Vol. 172, No. 7JOURNAL OF BACTERIOLOGY, JUlY 1990, p. 3974-39790021-9193/90/073974-06$02.00/0Copyright X 1990, American Society for Microbiology

Critical Regions of the Vibrio fischeri LuxR Protein Definedby Mutational Analysis

JAMES SLOCK,1t DEBORAH VANRIET,2 DANA KOLIBACHUK,2t AND E. P. GREENBERG'*Department of Microbiology, College of Medicine, University ofIowa 52242,1 and Department ofMicrobiology,

New York State College ofAgriculture and Life Sciences, Cornell University, Ithaca, New York 148532

Received 30 January 1990/Accepted 27 April 1990

Expression of Vibrio fischeni luminescence genes requires an inducer, termed autoinducer, and a positiveregulatory element, the luxR gene product. A plasmid containing a tac promoter-controled luxR was

mutagenized in vitro with hydroxylamine, and luxR mutant-plasmids were identified by their inability tocomplement a luxR deletion mutation in trans. Sixteen luxR mutant plasmids were obtained, ten of whichencoded full-length-but inactive luxR gene products as demonstrated by a Western immunoblot analysis. Theeffects of 1 of the 10 mutations could be overcome by the addition of autoinducer at a high concentration. Themutations in each of the 10 mutant plasmids that directed the synthesis of an inactive LuxR protein were

identified by DNA sequencing. Of the 10 proteins encoded by the mutant luxR plasmids, 9 differed from thenormally active LuxR in only a single amino acid residue. The amino acid residue substitutions in the proteinsencoded by the nine mutant luxR genes clustered in two regions. One region around the middle of thepolypeptide encoded by luxR was hypothesized to represent an autoinducer-binding domain, and the otherregion towards the carboxy terminus of the gene product was hypothesized to constitute a lux operatorDNA-binding domain or a lux operator DNA recognition domain.

The luminescent marine bacterium Vibrio fischeri can beisolated from seawater, and it also occurs as the bacterialsymbiont in the light organs of certain marine fishes (29,32-34). The luminescence of V. fischeri is inducible; theinducer, N-(3-oxohexanoyl)homoserine lactone, termed au-toinducer, is a diffusible metabolite produced by V. fischerithat accumulates at equal concentrations in the culturemedium and in cells during growth. When autoinducerreaches a concentration of a few molecules in a cell, induc-tion of luminescence commences (10, 11, 20, 28). At high celldensities, autoinducer can accumulate and induction ofluminescence will occur. Presumably this is the case in thelight organ symbiosis, in which V.fischeri occurs at densitieson the order of 1010 cells per ml of light organ fluid. Inlow-cell-density habitats such as seawater, where V. fischeriexists at less than 102 cells per ml, autoinduction of lumines-cence would not be expected (29, 32-34). Thus, autoinducerserves as a chemical signal in this cell density-dependentcontrol system that allows a rapid cellular response to anenvironmental change (10, 20).A 9-kilobase fragment of V. fischeri DNA that encodes all

of the functions for luminescence and contains regulatoryelements sufficient for their expression in Escherichia colihas been isolated. Seven lux genes have been defined, andthese genes are organized as two divergently transcribedunits (Fig. 1). One transcriptional unit contains luxR, whichencodes a protein (the LuxR protein) required for cells torespond to autoinducer. The other operon contains luxA andluxB, which encode the a and P subunits of luciferase; luxC,luxD, and luxE, which encode proteins involved in synthesisof the aldehyde substrate for luciferase; and luxI, which isthe only V. fischeri gene required for the synthesis of

* Corresponding author.t Present address: Department of Biology, King's College,

Wilkes-Barre, PA 18711.t Present address: Department of Microbiology, University of

Iowa, Iowa City, IA 52242.

