BIOSCIENCE, BIOTECHNOLOGY, AND a1OCHEMISTAY pseo.ong/101080/9168451 20191685371 BA ox: Identification of critical residues for the catalytic activity of ComQ, a Bacillus prenylation enzyme for quorum sensing, by using a simple bioassay system Kazutake Hirooka(*, Saki Shioda* and Masahiro Okada” Department ef Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama, Hirashima, Japan; "Department of Material and Life Chemistry, Kanagawa University, Yokohama, Hiroshima, Japan Bacillus ComQ participates in the biosynthesis of a quorum-sensing signaling molecule (ComX pheromone) through catalyzing the prenylation at a Trp residue of the precursor peptide (pre- Com) with geranyl diphosphate (Co type) or farnesyl diphosphate (Cis type). We hypothe- sized that several residues specifically conserved among either type of ComQs are important for their substrate specificities. Using a simple bioassay, we revealed that Phes3, Asn185, and, Gly190 In ComOxo.e2 (Cro type) were nondisplaceable to Ser63, Gly186, and Vall90, the corresponding residues in the C,.-type ComQ, respectively. A three-dimensional model sug- gested that the 186th and 190th residues are involved in the pre-ComX binding. In vitro analysis showed that substitution of Phe63 with Ser in ComQzo.e2 significantly reduced the {geranylation activity but substantially enhanced the farnesylation activity, whereas substitu- ton of Ser63 with Phe In ComOses (Cys type) reduced the farnesylation activity. Therefore, the (63rd residue was found to be significant for the prenyl-substrate preference, Received 5 September 2019 Accepted 11 October 2019 kerworos ecu subtis post- trensatonal moifaton; prenylaian; quorum sensing ‘Abbreviations: GPP: geranyl diphosphate; FPP: farnesy| diphosphate; IPP: sopentenyl dipho- sphate; GGPP: geranylgeranyl diphosphate; FARM: first aspartate-rich motif; SARM: second aspartate-rich motif; B-Gak: B-galactosidase; TBABG: tryptose blood agar base supplemented with glucose; X-gal 5-bromo-4-chloro-3-indolyl-f-D-galactoside Bacterial cells communicate with their cognate cells through quorum-sensing systems to coordinate their physiological activities depending on their cell density. ‘They individually secrete and share a quorum-sensing signaling molecule, called a bacterial pheromone (or autoinducer, or quormone), The released pheromones are sensed by the cognate cells, thereby measuring their own density. When the pheromone concentra- tion reaches a certain threshold with the increase in cell density, the corresponding cells synchronously induce the expression of associate genes for mediating phenomena, eg, bioluminescence, biofilm formation, genetic competence, and production of antibiotics, biosurfactants, and virulence factors [1-3] A Gram-positive bacterium, Bacillus subtilis, together with related species, possesses a regulatory system encoded on the comQXPA gene cluster to mediate ‘quorum-sensing responses [3-7] (Figure 1(a)), ComQ is involved in the biosynthesis of an oligopeptide-type bacterial pheromone called the ComX pheromone. The comX gene is initially translated as a precursor peptide, pre-ComX, which is converted to the ComX pheromone ComP is a membrane-bound receptor kinase to detect the ComX pheromone, ComA is a response reg- ulator for ComP, and both comprise a two-component regulatory system. Accumulation of the ComX phero- ‘mone activates the autophosphorylation of ComP, which subsequently phosphorylates ComA in the cytoplasm, leading to induction of many genes involved in the quorum-sensing responses [5,8,9]. In B. subtilis, the phosphorylated ComA induces the srf operon for the biosynthesis of surfactin, a major lipopeptide biosurfac- tant, and indirectly activates the acquisition of genetic competence through stabilization of the ComK compe- tence transcription-factor by ComS encoded within the srfA operon [10-12]. The production of surfactin also promotes biofilm formation by affecting the neighboring B, subtilis cells [2,3]. The degQ gene located adjacently upstream of comQis a direct target of the phosphorylated Coma, and degQ encodes a pleiotropic regulator of degradation enzymes [10,13]. DegQ contributes to the function of the DegSU two-component system, which regulates cellular processes including degradative enzyme production, genetic competence, biofilm forma- tion, and swarming motility, by interacting with the DegSU complex to promote the accumulation of the phosphorylated DegU [14]. The phosphorylated Deg response-regulator directly activates the pgsB operon for the poly-y-glutamate biosynthesis and represses the srfA. ‘operon whereas the unphosphorylated DegU, together CONTACT Kanutoe Hroola @ hootastukayaruacip Maahte lade @ otasatanagaweuacip ‘Tis paper I dedicated to the late Dr, Yous Sskagam, Supplemental data for this arte can be accessed ere © 209 pan Sot for Bcenc, tecndogy, and Agrchemisry2 © KiRooKa era. eS eer Rarer SPM Sorry Figure 1. Bacills ComQXPA quorum-sensing system. ‘Surfactin biosynthesis Gonetic competence acquisition Poly-yglutamato production, {a} Organization ofthe comQXPa gene uterin subi stain 168. The citer members and their neighboring genes are indicated