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Journal of Bacteriology, April 2007, p. 2945-2948, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01723-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Quorum-Sensing Hybrid Histidine Kinase LuxN of Vibrio harveyi Contains a Periplasmically Located N Terminus{triangledown}

Kirsten Jung,* Tina Odenbach, and Melanie Timmen

Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, München, Germany

Received 8 November 2006/ Accepted 17 January 2007


   ABSTRACT
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Hydropathy profile analyses of the amino acid sequence of the quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi predict a periplasmic location of the N terminus. To test this, two-hybrid proteins consisting of LuxN and an N-terminally fused maltose-binding protein with or without a leader sequence were analyzed with regard to the enzymatic activities of LuxN, protease accessibility, and complementation of an Escherichia coli malE mutant. The results strongly support a periplasmic location of the N terminus, implying that LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane.


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Vibrio harveyi, a gram-negative bioluminescent marine bacterium, regulates expression of multiple genes, including those for bioluminescence (luciferase) (1), siderophore production (14), a metalloprotease (18), and type III secretion (8), in a cell density-dependent manner. In contrast to most of the other bacteria, V. harveyi is able to respond to three autoinducers, HAI-1, AI-2, and CAI-1 (25). The autoinducers are recognized by their cognate membrane-integrated hybrid sensor kinases LuxN, LuxP/LuxQ, and CqsS (2, 7, 9, 19). In a recent biochemical study, it was clearly shown that HAI-1 downregulates the autokinase activity of LuxN (22). Nevertheless, the HAI-1 binding site in LuxN is still ill defined. The only known residue that might be involved in HAI-1 binding is Leu166 (7). Hydropathy analyses of LuxN by the use of TopPred (3), TMpred (11), and SOSUI (10) suggested that the membrane-integrated part contains nine transmembrane domains (Fig. 1). This model is well supported by the distribution of positive-charged amino acids according to the positive inside rule (24) (Fig. 1). Since a periplasmically located N terminus of a membrane-integrated protein is not frequently found, we tested the location of the N terminus in LuxN experimentally by applying a MalE fusion strategy.


Figure 1
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  		FIG. 1. Secondary structure model of LuxN of V. harveyi. The model comprises in detail the transmembrane part, whereas the cytoplasmic domains are represented by two circles labeled with the phosphorylation sites at positions H471 and D771. The transmembrane domain model is based on the hydropathy plot prediction according to TopPred (3). Transmembrane domains are represented as rectangles and are numbered with Roman numerals I to IX. Charged amino acid residues are highlighted by black rectangles. Amino acid 166, predicted to be involved in HAI-1 binding (7), is highlighted in the transmembrane domain.

 
Use of MalE as reporter protein. Native MalE is a periplasmic protein, and it contains a leader sequence to translocate the protein through the cytoplasmic membrane via the Sec system. In contrast, MalE with an exact deletion of the signal sequence is produced as a cytoplasmic protein (23). Both malE genes were fused to the 5' ends of luxN, encoding hybrid proteins with either a putative periplasmically located maltose-binding protein (MBPp-LuxN) or a cytoplasmically located MBP (MBPc-LuxN). To determine the location of MBP in each hybrid protein, complementation studies were performed with an MBP-deficient Escherichia coli mutant. E. coli MM39 producing MBPp-LuxN was able to grow on a medium with maltose as the sole carbon source (solidified M9 minimal medium containing 0.1% [wt/vol] maltose), whereas the strain producing MBPc-LuxN was not (Table 1). Since growth was detectable only when the MBP moiety of the chimera was located in the periplasm (see the control experiment with E. coli MM39 and pMAL-p2X), this result suggested that only MBPp-LuxN contains a periplasmically located MBP.


