INFECTION AND IMMUNITY, Aug. 2004, p. 4895–4899 Vol. 72, No. 80019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.8.4895–4899.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inactivation of the ciaH Gene in Streptococcus mutans DiminishesMutacin Production and Competence Development, Alters

Sucrose-Dependent Biofilm Formation, and ReducesStress Tolerance

Fengxia Qi,* Justin Merritt, Renate Lux, and Wenyuan ShiDepartment of Oral Biology and Molecular Biology Institute, UCLA School of Dentistry,

Los Angeles, California 90095

Received 23 December 2003/Returned for modification 1 March 2004/Accepted 26 April 2004

Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin produc-tion may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of ahistidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminishedcompetence development, stress tolerance, and sucrose-dependent biofilm formation.

Streptococcus mutans is considered the major etiologic agentin causing human dental caries (21, 22). In addition to theknown virulence properties, such as biofilm formation and acidproduction and tolerance, most clinical isolates of S. mutanselaborate antimicrobial peptides called mutacins (5, 12).Mutacins exhibit antimicrobial activity against closely relatedstreptococcal species and other gram-positive bacteria. Theability to produce mutacin may play an important role in thesustained existence of S. mutans in dental plaque (11, 14). Sofar, two types of mutacins have been characterized at themolecular level: the lantibiotics, represented by mutacins I, II,and III (25, 28, 29), mutacin JH1140 (15), and mutacin B-Ny266 (24); and the nonlantibiotic bacteriocins, represented bymutacin IV (27) and mutacin N (1). The lantibiotics are smallpeptides that are ribosomally synthesized and posttranslation-ally modified (31) and, in general, have a wider spectrum ofactivity than the nonlantibiotic bacteriocins.

The regulation of lantibiotic production has been shown tobe mediated by a two-component signal transduction system(TCSTS) for nisin, subtilin, and related lantibiotics (9, 18). ATCSTS comprises two proteins, a histidine kinase sensor andits cognate response regulator (33). The histidine kinase sensorbinds to a specific signal molecule, which triggers autophos-phorylation. Signal transduction from the phosphorylated ki-nase sensor to the response regulator enables it to activate orrepress transcription of its target genes. In previous studieswith the lantibiotic mutacin II, Qi et al. demonstrated that aspecific transcription activator, MutR, is required for transcrip-tion activation of the mutacin operon, and no other regulatorsappeared to be required (26). In contrast, regulation of muta-cin I production appeared to be more complicated. In additionto a requirement for MutR, mutacin I production was alsodependent on culture conditions that produced large bacterialaggregates (27; C. Bordador and F. Qi, unpublished data). In

our efforts to find additional regulators for mutacin I produc-tion, we found a gene, ciaH, whose inactivation abolishedmutacin I production. Surprisingly, the same mutation alsoaffected other cellular functions.

Inactivation of ciaH abolished mutacin I production. S. mu-tans strain UA140 was a clinical isolate from a severe carieslesion. Our investigators demonstrated earlier that UA140produces two mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV (27). Previous studies have shown thatproduction of mutacin I appears to be regulated by cell density,suggesting that signal transduction systems may be involved(27, 29). In order to find potential regulators, a BLAST searchof the S. mutans UA159 genome sequence database was per-formed using known TCSTS sequences as queries. This searchrevealed 13 pairs of TCSTS sequences; 5 of these were chosenfor further characterization based on their homology to otherTCSTS sequences known to be involved in various stress re-sponses. As an initial screen, only the histidine kinase genefrom each pair was inactivated. Because these genes were thelast gene of their respective operons, they could be disruptedby a single crossover integration without complications of polareffects. Typically, a �300-bp internal fragment close to the 5�end of the gene was amplified by PCR using an upstreamforward primer with an EcoRI site incorporated at its 5� endand a downstream reverse primer with a BamHI site incorpo-rated at its 5� end. The fragment was digested with EcoRI andBamHI and cloned into the suicide vector pJY4164 digestedwith the same enzymes. The plasmid was transformed intoUA140 using standard transformation procedures (32). Tenerythromycin-resistant transformants were randomly selectedand tested for mutacin production on trypticase soy broth plusyeast extract (1%) plates by the deferred antagonism assay (5)using OMZ176 and NY101 as indicator strains. OMZ176 is aStreptococcus sobrinus strain which is sensitive only to the lan-tibiotic mutacins, and NY101 is a Streptococcus sanguis strainwhich is sensitive to both the lantibiotic mutacin I and thenonlantibiotic mutacin IV (F. Qi et al., unpublished data). Ofthe five histidine kinase genes (ciaH [SMu1031], spaK[Smu0602], phoR [Smu0946] (6), scnK [SMu1652] (17), and

* Corresponding author. Mailing address: Department of Oral Bi-ology and Medicine, UCLA School of Dentistry, P.O. Box 951668, LosAngeles, CA 90095-1668. Phone: (310) 267-2767. Fax: (310) 794-7109.E-mail: fqi@dentnet.dent.ucla.edu.