by large open arrows and the promoters and harp stucures ely funconing os Wanscriton terminators are nates by Ben srtows and ster Toop, respectively (6), was reported that hve transcripts, respectively composed of comONPAyand, comaXA, and comOXP, were obtained by the anseaton fom the com promoter [7]. an amino ais (b) Scheme af quorumrsensing responses through the CmOXPA system bre-Com i converted tothe ComX pheromone ‘a renyl modfation by Com, ComP detects the secreted ComX pheramane to actvate Coma by phosphoryl wanse.The phosphorylated Com Induces the expression af astcate genes for mediating phenomena such as srfctinbiosynthess, acquisition of genelc competence, and poly slutamate precucton. with ComK, stimulates the comK transcription [15-17] Interestingly, B. subtilis strain 168, a domesticated laboratory strain, possesses a mutation in the degQ pro- moter causing decrease in the degQ expression, which ‘enhances the genetic competence and impairs the poly- y-glutamate production [16,18] (Figure 1(b)). ‘The comQX genes and 5'-part of the comP gene are not highly conserved among B. subtilis and Bacillus isolates [19-21]. Consequently, the C+terminus of pre-ComX exhibits polymorphism, except that only a Trp residue is conserved (Figure 2). The Trp residue is post-translationally pre nylated by ComQ to form a characteristic tricyclic structure, and the mature ComX pheromone is gener~ ated by cleavage processing. The prenylation of pre: ComXs in various B. subtilis and B. mojavensis isolates is dassified into two groups: geranylation (Cio type) and farnesylation (C,s type). These prenylations are attributed to the specificity of the respective ComQs to prenyl donors, ie. geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) [22-24]. For example, B. subtilis strain RO-E-2's ComQ (ComQuo.x-2) Uses GPP for the geranylation of this strain’s pre-ComX (pre-ComXzo r 2) [25], while ComQs from B, subtilis strain 168 and B. mojavensis strain RO-C-2 (ComQues and ComQko.c2) use FPP for the farnesylation of their target pre-ComXs (pre-ComKige and pre- ComXgo.c-2 respectively) [26]. Such different chain lengths of the prenyl modifications confer the struc: tural diversity of ComX pheromones as well as the difference in the amino acid sequence (Figure 2). It should be noted that the length of a lipophilic side chain of quorum-sensing pheromones is. generally responsible for their group-specific biological activ- ities to the corresponding bacteria as a quorum-sen- sing pheromone (1,21,23.27] ComQ shows the sequence similarity to trans-type prenyl diphosphate synthases [22,23], which catalyze the consecutive condensation of isopentenyl dipho- sphate (IPP) with allylic diphosphate, e.g. GPP and FPP, to produce clongated preny! diphosphates, eg. FPP and geranylgeranyl diphosphate (GPP, C39). 18 these synthases, two aspartate-rich motifs are crucial for the binding of the two substrates; the first aspar- tate-rich motif (FARM) is for allylic diphosphate, and the second aspartate-rich motif (SARM) is for IPP. ‘The carboxyl groups of Asp residues in each motif bind to the diphosphate moiety of each substrate via divalent cations [28]. ComQ also possesses the FARM, whereas the region corresponding to thecamicAL esibuts oF sxcruscoma @ 3 Nx gMDYLARPOVETRLENGEASLIGIPORLIPSIVDIFURKNTLSKKCKGIEWEQ- 58 ~nomveyLixyenviRevarcnacuicvoxDgsECri~---norKoLezYsimowsr 53 |Gerenylation SNQEIVGYLRNPEVEDEVAKGRASLLNTDKDQLKSIV--—-DAFCOLOTYTNGMVPS 54 QOL TYFLAYPEALKRLENNEACLIGFDVQETETIT--KATNDYYLADDETAQHGD- 55 Qo THYFL.SYPEVEKRLINREACLIGFSSNETETI2~ KAYHDYELSEPTTREWEG. (Come pheronone rarnesylation 1n=1: Geranylation HN. 1n=2: Famesylation > HN yr a Figure 2 Cmpation of amino a sequences of preComks and stu of the preytnelied Tip ree of Com pheromones (a srger cle bomen Crs dette nh a 90624525 1d a moet eee ate anise Bu Drege OTe Are Fae eee ee en me, aes oe ears? red eet ee reece wr se tam (ae moc we ree ee Me ee ee ee ean ee een same ees ea ane sa SARM is less conserved and thus is designated as, a pseudo-SARM [29] (Figure 3). A previous study fon the in vitro geranylation activity of ComQno.s-2 showed that Mg’ was required for the activity [25]. ‘Thus, the FARM in ComQ was estimated to serve as, via Mg” similarly to trans-type prenyl diphosphate synthases. By contrast, the pseudo-SARM in ComQ ‘was inferred to function in the binding of pre-ComX instead of IPP 29]. However, the precise recognition mechanisms for both substrates remained to be a binding site of prenyl diphosphate (GPP or FPP) elucidated. RO-E-2 1 NRETVHEKIONLOLINEYLINGIDERIRYSFGILSPRAVALSGURSSHILTLAGGTELLILAFOIEDDLEDEDNTEIIO 60 RS-B-1 1 HRETVSOKIAIDGIRYLQS® TESROTTEFADLAPHRYLATRGQORKAELLAAGTELLITSPOTYDBLEDRONVRATION 60 1 MRE VSRRIMROLERYL:QN® TESKDTTEPADLALHHYLAPNCQORRATELLANGTELLTLSPDTYDDLEDRONRNAVWH 80 1 NREL VEQuIPNEDLSQLLYSP IOSKBTY SPAESTILMYWFCCENLIVATRLCAGIETLILSSDINQDLEDEONIHALWH 80 1 REE RQUSPWED:.SQ:LYSPIOSKETFSPARSATLAYWPCGENERVATRLGAGIES1TLSSD20QDEEDRONTORLIN 80 81. KIDPSLALNAARTLYTUGLETICSISNSAEFHRLTLRYAL.UANQOQHEDLANSPETERECIONMRQRAGSLEANSAVLAR 160 81 KIDSSIALNAVEALYTLSTQVNCOASHEPEFSQKIL/TPALOSIQCONDDIVHAPRTEEACLENIRNKSGALEALPCYMNCY 160 