Quorum sensing in Yersinia enterocolitica controls swimming and swarming motility

Abstract

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26 degrees C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) or fliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

FIG. 1.

Analysis of swimming motility in Y. enterocolitica and the isogenic yenI mutant on 0.3% swim agar plates which were point-inoculated and grown at 26°C. (A) The parent and the yenI mutant after 17 and 24 h showing that the yenI mutant is unable to swim when compared to the parent but begins to swim after 24 h. (B) The complemented yenI mutant containing either pSU18 or pSUyenI after 17 and 24 h showing that swimming motility is restored to parental levels in the yenI mutant when a functional copy of yenI is supplied in trans.

FIG. 2.

Analysis of swarming motility in Y. enterocolitica and the isogenic yenI mutant on 0.6% swarm agar plates grown at 26°C and monitored after 24 h. (A) The yenI mutant is unable to swarm compared to the parent. (B) When the yenI mutant is complemented with a functional copy of yenI in trans (pSUyenI) swarming motility is restored to parental levels whereas the yenI mutant complemented with the vector alone remains nonmotile.

FIG. 3.

SDS-PAGE of flagellar proteins isolated from Y. enterocolitica and the isogenic yenI mutant after 17 h of growth in liquid culture. (A) The ∼45-kDa band is present in flagellar extracts taken from the parent but absent from the yenI mutant. (B) There is no band visible for the yenI mutant, indicating that the mutant is incapable of producing flagellin.

FIG. 4.

RT-PCR analysis of the presence of flhDC (A), fliA (B) and fleB (C) transcripts in the Y. enterocolitica parent and yenI mutant grown in liquid culture at 26°C for 14 and 17 h, respectively. In each case lane 1 is the positive control, lanes 2 and 3 are the PCR using RNA templates isolated from the parent and yenI mutant, respectively, prior to first-strand cDNA synthesis, lanes 4 and 5 are the PCR using the newly synthesized cDNA templates isolated from the parent and yenI mutant, respectively. The size of the PCR products from primers Mot14 and Mot15 (flhDC) is 832 bp, that of Mot16 and Mot17 (fliA) is 435 bp, and that of Mot18 and Mot19 (fleB) is 778 bp.

FIG. 5.

TLC chromatograms of the AHL profiles from supernatant extracts of Y. enterocolitica strains grown at 26°C for 3 h. (A) Using the AHL biosensor E. coli[pSB1075], lane 1, synthetic standards (from bottom) 3.0 × 10⁻¹² mol of 3-oxo-C14-HSL, 3.3 × 10⁻¹³ mol of 3-oxo-C12-HSL, and 3.7 × 10⁻¹² mol of 3-oxo-C10-HSL. Lanes 2 to 4, AHLs extracted from Y. enterocolitica, the yenI mutant, and the yenI mutant complemented with pSUyenI, respectively. (B) Using the C. violaceum CV026 AHL biosensor, lane 1, synthetic standard 4.7 × 10⁻¹⁰ mol of 3-oxo-C6-HSL. Lane 2, synthetic standards (from bottom) 4.4 × 10⁻⁹ mol of C8-HSL and 5.0 × 10⁻¹¹ mol of C6-HSL. Lanes 3 to 5, AHLs extracted from Y. enterocolitica, the yenI mutant, and the yenI mutant complemented with pSUyenI, respectively. Migration of AHLs can vary by as much as ±5% compared to synthetic standards.

FIG. 6.

Mass spectra of three long-chain AHLs purified from spent culture supernatants of E. coli JM109 transformed with yenI on pYeI7 which correspond with those of synthetic (A) 3-oxo-C10-HSL ([M+H] 269), (B) 3-oxo-C12-HSL ([M+H] 298), and (C) 3-oxo-C14-HSL ([M+H] 326).

FIG. 6.

Mass spectra of three long-chain AHLs purified from spent culture supernatants of E. coli JM109 transformed with yenI on pYeI7 which correspond with those of synthetic (A) 3-oxo-C10-HSL ([M+H] 269), (B) 3-oxo-C12-HSL ([M+H] 298), and (C) 3-oxo-C14-HSL ([M+H] 326).

FIG. 6.

Mass spectra of three long-chain AHLs purified from spent culture supernatants of E. coli JM109 transformed with yenI on pYeI7 which correspond with those of synthetic (A) 3-oxo-C10-HSL ([M+H] 269), (B) 3-oxo-C12-HSL ([M+H] 298), and (C) 3-oxo-C14-HSL ([M+H] 326).

FIG. 7.

(A) Uptake of tritiated 3-oxo-C12-HSL by the Y. enterocolitica parent, yenI mutant, and E. coli JM109[pSB401]. (B) Induction of bioluminescence after 8 h in the Y. enterocolitica yenI mutant containing the AHL biosensor pSB401 when supplied with an exogenous source of 3-oxo-C6 HSL or 3-oxo-C12 HSL.