autoinducer in E. coli (12, 13). Autoinducer together withthe LuxR protein serves to activate transcription of theluxICDABE operon. Thus, autoinducer controls expressionof luxI, creating a positive autoregulation of autoinducersynthesis (12, 17). Furthermore, it has been reported thatluxR is negatively autoregulated at the level of transcription(8, 9) and at a posttranscriptional level (14). Induction ofluminescence has also been shown to require the cyclic AMPreceptor protein and cyclic AMP (6, 7, 8, 17). Cyclic AMPreceptor protein and cyclic AMP are required to activatetranscription of luxR such that sufficient quantities of theLuxR protein are present within cells (8).The luxR gene product has been considered the autoin-

ducer receptor, since it is the only V. fischeri gene productrequired in E. coli for a response to autoinducer (12, 13);however, a direct interaction of the LuxR protein withautoinducer has not yet been demonstrated (21). The luxRgene has been sequenced, and the protein predicted from theDNA sequence is either 250 or 252 amino acid residues inlength, depending upon the exact location of the start oftranslation (4, 5, 15, 21). The carboxy-terminal one-third ofLuxR shows sequence similarity to a group of known orsuspected transcriptional-regulator proteins (19). This groupincludes members of the so-called two-component environ-mental-sensing systems, such as the transcriptional activa-tors UhpA, FixJ, and NarL (3, 16, 19, 39). The amino-terminal region of LuxR does not, however, show homologywith the amino-terminal regions of UhpA, FixJ, or NarL; itshows sequence similarity only to a protein of suspectedregulatory function that is encoded by a gene upstream of theE. coli uvrC gene (19, 37). Because the sequence similaritybetween LuxR and the other regulatory proteins occurs in aregion thought to be involved in the binding of these otherproteins to DNA (1, 19, 22, 27), we suspect that thecarboxy-terminal one-third of LuxR is involved in binding tothe lux operator DNA. The amino-terminal regions of pro-teins such as UhpA and FixJ are thought to serve assubstrates for their sensor-kinase counterparts (1, 27). Per-

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MUTATIONAL ANALYSIS OF LuxR 3975

IluxIG>H luxc H uxD HluxAwas solubilized as described elsewhere (21). Antiserum topurified, solubilized LuxR was raised in a Flemish Giantrabbit. On day 1, the rabbit was inoculated subcutaneously

0 1 2 3 4 5 6 7 8 with 200 jig of LuxR protein in Freund complete adjuvant.

The rabbit was boosted subcutaneously on days 14 and 32Kilobase Pairs with 400 ,ug of LuxR and was bled on day 48. The resulting

antiserum was preadsorbed with boiled E. coli prior to use inFIG. 1. Organization of V. fischeri lux genes. The luxR operon immunoblotting experiments (see below).and the luxICDABE operon are transcribed divergently. Gel electrophoresis and Western immunoblotting. Sodium

dodecyl sulfate-polyacrylamide gel electrophoresis was car-haps in the case of LuxR, the amino-terminal two-thirds of ried out by the procedure established by Laemmli (25) asthe protein is designed as an autoinducer-binding domain described elsewhere (24); the resolving gel contained 12.5%rather than a site of phosphorylation by a sensor-kinase. The acrylamide. Western immunoblotting involved the proce-mutational analysis described here was initiated as a first dure described by Brahamsha and Greenberg (2) using astep towards defining the functional domains of the LuxR 1:200 dilution of preadsorbed LuxR antiserum to probe theprotein. nitrocellulose blot.

DNA sequencing. The luxR gene in pHK724 and the luxRMATERIALS AND METHODS mutant genes in the pDV700 series were sequenced by the

chain termination method described by Sanger et al. (35)Bacterial strain, plasmids, and culture conditions. The E. with the Sequenase 2.0 reagents (U.S. Biochemical Corp.,

coli strain used was JM109 (41). The plasmids used were Cleveland, Ohio). Single-stranded template DNA was pre-pHK555, which contains a P1SA replicon, a chlorampheni- pared as recommended by U.S. Biochemical Corp. (U.S.col resistance marker, and a functional luxICDABE operon; Biochemical Corp. Editorial Comments, vol. 14, p. 22).pHK724, which contains a ColEl replicon, an ampicillin Primers were annealed to template DNA by incubating theresistance marker, and luxR under control of the tac pro- mixture at 65°C for 2 min and then allowing it to cool to roommoter (21); and the pDV700 series plasmids, which were temperature, slowly. Nine luxR primers based on thosederived from pHK724 as described below. described by Devine et al. (5) were used: 5'-AATGCCGAC