81 KIDSSIALNAVEALYTLSIQVAROASHEPEFSQKILNFALOSIQGQHDDIVIAPOTEETCLEMIRNRSGALRALPCVNGY 160 81. KINRSESLNAALSLYTVGLES1YSLIOINPL IPA YVLRYVMEANQGDIDDITURSRTEDESLEVIRLACGSLEALANVAGY 160 81. OMRSESLNAALPLYTIGLESIHSNDINELTPRYVLRYVRDANQOQHDDITURSRTEDECLEVIRLRCOSLEALANVTGY 160 oa : a 161 MLANGERNgTTEDYAYKCxIKOLEUDYYGLVNDQRSDIRXKRKTLIVLPLARKPNEASERILKLINSHTSYHBYISDSS 240 161 MLATGKYDPIVASYSYELCIMSOTDEDISGL#YLN-NDFVORRNTLAYLYLRKOPNDASVELLSUYORPEEFVBINTRAL 239 161 MUATGKYH?IVASYSYELGIIAQIENDYOGLYYLN-NDF VORRUTLAYLYLNKRENEASEEILSWYENAALFLSLOPRYI 239 161 LLATGEYNETVERYSYYKGr1AQ1SGDYXVLLSCURSD IEXMRITLIYLYLERLPNDASEDLLYLISURDLYYRSLLDKE 240 161 TLARGEYNEIVERYSYYEGIVAQT SOVHVLPSCURSDIERAKQTLIVLYLERVPNEASKELLYLESNRDLYSRALLARD 240 BeeudonsaR 241. RPDELLPEAGLUQWSMLIELYEEEITASMNOLNINIKL- 209 240-R--QRUTEAGVQYLBVHRHLSUARTRRENSQUALEDORTERLLSU 286 240. R_-AXLTEACWVOYSLVAKELSUQRLRRETARLELQENRIEELITGTI 286 241. RPQEXLIRAGUTOYISVLLEIYRQRCTSATEOLNLDREFKELTRECLLSYTRGDTRCRT-~ 209 RO-C-2. 241. KPARELIRAGUSQVSVLLQIYKQRCPSATEQLLYKERKDLLREHLLNYREGDENDARPH aor Figure 3. Comparison of amino acid sequences of ComQ3s derived from three 8. subtilis strains (RO-E-2, RS-B-1, and 168) and two 8. mojavensis trains (RO-B-2 and RO-C-2) The ComQs tam the tv stains {ROE-2, RBI, and RO-B-7] re he Cye type and thas rr the two sans (168 and RO-C2} 3 the Cys type The FARM and pzeudo-SARM are uncerined,ané four Asp residues in these two mots are inate by number signs. Twenty-five amino acid recive: that ae ‘Conserved among eer CaF Cie Comsat inated by asterisks and the potions of 1 amino aid residues, which wetesubsttuted nthe Coma. point mutants for the in vo bioassay, are indicated by boeface type. The accession numbers ofthe ComOs are AALT738(RO-E2), ARFA2172 (R581), AAL67730 (ROB, AN9A4SS (68), and AALS7727 RO“2},In this study, we focused on type-specific amino acid residues in ComQs based on a multiple-sequence align- ment of five ComQ variants. To investigate whether those residues are important for the specificity to each substrate or the catalytic activity, we constructed a three dimensional model of ComQgo.s.2 (Cio type) based on. a known GGPP synthase, and the spatial arrangement of the relevant residues was estimated. Then we evaluated ComQrox2 mutants with each single substitution of those residues through an in vivo bioassay system, together with the wild-type ComQno.r.2 and ComQues (Cis type). In this system, the Escherichia coli transfor- ‘mant carrying each of the wild-type and mutated comQX gene clusters was co-cultivated with two B. subtilis tester strains that respond to the ComXno.x- pheromone (Cio modified) or ComXig pheromone (Cis modified), respectively. Furthermore, we measured the in vitro pre- nylation activity of ComQ mutants as well as that of the wild types. Materials and methods B. subtilis tester strains B. subtilis strain BD3020 was constructed from a deriva. tive of strain 168 in the previous study (20,21,23], in which the comQXPyes genes were replaced by the comQXPpo.r 2 genes followed by the comQro.x 2 disrup- tion by plasmid insertion, and the lacZ gene was inte- grated into the sfA operon on the chromosome (Table 1). Thus, this strain is incapable of producing the ComXyeq pheromone and able to induce the -galac- tosidase (B-Gal) activity in response to the exogenously added ComXzo 2 pheromone. Strain BD3020 and a plasmid pED413 (mentioned below) were kindly pro- vided by Prof. D. Dubnau from Rutgers University. To detect the extrancous ComX,es pheromone, B. subtilis strain FU1086 was constructed as follows A DNA fragment corresponding to a region around the srfA promoter (bases -313 to 406; base 1 is the transcription start base) [10] was amplified by PCR with the genomic DNA of strain 168 (Table 1) and a primer pair of PsrfA_XF/Psrf_BR (Table $1), fol- lowed by trimming with Xbal and BamHI digestion It was then cloned into the pCRE-test2 vector at the same restriction sites [30]. The resulting plasmid, PCRE-PsfA (Table $2), was linearized by PstI Table 1. B. subtilis strains used in this study. Source or Stain Genotype reference ss pC Tabratony stock 803020. hi eu mer Palace amyEaniR Pay (21,23) cork eat (com@pEDS75 comi replaced by genes rom RO) 1asi2 mpc upp ii) Bosc FUIO8S UpC2 Bupp amyE:Psthec (cot This study FUIO8S C2 upp LeomOX anye-Patlacz (ct) This study HGSC:Bacilus Genetic Stock Cente. digestion and then integrated into the amyE locus of strain 1A832, a upp gene disruptant (31] (Table 1, provided by the Bacillus Genetic Stock Center, Columbus, OH, USA), to obtain chloramphenicol resistance (5 g/mL) on a plate of tryptose blood agar base (Difco, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.18% glucose (TBABG), which resulted in strain FU1085, carrying the srfA promoter-lacZ fusion (Table 1). In- frame deletion of the comQXrgy genes from this strain's chromosome was performed according to the ‘method previously reported [31]. The flanking regions upstream of comQjge and downstream of comX es were respectively amplified by PCR with the genomic DNA of strain 168 and the primer pairs of comQ_P1/ comQ_P2 and comX_P3/comX_P4 (Table $1). The uupp-K7 cassette was amplified by PCR with a