Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N‐acylhomoserine lactone signal molecules

Abstract

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.

Quorum Sensing and the Lifestyle ofYersinia

“…However, in Y. enterocolitica, yenI is not subject to such autoinduction since inactivation of yenI did not abolish production of yenI mRNA (Throup et al, 1995). This observation was confirmed by examining the expression of yenI'::luxAB transcriptional fusions as chromosomal insertions in both the parent and yenI mutant respectively.…”

“…All of these strains induced bioluminescence in the reporter offering preliminary evidence that Yersinia spp. were AHL producers (Throup et al, 1995). Subsequently, Yersinia pestis and Yersinia ruckeri (unpublished data) were also confirmed as AHL-producers using the same strategy.…”

“…These AHLs were identified as 3-oxo-C6-HSL and N-hexanoyl homoserine lactone (C6-HSL) respectively ( Fig. 1) (Throup et al, 1995) and are easily separated and visualized by thin layer chromatography (TLC) as purple spots on a white background using the Chromobacterium violaceum CV026 biosensor in an agar overlay (see Fig. 2 for an example of this TLC overlay originally described by McClean et al, 1997).…”

“…Since AHL synthesis depends on the presence of an AHL synthase, a plasmid-based in trans complementation strategy in which a Y. enterocolitica chromosomal gene library was transformed into E. coli [pSB401] was devised (Swift et al, 1993;Throup et al, 1995). This approach yielded a highly bioluminescent clone indicative of an insert carrying a gene whose product was capable of inducing the AHL reporter fusion.…”

“…However, since they lack good chromophores, relatively high concentrations are required for detection via their ultra violet spectroscopic properties following separation by HPLC. As a consequence, a number of sensitive bioassays have been developed which facilitate detection of AHL production by bacterial colonies on plates, in microtitre plate assays, on thin layer chromatograms (TLC) and in fractions collected after HPLC-based separation (Bainton et al, 1992;Throup et al, 1995;McClean et al, 1997;Shaw et al, 1997;Winson et al, 1998). For example, Throup et al (1995) constructed a recombinant AHL reporter plasmid which coupled luxR and the luxI promoter region from Vibrio fischeri to the luxCDABE structural operon of Photorhabdus luminescens.…”