Cultures were grown in L broth or on L agar (38) contain- GACACTTACAG-3 (nucleotides 13 to 32), 5'-ATCTGAing the appropriate antibiotic for plasmid screening or main- TATTTCAATTCTAG-3 (nucleotides 159 to 178), 5'-GCTGtenance (80,g of ampicillin or 50 ig of chloramphenicol per TAAATAAAAAATCTCC-3' (nucleotides 301 to 320), 5'-ml) as described previously (21). Where indicated, isopro- GATAGTTTATTTTTACATGC-3' (nucleotides 442 to 461),pyl--3-D-thiogalactoside (IPTG; final concentration, 1 mM), 5'-TCTTGGG-3' (nucleotides 597 to 604), 5'-GGGCAATCautoinducer (final concentration, 200 nM), or both were AATTGCTCCTG-3' (nucleotides 737 to 718), 5'-CTCTTTadded to the culture medium (8, 21). TGGTTAAATCGT-3' (nucleotides 559 to 542), 5'-GGGAAPlasmid purification and isolation of luxR mutant deriva- ACTAAACCCAGTG-3' (nucleotides 374 to 357), and 5'-GC

tives of pHK724. Plasmids were purified by the procedure CTCCATTTTTTAGGGT-3' (nucleotides 202 to 185). Thesedescribed by Kraft et al. (23). Manipulations of plasmids primers were synthesized by the University of Iowa DNAwere performed according to the methods of Maniatis et al. Core Facility. The nucleotide number is based on the as-(26) and Struhl (40). The transformation procedure used was sumption that the gene product is 250 rather than 252 aminothat described by Hanahan (18). acid residues in length (5, 15) as indicated by the amino-

Mutagenesis of plasmid DNA was according to the proce- terminal sequence of LuxR purified from E. coli containingdure described by Engebrecht and Silverman (13). To gen- pHK724 (21).erate the pDV700 series plasmids, 5 ,ug of pHK724 wasincubated in 50 ,ul of a mixture containing 0.5 M hydroxyl- RESULTSamine, 0.5 mM EDTA, and 5 mM Tris (pH 6.0) for 8 h at37°C. The reaction was stopped by the addition of 950 RI of Isolation of pHK724 derivatives with luxR mutations and100 mM CaCl2, and the hydroxylamine-mutagenized DNA phenotypic characterization of E. coli containing these plas-was then used to transform E. coli JM109 containing mids. Sixteen dark mutants were isolated after transforma-pHK555. Because the basal level of LuxR synthesis directed tion of E. coli containing pHK555 with mutagenizedby pHK724 is sufficient for in vivo activity, cells containing pHK724. These dark mutants represented less than 1% ofboth pHK724 and pHK555 are luminous even in the absence the total transformants. Because mutagenesis was in vitroof IPTG (21). Thus, the transformants were plated on L agar and plasmid replication could not occur, each mutant wascontaining ampicillin and chloramphenicol, and colonies that assumed to have resulted from an independent mutation.did not appear luminescent after 2 days at 30°C were picked We were interested in sequencing luxR genes containingfor further study. Plasmid DNA from each of the dark strains missense mutations: luxR genes that encode full-lengthwas used to transform E. coli JM109, and ampicillin-resis- LuxR proteins with substitutions in single amino acid resi-tant, chloramphenicol-sensitive transformants were se- dues. As a screen for such mutations, the synthesis of LuxRlected. Plasmids from these transformants showed BamHI by E. coli containing each of the 16 mutant plasmids wasand SalI restriction patterns that were identical to those for assessed by Western immunoblotting (Fig. 2). Of the 16pHK724. To ensure that these pDV700 series plasmids mutant plasmids, 10 appeared to direct the synthesis of ancontained mutations in luxR, each was used to transform E. antigen that was indistinguishable from LuxR synthesized bycoli JM109 containing pHK555 and the nonluminescent E. coli containing pHK724. Seven of the ten plasmidsphenotype of the transformants was confirmed. directed the synthesis of LuxR at levels comparable to that

Preparation of anti-LuxR serum. The LuxR protein was in E. coli containing pHK724. The other three plasmids,overproduced in E. coli containing pHK724, the resulting pDV756, pDV747 and pDV754, directed the synthesis ofLuxR inclusion bodies were purified, and the LuxR protein relatively low levels of LuxR. In addition, pDV756 directed

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FIG. 2. Western blot analysis of whole-cell extracts of IPTG-induced E. coli containing pHK724 or a pDV700 series plasmid. Lane 1,pHK724; lane 2, pDV756; lane 3, pDV751; lane 4, pDV740; lane 5, pDV755; lane 6, pDV747; lane 7, pDV731; lane 8, pDV743; lane 9, pDV754;lane 10, pDV728; lane 11, pDV752. R, The 28-kilodalton luxR product; R', a 25-kilodalton luxR product. Synthesis of this product in E.coli(pHK724) is coinduced with R by IPTG (D. Kolibachuk and E. P. Greenberg, unpublished data). Unlabeled arrowheads indicate an

antigen of about 11 kilodaltons produced by E. coli(pDV756) (lane 2) and a 21-kilodalton antigen produced by E. coli(pDV747) (lane 6). Thepolypeptide above R in each lane was detected in E. coli JM109 without pHK724 or any of its pDV700 series derivatives (data not shown).Thus, it is not encoded by luxR.