pUCI9-upp-phleo plasmid [31] as the template and a primer pair of Phleo3/PhleoS (Table $1). These three fragments were combined by the joining PCR, and the resultant fragment was used for transformation of strain FU1085 to obtain phleomycin resistance (5 Hg/ mL) on the TBABG plate. After checking the chlor- amphenicol resistance (5 jig/ml) and 5-fluorouracil sensitivity (25 uM) of the obtained strain, the upp-K7 cassette of this strain was removed through pop-out recombination; since the Rec activation via ComK was impaired by the comQXiss disruption [11,12,32], the RecA activity was induced via Lex inactivation by UV radiation [32,33] to the culture in a minimal med. ium [31] on a clean bench (MCV-BIGIE, PHC Holdings Corporation, Tokyo, Japan), followed by cultivation for another 1h. The cassette-lacking clone was selected on an agar plate of minimal med- ium containing 25 4M 5-fluorouradil, and its phleo: mycin sensitivity was checked to obtain strain FU1086 (Lable 1). Correct cassette integration and pop-out event were also confirmed by PCR with the genomic DNA ofeach clone as the template and primer pairs of comQ Pl/comX_P4. By this in-frame deletion, the short part of the comQzzx coding sequence was fused to the 3/-short part of the comXiss coding sequence, which resulted in a fusion of the N-terminal 66 amino acid residues of ComQieq with the C-terminal 10 amino acid residues of ComXex having no catalytic function, together with the intact ComPAyc two. component system indispensable for detection of the ‘ComX6s pheromone. Construction of co-expression systems in E. coli To construct plasmids for co-expression of the comQXro.s2 gene cluster and for co-expression of the com@Xga gene cluster inthe E. coli cells, DNA fragments covering the comQX genes of each strain were amplified by PCR with the plasmid pED413 [23] carrying the com@Xgo.e2 genes and strain 168's genomic DNA as,the template and primer pairs of uni_comQ_BXF/ ROE2_comX_HSR and uni_comQ_XF/168_comX_SR (Table $1), respectively. The comQXzo.x.2 fragment was cloned into the pUCI8 vector (TaKaRa Bio, Shiga, Japan) that had been treated with Xbal and Sphl using an In- Fusion HD dloning kit (Clontech, TaKaRa Bio) to pro- duce a plasmid pUC-ROE2-comQX, and the comQX ies fragment was cloned into a pCR21 TOPO vector (nvitrogen, Thermo Fisher Scientific, Waltham, MA, USA) by TA cloning to produce a plasmid pCR-168- comQX. In these two plasmids, the comQX genes of each strain are under the control of the lac promoter (Table $2). To construct plasmids for co-expression of each mutated comQroxe gene with the wild-type comXgo.x.2 gene in the E.coli cells site-directed muta- genesis was applied to the comQXroe2 fragment through a two-step overlapping PCR method as follows The Spars of comQno s-2 were amplified by PCR with pUC-ROE2-comQX DNA as the template, oligonucleo- tide uni_comQ_XF as the forward primer, and oligonu- cleotides 149A_R, F63A_R, F63I_R, FO3S_R FOSV_R, T9ILR, PIMSR, TIS2LR, N186G_R, GI90V_R, K202N_R, $234K_R, and E248K_R as the respective reverse primers (Table $1). Similarly, the 3parts of comQxo-e2 with the entire comXro.e2 were amplified using oligonucleotides 49A_F, F63A_F, F631_F, F63S_F, FOVE Tol_F PIMSF, TIS2F, N186GF, G190V_E, K202N_F, $234K_F, and E248K_F as the respective forward primers and oligonucleotide ROE2_comX_HSR as the reverse primer (Table $1). Each pair of mutation-introduced PCR products was combined by the joining PCR, and the resultant frag- ments were individually cloned into the pUCI8 vector using the In-Fusion HD cloning kit as in the case of PUC-ROE2-comQX construction to yield 13 plasmids (pUC-ROE2-comQX_H9A, _F63A, _F631,_F63S, FOV, _T92L, _P1348, T1521, _N186G, _GI90V, ~K202N, _$234K, and _E248K) for the production of each ComQno.z2 mutant carrying the indicated single substitution together with the wild-type pre-ComXso.n2 (Table $2), Bioassay of the ComQno.c.2 mutants B. subtilis tester strains BD3020 and FULO86 were grown at 37°C overnight on TBABG plates, which contained both chloramphenicol (5 jig/mL) and tetra- cyeline (10 ug/ml) or only chloramphenicol (5 g/mL), respectively. E.coli strain DHSa was transformed with cach of the 15 plasmids constructed as described above and grown at 37°C overnight on an LB agar plate containing ampicillin (50 ug/ml). Subsequently, each E.coli transformant and the two B, subtilis tester strains were inoculated on one TBABG plate containing 5-bromo-4-chloro-3-indolyl-f-D-galactoside (X-gal) (80 jigimL) and grown at 37°C overnight to form camcat ResiouES oF aacucus coma © s three compartments of the individual strain’s cells. To claify the blue coloration caused by the induced 8-Gal activity, the plates with the grown cells were held at room temperature for a few days (Figure SU). Preparation of the wild-type and mutated ComQs A plasmid pET15-ROE2-comQ for the overexpression of ComQzo.n.2 in the E.coli cells was constructed in the previous study [25]. For site-directed mutagenesis into the comQro.p.2 in pET15-ROE2-comQ, a KOD. Plus-Mutagenesis kit (Toyobo Life Science, Osaka, Japan) was used with primer pairs of ROE2_F63S_F/ ROE2_F63S_R and