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hk11 [SMu1407]) (3), only ciaH inactivation resulted in a totalloss of mutacin I production (Fig. 1A). Since the same muta-tion did not affect mutacin IV production (Fig. 1B), this resultsuggested that ciaH may have specifically affected lantibioticmutacin production. To test this, the same mutation was in-troduced into T8, a lantibiotic mutacin II-producing strain(25), to yield T8ciaH. As shown in Fig. 1, the ciaH mutation didnot have a significant effect on mutacin II production. Takentogether, these results suggest that the production of mutacinI and mutacin II is probably controlled by different regulatorymechanisms and that ciaH is required specifically for the pro-duction of mutacin I.

Inactivation of ciaR did not exert a significant effect onmutacin I production. Given the fact that most (if not all) ofthe TCSTS were arranged as two-gene operons with the histi-dine kinase and the response regulator occurring as cognatepairs, we were interested in determining the function of ciaR,the putative response regulator of ciaH in the ciaRH operon.The ciaR gene was inactivated by using a terminatorless kana-mycin resistance gene cassette insertion to prevent a polareffect on the downstream ciaH gene. A 1.38-kb DNA fragmentencompassing regions upstream of ciaR and part of ciaH wasgenerated by PCR and cloned into a TA cloning vector, pCRII(Invitrogen Co., San Diego, Calif.). The terminatorless kana-mycin resistance gene cassette (aph III) (35) was inserted intothe ciaR gene at a unique HincII site in the middle of the genein the same orientation. The plasmid was linearized and trans-formed into UA140. Upon integration into the chromosomethrough homologous recombination at the ciaR locus, the re-combinants were selected on Todd-Hewitt (TH) plates with800 �g of kanamycin/ml and the insertion was confirmed byPCR. Reverse transcription-PCR was also performed to verifythat the insertion had no polar effect on transcription of thedownstream ciaH gene (data not shown). Ten isolates werethen tested for mutacin production; one of them is shown in

Fig. 2. It is apparent that the ciaR mutation did not exert mucheffect on production of any of the mutacins tested, although aslight increase in inhibition zone was observed for mutacin I(Fig. 2A). This result indicates that the ciaR gene is not re-quired for mutacin biosynthesis.

ciaH gene inactivation diminished competence development.One of the major effects of ciaRH mutations in Streptococcuspneumoniae occurs with competence development (8, 10, 23).Insertional inactivation of ciaR and ciaH results in derepres-sion of competence both in aerobic and microaerobic cultures(7). Since the CiaR and CiaH proteins in S. mutans share ahigh degree of similarity with the CiaR and CiaH proteins,respectively, in S. pneumoniae (89% identity and 93% similar-ity for CiaR; 55% identity and 72% similarity for CiaH), it waslogical to test if the S. mutans ciaR and ciaH genes were alsoinvolved in competence development. We performed transfor-mation assays using chromosomal DNA isolated from aUA140 derivative strain carrying a tetracycline resistancemarker. Transformation assays were performed following stan-dard procedures (26, 32) in competence development medium(TH broth [THB; 0.9% beef heart digest, 1.1% pancreaticdigest of casein, 0.3% soybean peptone, 0.2% glucose, 0.25%sodium carbonate, 0.2% sodium chloride, and 0.05% monoso-dium phosphate] plus 0.2% bovine serum albumin). The ex-periments were repeated three times, and each time the trans-formation efficiency of the wild-type (wt) strain was arbitrarilyassigned as 100%. The transformation efficiency of the mutantswas calculated as the ratio of the number of transformants permilliliter of competent cells of the mutant versus that of the wt,times 100. The results showed a dramatic reduction in trans-formation efficiency for the ciaH mutant strain (�0.1% �0.01% of the wt level). Surprisingly, the transformation effi-ciency for the mutR mutant strain did not show a significantreduction (73% � 32% of the wt level).

ciaH gene inactivation altered sucrose-dependent biofilmformation. In the oral cavity, S. mutans mostly exists in biofilmsknown as dental plaque. Biofilm formation by S. mutans in-volves two processes: sucrose-independent initial attachmentmediated by surface binding proteins, and sucrose-dependentbiofilm formation mediated by glucans synthesized by the glu-cosyltransferases (Gtfs) from sucrose (36, 37). In vitro, espe-

FIG. 1. Deferred antagonism assay to determine the effect of ciaHmutation on mutacin production. A single colony from a fresh THplate was stabbed onto a trypticase soy broth plus yeast extract plateand grown anaerobically for 1 day. The plate was then heated at 80°Cfor 30 min to kill the producer cells and overlaid with 3 ml of soft agarmixed with 0.4 ml of overnight culture of the indicator strain, afterbeing cooled to room temperature. The zone of inhibition was in-spected after an overnight incubation at 37°C anaerobically. (A) S.sobrinus OMZ176 was used as the indicator. (B) S. sanguis NY101 wasused as the indicator.