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  		TABLE 1. Complementation of E. coli MM39 with different MBP-LuxN derivativesa

 
Autokinase and phosphotransfer activities of MBP-LuxN hybrid proteins. Next, we tested the autokinase activity of each MBP-LuxN hybrid protein. Our prediction was that only the hybrid protein in which the N terminus of LuxN was located at the correct side of the membrane would result in a correct membrane topology, as indicated by an enzymatically active protein. Inverted membrane vesicles were prepared, and enzymatic activities were tested as described previously (22). As shown in Fig. 2A, MBPp-LuxN had autokinase and phosphotransfer activities to LuxU, as indicated by time-dependent LuxU phosphorylation. As shown previously (22), phosphorylated LuxN was barely detectable in such an assay. By taking into account the somewhat unequal amounts of LuxN derivatives in the membrane vesicles (Fig. 2B), the activity of MBPp-LuxN was found to be in the range of that of the wild-type LuxN. MBPc-LuxN was produced in sufficient amounts and found to be located in the membrane fraction (Fig. 2B); however, this hybrid protein was enzymatically inactive (Fig. 2A). It is important to note that the smear observed in the corresponding lanes of MBPc-LuxN is unrelated to phosphorylated LuxU (LuxU~P) (Fig. 2A), because the Western blot indicates that LuxU runs as a distinct band (Fig. 2B). These results indicate that only LuxN with a periplasmically fused MBP is correctly integrated into the membrane.


Figure 2
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  		FIG. 2. Autokinase and LuxU phosphotransfer activities of MBPp-LuxN-His6, MBPc-LuxN-His6, and LuxN-His6. (A) Autokinase and phosphotransfer activities were tested with inverted membrane vesicles containing MBPp-LuxN-His6 (no. 1 lanes, 1.5 mg protein/ml; 9.6 µg membrane protein/lane), MBPc-LuxN-His6 (no. 2 lanes, 11 mg protein/ml; 70 µg membrane protein/lane), and LuxN-His6 (no. 3 lanes, 4 mg protein/ml; 26 µg membrane protein/lane) in phosphorylation buffer (50 mM Tris-HCl [pH 8], 10% [vol/vol] glycerol, 500 mM KCl, 110 µM MgCl2, 2 mM dithiothreitol). The reaction was started by the addition of 100 µM [{gamma}-32P]ATP (0.94 Ci/mmol) and run for 1 min before purified His6-LuxU (0.1 mg/ml; 2 µg/lane) was added. The amount of total protein used per assay was adjusted to test comparable amounts of the LuxN derivatives. The reactions were stopped after 5 and 30 min. The phosphorylated proteins were separated by 15% SDS-polyacrylamide gel electrophoresis (PAGE), and radioactivity was detected by autoradiography. (B) Membrane vesicles containing the same amounts of membrane proteins as reported for panel A were separated by 15% SDS-PAGE, immunoblotted, and probed with a penta-His antibody. As a control, membrane vesicles of E. coli TKR2000 (lane 4, 25 µg membrane protein/lane) were separated and immunoblotted.

 
Protease accessibility of the periplasmic domains of MBP-LuxN hybrid proteins. To test the accessibility of the periplasmic domains of the MBP-LuxN hybrid proteins, spheroplasts bearing the different derivatives were treated with trypsin. This study would further determine the location of MBP in the two-hybrid proteins. Spheroplasts were prepared from E. coli strain TKR2000 ({Delta}kdpFABCDE trkA405 trkD1 atp706) (13) transformed with the plasmid pMALp-luxN, pMALc-luxN, or pKKLuxNb-His6. Cells were grown aerobically at 37°C in KML complex medium (1% tryptone, 0.5% yeast extract, and 1% KCl) supplemented with ampicillin (100 µg/ml). At mid-logarithmic growth phase, luxN gene expression was induced with 0.5 mM isopropyl-1-thio-ß,D-galactoside for 2 h. Cells were harvested at an absorbance at 600 nm of ~1.0. For spheroplast preparation, a protocol described by Mendrola et al. was used (16). Spheroplasts were collected by centrifugation at 500 x g for 5 min, resuspended in 90 µl resuspension buffer (100 mM Tris-HCl [pH 8], 500 mM sucrose, 0.5 mM EDTA, 2.5 mg/ml lysozyme), and divided into three aliquots. To one aliquot, 6 µl of trypsin (Sigma, Deisenhofen) in resuspension buffer (trypsin:protein, 1:10 [wt/wt]) was added; to the second aliquot, 3 µl of 10% (vol/vol) Triton X-100 and trypsin in resuspension buffer was added. The third aliquot remained untreated (except for the addition of resuspension buffer to maintain volume). Proteolysis was carried out at 37°C for 30 min. Then, proteins from each sample were precipitated by the addition of 3.5 µl of 100% trichloric acid, and pellets were resuspended in sodium dodecyl sulfate (SDS) loading buffer for electrophoresis and Western blotting. LuxN fragments were detected by Western blotting using antibodies directed against the His tag (22). MBP was completely degraded by trypsin in spheroplasts containing MBPp-LuxN but remained untouched in spheroplasts bearing MBPc-LuxN. Trypsin treatment of spheroplasts permeabilized with Triton X-100 resulted in a complete degradation of both hybrid proteins (Fig. 3). These results further confirm the periplasmic location of MBPp when it is fused to the N terminus of LuxN.