ROE2_D68E_F/ROE2_D68E_R (Table $1), which resulted in two plasmids of pETIS ROE2-comQ_F63S and _D6BE for the overexpression of [F638] and [D68E]ComQro 5.28 (Table $2). The comQues coding region was amplified by PCR with the genomic DNA of strain 168 and a primer pair of 168_comQ F/168_comQ_R (Table St), which was then cloned into the pET-15b vector (Novagen, Sigma-Aldrich, Merck, Darmstadt, Germany) that had been treated with Neol using the In-Fusion HD cloning kit to yield a plasmid pET15-168-comQ for the ComQioe overexpression. Site-directed mutagen. esis into the comQuse in pETI5-168-comQ was per- formed using the KOD-Plus-Mutagenesis kit with a primer pair of 168_S63F_F/168_S63F_R (Table S1), and the resultant plasmid pET15-168-comQ_S63F was used for the [S63F]ComQjs5 overexpression (Table 82). ‘The wild-type and mutated ComQs were prepared according to the procedure previously reported [25] Cells of E. coli strain BL21(DE3) (Novagen, Sigma- Aldrich, Merck), transformed with each of the pE 15b-based plasmids described in the preceding section, were grown in a M9 minimal medium supplemented with ampicillin (400 jig/mL) and a mixture of amino acids [25] at 37°C until an optical density at 620 nm reached 0.6. After isopropyl-B-D-thiogalactopyranoside was added to a final concentration of 1 mM, the cells were cultivated for another 4h, The cultured cells were harvested by centrifugation, resuspended in 25 mM. Tris-HCl buffer (pH 7.4) containing 0.1 mM MgCl and 0.1 mM EGTA, and then disrupted by sonication, Afier ultracentrifugation of the slurry, the precipitate ‘was collected and resuspended in the same buffer except for the addition of n-dodecyl-§-D-maltoside at 2 mg/ ml, wich was used as the crude enzyme. The produc: tion of each ComQ was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure $2). In vitro prenylation reaction and detection of prenylated peptides ‘The synthesis of pre-ComXs, prenylation of these pep- tides using wild-type and mutated ComQs, anddetection of the prenylated peptides were performed according to the methods previously reported (25]. In 50 wL of 50 mM_N-Tris(hydroxymethyl)methyl- 3-aminopropanesulfonic acid (TAPS) -NaOH butfer (pH 8.5) containing S mM MgCl, 2.5 pmol of pre ComX (["*C;]pre-ComXgo.x2 oF pre-ComXi¢s), 10 pmol of prenyl diphosphate (GPP or FPP, Sigma- Aldrich, Merck), and each ComQ_ suspension (7.5 ml broth equivalent) were incubated at 37°C for 2h, After the addition of 200 ul. of CH,CN, the reaction mixture was centrifuged, and 10 iL of the obtained supernatant was combined with 30 of the solution containing 2.5 pmol of the synthetic Ala ComXgo.z2 pheromone as the internal standard, was subjected to LC-MS analysis (HCTplus, Brucker Daltonics, Bremen, Germany). The amount of prenylated peptide was calculated from the integra- tion of the peak area (Figure $3) whi Results and discussion Selection of candidate residues responsible for prenyl donor selectivity in ComQ A multiple-sequence alignment was performed among five ComQs, which were derived from three B. subtilis strains (RO-E-2, RS-B-l, and 168) and two B. mojavensis strains (RO-B-2 and _RO-C-2) (Figure 3), The three strains, RO-E-2, RS-B-1, and RO-B-2, produce the Cy-modified ComX phero. mones, and the two strains, 168 and RO-C-2, produce the Cys-modified ComX pheromones [5,23,24,26]. Strains RO-E-2, RS-B-1, RO-B-2 and RO-C2 are Figure 4, Three-dimensional structural model of ComQo.2 undomesticated isolates from Mojave desert, Rosamond, CA, USA [34]. The alignment showed that the region including the FARM is relatively well conserved among the five ComQs, suggesting that this region plays a central role in the prenyl transfer reac: tion common to all ComQs, Notably, no species-spe- cific conservation was found in the five ComQs; however, 25 amino acid residues were found to be type-specifically conserved. They are individually con- served among either the Cio or Cis type of ComQs but are different between the two types. It was assumed that some of these residues play a eritial role in the specific recognition of the chain length of the preny! donor. We focused on 10 such residues to elucidate their functions in the substrate specificity because those residues are quite different in bulkiness or charge between the two types of ComQs. Prediction of the spatial arrangement of candidate residues in ComQro.e2 ‘To explore the spatial arrangement of the 10 residues and essential regions for the ComQ activity, a three dimensional model of ComQso x.2 (Cio type) was constructed through SWISS-MODEL homology mod. cling based on a crystal structure of the GGPP synthase from Thermus thermophilus strain HBS (PDB ID: 1WMW) [35,36] (Figure 4{a)). The model structure of ComQao.1.2 showed that the FARM resi ues, including three Asp residues (Asp67, Asp68, and Asp71), and the pseudo-SARM residues, including Asnl86 and Gly190 residues of the 10 residues as, a) Overal structure calculated trough the SWISS-MODEL homology modeling [36] based on 2 dimer stucure of the T. thermophilus stain HBS GGPP sythase PDB ID: TW) (b) Magne view ofa region around the putative cata centr. Four Asp residues inthe FARM and pseude-SARM