FIG. 2. Deferred antagonism assay to determine the effect of ciaRmutation on mutacin production. The experiment was performed asdescribed in the legend for Fig. 1.

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cially on glass surfaces, the latter process appears to play amore important role; without sucrose, the biofilm remainsweakly associated with the surface and is very sensitive to shearforce. Since biofilm formation was found to be regulated by thecompetence genes comCDE (20), we performed assays to test

if the ciaH gene also was involved in biofilm formation due toits role in competence. UA140 and the mutant derivatives weregrown overnight in THB (Difco Laboratories, Detroit, Mich.)at 37°C anaerobically. The culture was diluted 1:20 into freshTHB and further incubated until the culture reached an optical

FIG. 3. Effect of ciaH gene inactivation on biofilm formation. UA140 wt (A, D, and G), ciaH mutant (B, E, and H), and ciaR mutant (C, F,and I) biofilms were grown in THB plus 1% sucrose (A to F) or in sterile saliva supplemented with 20% THB and 1% sucrose (G to I). The biofilmswere gently rinsed first (A to C) and then vigorously washed (D to I). Side frames in panels A to C are the z plane of the confocal image, whichshows the thickness of the biofilm and the architecture as viewed from the cross-section. The thin line on the z plane indicates the level at whichthe photograph of the x-y plane was taken. (H) To show the marks left by the detached microcolonies of the ciaH mutant grown in saliva, thephotograph was deliberately overexposed. Photographs were taken at a magnification of �100.

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density at 600 nm of 0.5. The culture was then diluted 1:1,000into THB containing 1% sucrose, and 0.4 ml of this cell sus-pension was added to each well of an eight-well Lab-Tek IIchamber slide system (Nalge Nunc International, Naperville,Ill.). The chamber was incubated at 37°C for 20 h as a staticculture to allow for biofilm formation. The supernatant wasremoved, and a labeling solution containing TH plus 0.2 mMCellTracker Orange (Molecular Probes) was added to the bio-film. The labeling proceeded for 2 h to allow live cells tometabolize the dye and develop fluorescence.

Biofilms were analyzed by confocal laser scanning micros-copy. In our initial studies, we noticed that without disturbancethe biofilm formed by 140ciaH appeared thicker than the oneobserved for the wt. However, this mutant biofilm did notattach tightly to the surface and was easily disrupted whenrinsed. To get a clear picture of the apparent sensitivity of thisparticular biofilm to shear forces, we imaged the biofilm in twosteps. First, biofilms were rinsed by gently adding and thenremoving 0.5 ml of phosphate-buffered saline, using a pipettor.After the image was taken (Fig. 3A to C), the biofilm wasrinsed again with a squirt bottle to wash off loosely attachedcells and a second image was taken (Fig. 3D to F). To minimizepossible inconsistencies resulting from washing, biofilms werealso washed with running water at a constant flow rate, andsimilar results were obtained (data not shown). As shown inFig. 3, both the wt and the mutant strains formed similardome-like microcolonies. However, despite a similar micro-colony architecture, the gently rinsed biofilm of 140ciaH wasmuch thicker than that of the wt (Fig. 3A to C). A closerinspection of the cross-sections of 140ciaH revealed that themajority of the dome-like microcolonies were not attached tothe glass surface (Fig. 3B, side panels). Instead, they attachedto each other to form a multilayered sheet that was looselyattached to the surface. In contrast, the biofilms of the wt andthe ciaR mutant consisted of a single layer of microcoloniesthat were all attached to the surface (Fig. 3A and C). When thewash was applied more forcefully by using a squirt bottle orrunning water, the biofilm formed by 140ciaH came off withonly a few microcolonies remaining attached (Fig. 3E). Incontrast, the biofilm formed by the wt and the ciaR mutantremained intact even after such a vigorous wash (Fig. 3D andF). To see if this phenotype was caused by the artificial growthmedium, we tested biofilm formation in sterile saliva supple-mented with 20% THB and 1% sucrose. As shown in Fig. 3Gto I, the same results were obtained. More interestingly, on thesaliva-coated surface, clear marks were shown on the slidesurface of the 140ciaH biofilm (Fig. 3H). These marks wereprobably left by detached microcolonies taking with them sa-liva deposits on the glass surface. Taken together, these resultsindicate that the ciaH mutation diminishes sucrose-dependentsurface attachment for biofilm formation.