Figure 3
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  		FIG. 3. Protease treatment of MBP-LuxN-His6 and LuxN-His6 in spheroplasts. Spheroplasts containing MBPp-LuxN-His6 (lanes 1 to 3), MBPc-LuxN-His6 (lanes 4 to 6), and LuxN-His6 (lanes 7 to 9) were incubated with or without trypsin (1:10 [wt/wt]) at 37°C for 30 min and permeabilized when indicated with Triton X-100. Proteins (30 µg/lane) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted using a penta-His antibody for detection. Lanes 1, 4 and 7, spheroplasts without any treatment; lanes 2, 5 and 8, spheroplasts digested with trypsin; lanes 3, 6 and 9, spheroplasts digested with trypsin and permeabilized with Triton X-100.

 
In summary, our studies provide experimental evidence for a model according to which LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane, whereby the N terminus is periplasmically located. According to this model, Leu166 is embedded in the middle of the protein at the outer part of transmembrane domain V, allowing access of HAI-1 from the periplasmic side.

A periplasmic location of the N terminus of an integral membrane protein has not been reported often. The 13-helix motif was found to be a feature of members of the Na+/solute cotransporter family, e.g., PutP of E. coli (12). Most sensor kinases contain two transmembrane domains. For some sensor kinases with more than two transmembrane domains, the membrane topology was determined to be an even number (four to six), implying a cytoplasmic location of the N terminus (6, 20, 21, 26). The histidine kinase AgrC of Staphylococcus aureus is, to our knowledge, the only example for which an outside location of the N terminus has been proposed (15). Interestingly, AgrC and LuxN are similarly involved in quorum sensing.

Daley et al. analyzed the inner membrane proteome of E. coli (4). Using C-terminal tagging with alkaline phosphatase and green fluorescent protein, they were able to determine the C-terminal location of 502 membrane proteins, but the location of the N terminus remained unclear. Here, we applied the maltose-binding protein hybrid technique, which has already been used for other membrane proteins (5, 17). This fusion technique seems to be the most suitable for the determination of the location of the N terminus in membrane proteins.


   ACKNOWLEDGMENTS
 
We thank B. L. Bassler (Princeton University, NJ) and H. Jung (LMU, München, Germany) for providing cosmids or plasmids and many critical discussions. In addition, we thank J. Beckwith (Harvard University, Cambridge, MA) for providing E. coli MM39.

This work was financially supported by the BMBF-Verbundvorhaben MetaGenoMik.


   FOOTNOTES
 
* Corresponding author. Mailing address: Ludwig-Maximilians-Universität, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München, Germany. Phone: 49 89 2180 6120. Fax: 49 89 2180 6122. E-mail: kirsten.jung{at}lrz.uni-muenchen.de. Back

{triangledown} Published ahead of print on 26 January 2007. Back


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Journal of Bacteriology, April 2007, p. 2945-2948, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01723-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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