are epicted by pink socks, The 10 amino acd fesidues of intrest are depicted by green sti. The putative GPP-binding domain an the purate pre Coma nding domain are indicated by yelow dashed ovalswell as one Asp residue (Asp187), ate arranged to be proximal to each other (Figure 4(b)). The three Asp residues in the FARM were predicted to bind to the diphosphate moiety of GPP via the Mg” bridge [22,28]. A previous study revealed that the Asn186 and Aspl87 residues were necessary for the in vitro geranylation activity and suggested that the pseudo- SARM serves as the pre-ComXao 2 binding site [29] ‘Therefore, the residues in the FARM and the pseudo- SARM and their peripheral residues probably form the binding domains for the respective substrates to be assembled into the major components of the catalytic center. In addition, another of the 10 residues, Phe63, is situated at the 4th position before the FARM. In trans-type FPP synthases, an aromatic residue at the Sth position or two aromatic residues at the 4th and Sth positions before the FARM are critical for the determination of the product chain length by direct interaction with the w-terminus of the farnesyl chain of FPP [28]. Hence, the Phe63 residue in Cyo-type ‘ComQs was assumed to interact with the gerany! chain of GPP. The other seven candidate residues appeared apart from the two motifs, and thus they were not likely to have a catalytic function, Construction and in vivo analysis of ComQno.e2 mutants ‘To examine whether each of the 10 Cyo-type-specific residues of ComQno.r-2 is displaceable to the corre- sponding residue in the Cys-type ComQ or whether the prenyl donor specificity of Como x. can be chan- ged by either of such substitutions, 10 Como z2 mutants ((149A], [F638], [T92L], (P1345), [T1521], INI86G], [G19OV], [K202N], [S234K], and [E248K] ComQro.r28), were individually produced together with the wild-type pre-ComXgo.r2 in the E. coli cells by introducing the corresponding mutated comQXro.2-2 gene cluster. We also prepared the E. coli transformant carrying each of the wild-type comQXrox2 and comQX ree ene clusters To evaluate the in vivo activity of ComQzo.r2 mutants, we established a simple tri-partitioned bioas- say system, In this system, each E. coli transformant carrying the wild-type or mutated com@QX gene cluster was co-cultivated with two B. subtilis tester strains (D3020 and FU1086) on a solid medium containing X-gal. Strains BD3020 and FU1086 were constructed to be able to induce the B-Gal activity in response to extraneous ComXgo.n2 and ComXie, pheromones, respectively [10.21,23] (Table 1, Figure 5). When the E.coli transformant carrying the _ wild-type comQXeo.s.2 gene cluster was co-cultivated with the two tester strains, the BD3020 cells near the E. coli cells responded to the ComXzo.22 pheromone secreted, resulting in blue coloration, whereas no col- oration was observed in the FU1086 cells. By contrast, cameat RESIDUES oF aacucus coma @ 7 only FU1086 cells showed blue coloration in response to the ComX;4q pheromone when the E, coli transfor- ‘mant carrying the wild-type comQX6s gene cluster was co-cultivated (Table 2 and Figure $1). We confirmed that, in the absence of the specific pheromone, the f Gal activity under the control ofthe srf promoter was sufficiently repressed when each tester strain was culti- vated on the TBABG plate, but not on the minimal medium plate [31], and we found that the blue colora- tion by the induced -Gal activity became clear when X-gal was added to the TBABG plate at high concen- tration (80 pg/mL) (data not shown). Thus, all bioassay results presented here were obtained under this med- ium condition, In the previous study, for detection of the ComX pheromone, the sterile pheromone-contain- ing supernatant was prepared from the liquid culture of the E. coli producer strain, and then the supernatant was added to the liquid culture of each of the B. subtilis tester strains such as BD3020, followed by measure- ment of the B-Gal activity [20]. Compared to that method, our bioassay system can be easily performed in a short time although the induced §-Gal activity is not quantitatively evaluated Using the bioassay system that we devised, the effect of each substitution on the ComQao.r.2 activity was investigated. The results showed that seven mutants (G49A], (T92L}, (P1348), [T1521], [X202N), [$234K], and [E248K]ComQso.x.28) mediated the blue colora- tion of strain BD3020 (ComXso.z.2-specific tester) but not that of strain FU1086 (ComX,ge-specific tester), which was the same as the wild-type ComQzo.xa (Table 2 and Figure $1). These results indicated that none of these substitutions affected the production or secretion of the ComXgo-s.2 pheromone and that seven replaced residues are not essential forthe catalytic func: tion of ComQzo.x.2. However, when the E. coli trans- formants corresponding to [F638], [N186G], and [G190V]ComQro 5-28 were tested, neither tester strain responded (Table 2 and Figure S1). Since the Asn186 and Gly190 residues are included in the pseudo-SARM, each mutation into these residues probably affected the pte-ComXo x2 binding, The previous study showed