”