Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi

Abstract

Vibrio harveyi regulates the expression of bioluminescence (lux) in response to cell density, a phenomenon known as quorum sensing. In V. harveyi, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.

FIG. 1

Genetic organization of the luxO-luxU region of the V. harveyi chromosome. The region encompassing luxO and luxU in the V. harveyi chromosome is shown. Sequence analysis suggests that a partial ORF encoding a V. harveyi uvrB homologue is located upstream of luxO and extends 692 bp into the sequenced fragment. The luxO gene is encoded by bp 1084 to 2445, and the luxU gene extends from bp 2442 to bp 2787. Two other ORFs, designated ORFA and ORFB, reside downstream of luxU and extend from bp 3302 to 3655 and from bp 3682 to bp 4312, respectively. Arrows designate the direction of transcription of each ORF. The extent of the chromosomal deletion in strain JAF78 is also depicted, showing that it encompasses both luxO and luxU. The PstI and MfeI sites used to insert Kn^r and Cm^r cassettes are also shown (see Materials and Methods).

FIG. 2

LuxU is involved in quorum sensing in V. harveyi. Cultures of wild-type and mutant V. harveyi strains were grown overnight in AB medium at 30°C. The strains were diluted 1:5,000 into fresh AB medium, and light production was subsequently measured at 30-min intervals during growth of the cultures. Symbols: ■, BB120 (wild type); •, JAF78 (ΔluxOU-Cm^r); ▵, JAF483 (luxO D47A); □, JAF536 (luxU::Kn^r); ○, JAF553 (luxU H58A). Relative light units are defined as 10³ counts per minute per milliliter divided by CFU per milliliter.

FIG. 3

Sequence analysis of luxU and alignment of LuxU with other phosphorelay proteins. The nucleotide and deduced amino acid sequences of LuxU are shown in panel A. The initiation and termination codons of the LuxU ORF are in boldface. The termination codon for the LuxO ORF is underlined. Panel B shows an alignment of a portion of LuxU with other phosphorelay modules over the 20-amino-acid conserved phosphorelay region (, –27, 31, 35, 39, 41, 44). The most highly conserved residues in this region are in boldface. A theoretical consensus sequence is shown in the bottom line of the alignment (24). In the consensus sequence, the conserved histidine is represented by H, the letter h denotes a hydrophobic residue, and a plus sign denotes a positively charged residue.

FIG. 4

Epistasis analysis to determine the Lux signalling pathway. V. harveyi cultures were grown overnight in AB medium and diluted 1:5,000 on the next morning. The diluted cultures were subsequently grown to a high cell density (∼10⁸ CFU/ml). The light production per cell of each strain was quantified as described in the legend to Fig. 2. The strains are BB120 (wild type), JAF536 (luxU::Kn^r), JAF549 (luxN L166R), JAF558 (luxN L166R, luxU::Cm^r), JAF548 (luxO D47E), and JAF552 (luxO D47E luxU::Kn^r).

FIG. 5

Model for the regulation of quorum sensing in V. harveyi. (A) Genetic analysis (18) suggests that under conditions of low cell density, in the absence of autoinducers, the two hybrid sensor kinases LuxN and LuxQ autophosphorylate at conserved histidine residues (H1). Intramolecular phosphotransfer from the sensor kinase domains to conserved aspartate residues (D1) in the response regulator domains of the hybrid proteins occurs next. Intermolecular transfer from D1 of both LuxN and LuxQ to His 58 (H2) of the phosphorelay protein LuxU occurs, with subsequent phosphotransfer to the conserved Asp 47 residue (D2) of the response regulator protein LuxO. Our evidence suggests that phosphorylation of LuxO activates its repressor function, luxCDABEGH transcription is repressed, and no light is produced. (B) Under conditions of high cell density, the presence of autoinducers stimulates the LuxN and LuxQ sensors to switch from kinases to phosphatases. This switch ultimately results in dephosphorylation of LuxO and inactivation of its repressor function. Dephosphorylation could occur by several different mechanisms, and these terminal reactions have not been characterized. Inactivation of LuxO allows transcription of the luxCDABEGH operon and light production. Outer membrane and inner membrane are abbreviated OM and IM, respectively. HTH, helix-turn-helix.