that each single substitution of the Asn186 residue with Ala and Asp ({N186A] and [N186D]ComQuo x28) si nificantly reduced the in vitro geranylation activity, which supports the importance of the Asn186 residue in the catalytic activity [29]. By contrast, the previous study also showed that single substitution of the Gly190 residue with either Ala or Asp ({G190A] and [G190D] ComQzo.r.28) did not severely impair the in vitro ger- anylation activity [29] It was supposed that interaction between pre-ComXno.x.2 and the pseudo-SARM was prevented due to a large steric hindrance of the Val residue at the 190th position. No activity of [F63S]ComQzo.x.2 suggested that the Phe63 residue played a critical role in the binding of GPP through a x-7 interaction. To clarify the Phe638 © KitRooKa era. E.coli producer Detection | B. subtilis strain FU1086 . [econ res (Cente: phrrene f= 2B. subtis strain 803020 Bao tig Figure 5. Scheme of production ofthe ComX pheromone inthe £. col cells and detection of the heterologously produced ComXies and ComYXno.z2 pheromones by the two 8. subtilis tester strains Both tester sans wer derived fom strain 168 Strain FUTO8 lacs the comOX;., genes bur itis abl o respond tothe extraneous Com. pheromone through the ComPAys two-component system to indice the lac exresion unde the contra thes promoter. Strain 303020 dees not posses ether came 9 comgo e304 the cM ug Genes af replaced with cmmXPan gin ti stan. Swain BDSO20 is ale to detect the exvaneaus COmMGaa Dheromone by ComPyas, which phosphonates Corns leading to induction of the lacZ gene integrated nto the sh operon. Production of pre Comigaa without prenyl modscaton inthe 803020 cls doesnot affect the pheromene detection [1,23 Table 2. Response of two B, subtilis tester strains tothe E. col transformant expressing each of the wild-type and mutated ComQs together with its target pre-ComX Pal nducton of seer aS TuI086 303020 Coma expressed inthe (ComMreespectic {ComKas.rspecife Eco cls ete ‘ite ‘Come id ype + Comes wildtype) - + MMBAIComOnce - “ Fess|ComQioce - - Ns2tkcomQioes - ” Pists}comdnoe - " Mszicomyo: - " INIBGICemaroe - - |s90v}comdngs> - - 2O2NIomQe: - + [S2saNicomOyees - # ‘ExtanicomQyees - " FBBAIComOso a - - ‘Festcomanas - + FFSsVIComGson + ets clearly induced 4, moderately induced not Induced. The photo (gf2PRS of thet partioned bioassay plates are presented in Figure Si residue’s role, the in vivo activities of three point- ‘mutants at this position ((F63A], [F631], and (F63V] ComQxo.#28) were also investigated. The results showed that. (F63I] and _—_[F63V] ComQro « 25 specifically mediated the blue coloration of strain BD3020 but not that of strain FU1086, although their inducing effects appeared slightly lower than that of the wild-type ComQo.s.2. By con: trast, [F63A]ComQso.n.2 mediated neither tester strain (Table 2 and Figure S1). These results indicated that Ie and Val residues are sufficiently functional as, alternatives to the Phe residue at the 63rd position in ComQzo-n2 and strongly suggested that the hydro- phobic interaction of GPP with a bulky side chain of the 63rd residue is essential for the catalytic activity of ComQxo 2. Nevertheless, in this in vive analysis, we could not exclude the possibility that either of the substitutions of Asn186 with Gly, Gly190 with Val, and Phe63 with Ser or Ala reduced the ComQuo x 2 stability leading to the formation of an inactive inclusion body or the protein degradation in the E. coli cells. Measurement of in vitro ComQ prenylation activity For further analysis of the Phe63 residue's role in the ComQuo-.2 activity, we prepared recombinant [F635] ‘ComQxo..2 using an E, coli overexpression system as well as the wild-type ComQuo x 2. We also prepared [D68E]ComQxo.s.2 in which the 2nd Asp residue inthe FARM was substituted with Glu (Figure $2). Geranylation and farnesylation activites of the wild- type and mutated ComQgoe2s were measured through the in vitro reaction followed by the LC-MS analysis by using chemically synthesized pre ComXso.x2 with GPP or FPP according to the pre- vious study (25] (Figure 2(a)). When GPP and pre- ComXgo.n2 were used as substrates, the wild-type ComQxo #2 in the membrane fraction showed signif- icant geranylation activity to produce the Cyg-modi- fied ComXno.x.2 C-terminal heptapeptide (from the 52nd to 58th position) predominantly, and the native form of the ComXyo.z.2 pheromone (from the 53rd to 58th position) was given in a low ratio, similar to the previous data [25]. By contrast, no farnesylation activ- ity was detected when FPP was used instead of GPP in the reaction mixture (Figure 6(a) and Figure $3(a,b)), and no dimethylallylation activity was detected when imethylallyl diphosphate (C,) was used (data not shown), ‘These results clearly demonstrated that ‘ComQxo.2. has strict prenyl-donor specificity. Remarkably, [F63S]ComQno.z2 showed substan- tial farnesylation activity when FPP was used, while its geranylation activity was severely reduced (Figure 6 (a) and Figure $3(c.d)). ‘This result indicated that the ‘63rd Phe residue is critica for the specific geranylation activity of ComQao 2. Itis likely that the substitution of Phe63 with Ser caused a loss of binding affinity to GPP through the hydrophobic interaction and enabled the accommodation of FPP in the widened cavity space (Figure 4(b)). Based on the in vitro property of [F63S]ComQso.n.2. we estimated that, in the in vivo analysis, the observation that (F638]ComQxo 2 did not mediate the B-Gal induction of ether tester strain ‘was attributed to the low prenylation activity of this ‘mutant for both GPP and FPP even though the activity for FPP was elevated by the substitution. Alternatively, the produced C,s-modified ComXgo.