the synthesis of low levels of an antigen with an apparentmolecular weight of approximately 11,000, and pDV747directed the synthesis of an appreciable level of a 21,000-dalton antigen (Fig. 2). Presumably, the mutations in the sixplasmids that did not direct the synthesis of detectable levelsof the luxR product were regulatory mutations, nonsense

mutations, or missense mutations that resulted in the syn-

thesis of an unstable, quickly degraded protein. At any rate,these six plasmids were not studied further.As mentioned in Materials and Methods, even without

IPTG the level of LuxR synthesis directed by pHK724 issiifficient for complementation of the luxR mutation inpHK555 (21). The pDV700 series plasmids were identifiedbecause they did not effect such a complementation. Never-theless, it was possible that high levels of a mutant LuxRprotein or high levels of autoinducer would allow comple-mentation of the pHK555 luxR deletion mutation. To testthis possibility, E. coli strains containing pHK555 and a

pDV700 series plasmid (or pHK724 as a positive control)were plated on L agar containing ampicillin, chlorampheni-col, and either IPTG or a saturating concentration of autoin-ducer or both. The plates were incubated at 30°C for 2 daysand were periodically examined in the dark for colonies witha luminescent phenotype. As expected, the strain containingpHK724 was luminescent in the absence of IPTG andautoinducer. This strain was also luminescent on platescontaining IPIG, autoinducer, or both IPIG and autoin-ducer. With eight of the pDV700 series plasmids, the nonlu-minescent phenotype was maintained even in the presenceof IPIG or autoinducer or both. When E. coli containedpHK555 and pDV740, however, a luminescent phenotypewas observed in the presence of IPTG, autoinducer, or bothIPTG and autoinducer, and when E. coli contained pHK555and pDV747, colonies were luminescent on plates containingboth IPTG and autoinducer but did not appear luminescenton plates that contained IPTG or autoinducer individually.DNA sequence analysis of luxR mutations. The specific base

changes in each of the 10 luxR mutant plasmids that directedsynthesis of detectable levels of inactive or partially activeLuxR (Fig. 2) were determined by DNA sequencing. Thenucleotide changes and the resulting amino acid substitu-tions in the encoded proteins are listed in Table 1. Eight ofthe ten mutant plasmids had single base pair changes in luxR;one (pDV751) had two base pair changes, only one of whichresulted in an amino acid substitution in the encoded protein;and one (pDV752) had three base pair changes in luxR,resulting in three amino acid substitutions in the encodedprotein. Each of the plasmids contained different mutations,

thus confirming our conclusion that each was the result ofindependent mutations and indicating that we have notsaturated luxR with missense mutations that encode inactiveproteins. Twelve of the thirteen mutations identified wereGC-to-AT transitions (Table 1), as expected from hydroxyl-amine mutagenesis.The mutation in pDV756 converted a tryptophan codon to

an amber codon. Because E. coli JM109 carries a knownamber suppressor (40, 41), a full-length LuxR with a glu-tamine residue substituted for the tryptophan at position 94is produced, albeit at low levels, and a protein with anapparent molecular weight of approximately 11,000 was alsodetected in the immunoblot (Fig. 2). This 11,000-daltonprotein is presumably the expected 93-amino-acid residueproduct of the pDV756 luxR. The mutation in pDV747changed the triplet coding a tryptophan at position 193 to anopal triplet. From the results of the Western immunoblotexperiment, it appears that there was a weak suppression ofthe opal codon. A very faint band corresponding to thepHK724 LuxR band was observed along with a much moreintense band with an apparent molecular weight of about21,000, corresponding to the predicted 192-amino-acid resi-due product of the pDV747 luxR gene. We are not aware ofany reported opal suppressors in E. coli JM109; thus, we