Sucrose-dependent biofilm formation plays a pivotal role inthe cariogenicity of S. mutans (36). In addition to the gtf genesthat are required for glucan synthesis and biofilm formation (4,16, 36), a number of other genes have been reported recentlywhich affect sucrose-dependent biofilm formation (13, 30, 34).Some of these genes encode proteins associated with the cellwall (WapA) (30), and others may be involved in cell mem-brane synthesis or cell surface structures (13, 34). While thesegenes can be called terminal or effector genes, no regulatory

gene has been reported to be involved in regulation of thisimportance function. Of the three TCSTS that have been re-ported to be involved in biofilm formation, all were studied inthe absence of sucrose (2, 19, 20, 37). To our knowledge, ciaHis the first regulatory gene reported to affect sucrose-depen-dent biofilm formation.

The ciaH gene is involved in acid tolerance. The resultspresented above suggest that the ciaH gene may act as a globalregulator controlling multiple cellular functions. Previous stud-ies have shown that some of the genes regulating competencedevelopment or biofilm formation in S. mutans were also in-volved in regulating acid tolerance (19). Therefore, we wereinterested in determining if the ciaH gene was also involved inacid tolerance. To measure the acid sensitivity of the ciaHmutant, we grew UA140, 140ciaH, and 140ciaR in THB over-night. The cultures were then diluted 1:40 into THB and THBpH 6.4 and incubated at 37°C as static cultures. Samples weretaken at designated time points, and the cell density was mea-sured as the optical density at 600 nm. As shown in Table 1, theciaH mutant exhibited a 60% reduction in growth rate whengrown at pH 6.4; in comparison, the wt and the ciaR mutantshowed only 20% reduction in growth rate under the sameconditions. This difference in growth rate under acidic condi-tions between the wt and the ciaH mutant was statisticallysignificant (P � 0.0006). These results suggest that the ciaHmutation greatly reduced acid tolerance in strain UA140.

In summary, we characterized a putative histidine kinasegene, ciaH, in S. mutans. Inactivation of ciaH abolished muta-cin production, diminished competence development, alteredsucrose-dependent biofilm formation, and significantly re-duced acid tolerance. Although ciaH and ciaR are in the samegenomic organization as the ciaRH operon in S. pneumoniae,unlike the ciaRH system in S. pneumoniae, inactivation of theputative cognate regulator ciaR did not reveal any conspicuousphenotype. A possible explanation is that CiaH may have asecond cognate regulator located at a different location whichis responsible for regulation of the above cellular functions.Assays for protein-protein interactions, such as the yeast two-hybrid system or in vitro biochemical analysis with purifiedproteins, will be required to resolve this question.

We thank J. Yother for providing the plasmid pJY4164 and F. Gufor assistance in statistical analysis. We greatly appreciate the publicrelease of the S. mutans sequence data from the Streptococcal mutansGenome Sequencing Project funded by a U.S. Public Health Service,National Institutes of Health (NIH) grant from the Dental Instituteand B. A. Roe, R. Y. Tian, H. G. Jia, Y. D. Qian, S. P. Lin, S. Li, S.

TABLE 1. Effect of low pH on growth of S. mutans UA140 wt andciaH and ciaR mutants

StrainDoubling time (min)a in: % Reduction

in growthrate (TH pH6.4 vs TH)TH TH pH 6.4

UA140 69.4 � 5.1 86.7 � 4.5 20140ciaH 74.5 � 7.5 119.5 � 3.5 60140ciaR 72.9 � 6.9 88.5 � 7.6 20

a Doubling time was calculated based on the formulas ln Z � ln Z0 � k(t � t0),where k is the growth rate, and g � 0.693/k, where g is the doubling time. Valuesare the mean � standard deviation obtained from three independent experi-ments.

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Kenton, H. Lai, J. D. White, R. E. McLaughlin, M. McShan, D. Ajdic,and J. Ferretti from the University of Oklahoma.

This work was supported in part by NIH grant R01 DE 014757 to F.Qi, NIH MPTG Training Grant T32-AI07323 to J. Merritt, and aBioStar/C3 Scientific Corporation grant and a Washington DentalService Grant to W. Shi.

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Editor: V. J. DiRita

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