TABLE 1. DNA sequence changes in the mutant luxR genesand amino acid substitutions in the encoded proteins

luxR mutant plasmid Base and substitution Amino acid residueand substitution

pDV756 G-281 to A Trp-94 to Gln"pDV751 G-361 to A Gly-121 to Arg

G-576 to A Ala-192 to AlapDV740 C-379 to T His-127 to TyrpDV755 C-551 to T Thr-184 to IlepDV747 G-579 to A Trp-193 to __bpDV731 G-585 to A Cys-195 to TyrpDV743 G-600 to A Gly-197 to ArgpDV754 G-605 to A Asp-202 to AsnpDV728 C-688 to T Arg-230 to CyspDV752 A-419 to C His-140 to Pro

G-485 to A Val-162 to IleG-689 to A Arg-230 to His

a The transition at base pair 281 converts the trp codon to an amber codon.Because E. coli JM109 carries a sup2 gene, the predicted sequence of theLuxR protein encoded by pDV756 contains a glutamine at position 94.

b The transition at base pair 579 converts the trp codon to an opal codon.We are not aware of any opal suppressors in E. coli JM109; thus, thesubstitution could not be predicted.

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MUTATIONAL ANALYSIS OF LuxR 3977

Sensing Domain

LuxR L

UvrC-28k F

FUxJ

UhpA_

DNA Binding Domain

~-'zImz¶z7zzIT 250]-fIIIIIIIIIIIIJj241H-L 1 16204|}1 1 ~~196

OmpR E-

PhoB [-

239

229

VirG _ =... 267

NtrC 469

FIG. 3. Schematic diagram indicating locations of amino acid substitutions in products of mutant luxR genes and indicating the complexrelationships among selected regulatory proteins involved in environmental sensing by bacteria. At the top are locations of amino acid residuesubstitutions in LuxR. Open symbols indicate mutations described in this report. Closed symbols indicate mutations reported by Shadel etal. (36). Symbols: U and Hl, mutations that could not be suppressed by IPTG, autoinducer, or both; * and 0, mutations that werephenotypically suppressed by autoinducer; A, a mutation that was phenotypically suppressed by autoinducer together with IPTG. Differentregions of similarity are indicated by different shadings, and numbers indicate protein lengths in amino acid residues.

cannot predict the substitution for tryptophan at position 193in the full-length suppression product encoded by pDV747(Table 1). The only other plasmid that directed the synthesisof markedly low levels of LuxR was pDV754 (Fig. 2). TheluxR mutation identified in this plasmid converted an aspar-tate codon to an asparagine codon (Table 1). Why pDV754directed the synthesis of low levels of LuxR is not known.Perhaps the amino acid substitution destabilizes the protein,or perhaps there is a second mutation outside of the luxRcoding region that affects regulation of LuxR synthesis.Nine of the ten luxR mutant genes that were sequenced

encoded proteins with a single amino acid substitution.Clearly, some of these mutations could grossly influence thestructure of LuxR, for example, the mutation resulting in thesubstitution of a tyrosine for a cysteine in the LuxR encodedby pDV731. Other mutations, for example, that in pDV755which resulted in the substitution of an isoleucine for athreonine, might be expected to have more subtle, perhapslocal, effects on LuxR structure. However, without anystructural information about LuxR, such speculation haslittle substance. Notably, however, all of the nine indepen-dent mutations in LuxR clustered in two regions: one region,defined by three mutations, spanned residues 94 to 127, andthe other region, defined by six mutations, spanned residues184 to 230. These regions constituted 13 and 18% of theLuxR polypeptide, respectively. Although in the LuxRprotein encoded by pDV752, one of the three amino acidsubstitutions was at position 230, obviously it cannot beconcluded that it is this substitution that results in the mutantphenotype.

DISCUSSION

The two clusters of luxR mutations we have describedsuggest that there are two domains critical for activity of theLuxR protein. One domain is towards the carboxy terminusof LuxR, in a region that shows sequence similarity to theknown or predicted DNA-binding sites of several transcrip-tional regulators involved in environmental sensing andresponse (1, 19, 27). The other domain is within the amino-terminal two-thirds region of LuxR. Only one other proteinhas been reported to show sequence similarity with this

region of LuxR: the UvrC-28K protein, a protein of sus-pected regulatory function that is encoded by a gene up-stream of the E. coli uvrC gene (19, 37). In agreement withthe hypothesis that there are two domains critical for LuxRactivity, Shadel et al. (36) report in the accompanying paperthat a number of other luxR missense mutations, obtained bysomewhat different methods, map to the same regions. Thetop of Fig. 3 depicts the locations of the amino acid substi-tutions in the LuxR proteins encoded by the mutant genessequenced by Shadel et al. (36) and by us.The addition of autoinducer to the culture medium re-

sulted in activity of two of the mutant LuxR proteinsdescribed by Shadel et al. (36) and of one of the mutantLuxR proteins described by us (Fig. 3). The positions of theamino acid substitutions in these mutant proteins defined theboundaries of the domain closest to the amino terminus ofLuxR (positions 79, 82, and 127). The reversal of the mutantphenotype by high concentrations of autoinducer suggestedthat these three mutations resulted in a decreased affinity forautoinducer. The obvious hypothesis is that the domaindefined by the mutations between residues 79 and 127 ofLuxR is responsible for sensing autoinducer concentrations;it is the presumptive autoinducer-binding region.Although the carboxy-terminal one-third of LuxR shows

sequence similarity to a subgroup of a large family oftranscriptional regulators with related amino-terminal do-mains (19), the amino terminus of LuxR does not appearrelated to this family of proteins (Fig. 3). This large family ofproteins with related amino-terminal domains constitutes thetranscriptional regulator component of two-component (sen-sor kinase and transcriptional regulator) environmental sens-ing systems (1, 27, 31). Apparently, the sensor kinasesactivate the transcriptional regulators by phosphorylatingsites within the conserved amino-terminal domain (22, 31).These two-component sensing systems are involved withdetection and response to external cues in the environment,for example, nitrogen source, phosphate source, osmolarity,products of other organisms, and alternate electron accep-tors (1, 3, 27, 31, 39). There is not absolute specificitybetween individual sensor kinase-transcriptional activatorpairs, such that molecular cross talk between some of these

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3978 SLOCK ET AL.

systems has been reported (1, 22, 30). In the case of LuxR,it appears that the amino-terminal domain is involved inautoinducer binding rather than phosphate binding. Becauseautoinducer [N-(3-oxohexanoyl)homoserine lactone] is spe-cific to V. fischeri (and other organisms containing the V.fischeri luxI gene), LuxR is designed for environmentalsensing of self: sensing of the density of V. fischeri in anenvironment (10, 12, 13, 28). Furthermore, cross talk withthe systems involving phosphorylation is not expected. Thisspecificity is a key element of a self-sensing system. Thesignificance of the similarity between the LuxR protein andthe E. coli UvrC-28K protein is unknown, but this informa-tion raises the possibility that E. coli possesses a celldensity-dependent control system similar to that of V. fis-cheri.The LuxR mutations between residues 184 and 230 (Table

1) define a region within the carboxy-terminal one-third ofthe protein that shows a particularly high level of similarityto a subset of transcriptional regulators involved in two-component regulatory systems (19). This, taken togetherwith the indication that the other of the two domainsdescribed (Fig. 3) is involved in activation by autoinducer,leads to the hypothesis that the region of LuxR betweenresidues 184 and 230 is involved in DNA sequence recogni-tion or DNA binding. One of the mutations, that in pDV747,which converts a tryptophan codon to an opal codon (Table1), had a curious phenotype that merits some discussion. Inthe presence of both IPTG and excess autoinducer (but noteither alone), E. coli containing pDV747 and pHK555 wasluminescent. Presumably, the IPTG was necessary becausethe opal suppressor was weak, and without full induction ofthe tac-luxR gene in pDV747, there was insufficient LuxR foractivity, but why was exogenous addition of autoinducernecessary for luminescence? Significant levels of an approx-imately 21,000-dalton protein, presumed to consist of thefirst 192-amino-acid residues of LuxR, were detected (Fig.1). Such a protein would retain the autoinducer-bindingdomain and could conceivably compete with the full-lengthLuxR for autoinducer at limiting concentrations. This hy-pothesis and, in fact, the hypothesis that the domain proxi-mal to the amino terminus of LuxR forms an autoinducer-binding region while the other domain forms a DNA-bindingregion can be tested if and when activity of LuxR in vitro canbe demonstrated.

ACKNOWLEDGMENTSWe thank G. S. Shadel, J. H. Devine, and T. 0. Baldwin for

providing data in advance of publication and for their advice andsuggestions.

This research was supported by the Sea Grant Office, NationalOceanographic and Atmospheric Administration, U.S. Departmentof Commerce (NA86AA-D-SG05), and by the Office of NavalResearch (N00014-80-K-0570).

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