¢.2 pheromone CRITICAL RESIDUES OF BACILLUS COMA might not be recognized by either ComPyss or ComPao.z2 although the previous studies showed that the prenyl-chain length of ComX pheromones ‘was an influential determinant for specific bioactivities, for the corresponding strains, rather than their amino acid sequences [21,23,27]. At least it is unlikely that the substitution of the Phe63 residue with Ser caused destabilization of the ComQgo.s 2 protein structure. When (D68E]ComQuo x2 was similarly tested, the prenylation activity of this mutant was found to be severely impaired. Since it was already reported that sub- stitution of either the Ist or Sth Asp residue with Glu in the FARM abolished the in vivo activity of ComQuea (221, all three Asp residues in the FARM are presumed to be finely coordinated for the prenyl substrate binding (Figure 4(b), Figure 6(a), and Figure $3(e)). We also prepared [S63F]ComQiss, a ComQues mutant with the substitution of the Ser residue with Phe at the 4th position before the FARM, and the in vitro prenylation activity of this mutant, as well as that of the wild-type ComQios, was measured using chemically synthesized pre-ComXygq together with GPP or FPP (Figure 2(a) and Figure $2). When FPP and pre-ComXgy were used as substrates, the wild- type ComQuss in the membrane fraction mainly pro- duced the Cys-modified ComX;e, C-terminal nona- peptide (from the 47th to 55th position) predominantly, and the native form of the ComXes pheromone (from the 46th to 55th position) was given in a low ratio (Figure $3(f)). When GPP was used as, the prenyl donor, the wild-type ComQusg unexpect- edly provided Cyo-modified ComXye peptides, though the prenylation activity of the wild-type ‘ComQis obtained with FPP was much higher than that with GPP (Figure 6(b)). When [S63F]ComQigs was tested, this mutant significantly reduced the far- nesylation activity using FPP as the prenyl donor, whereas its geranylation activity using GPP was b a x 8 ComOnoe2 ComQres 2 o8 gn z Bus qs : i in 2 : i o a. o wr FeSS DSBE Wr S83F Figure 6. Measurement of in vitro prenylation activities ofthe wild-type and mutated ComQs. Gray bas represent the geranyltion activites and white bar the fanesyation actives of the wipe, [F635], and [DSEEIComQq.25 (a) and the wl type and (S631 coma), which wete determined by quantifying the lls of he Cy {and Cis-modied Com. Ccerminl nonapeptdes respective. The bats and ever bas indicate the mean alues and the standard deviations 3) The nd" means thatthe farnesylitonactty was nt detected, andthe "n/a? means that he famesylaton activity was nat messed « and Csmodifed ComKaaca Ctermial heptapepties andthecomparable to that of the wild-type ComQues (Figure 6(b)). The Phe residue at the 63rd position probably acted as a steric hindrance, interfering with the farne- «yl chain of FPP but not with the shorter chain of GPP. Taken together, these in vitro data clearly demon: strated that a single substitution at the 4th position before the FARM significantly affected the preny! donor preference of both Cyo- and Cys-type ComQs. Conclusion The present study demonstrated that, in addition to the previously reported Asp67, Asp71, and Asp187 residues, the Phe63, Asp68, Asn186, and Gly190 resi- dues in ComQgoz2 are critical for the geranylation (Cio modification) of pre-ComXgo.x-2 [22,28]. Since the Asn186, Asp187, and Gly190 residues are included in the pscudo-SARM, these residues are probably involved in pre-ComXzo.x2 binding, and a void space around the Gly190 residue is also likely to be required for catalytic activity in ComQuo.t 2. In addi- tion, it was deduced that the Phe63 residue and all of the three Asp residues (Asp67, Asp68, and Asp71) in the FARM play a crucial role in the specific biding to GPP through the hydrophobic interaction and the Mg’* bridge, respectively. Moreover, it was demonstrated that a single substitution at the 4th position before the FARM in ComQ enables alteration of the prenyl donor preference. Although the preny! donner specificity of ComQ may not be solely deter- mined by the 63rd residue, this residue’s property is presumably one of the critical determinants. The other tical residues may be identified using high-through- put screening with the bioassay system established in this study. The prenyl-chain length of ComX phero- mones was a major determinant for specific bioacti- ities for the corresponding strains. The specificity of the ComX pheromone’s bioactivities may be exchangeable by a small number of point mutations at the 63rd position and the other unidentified posi- tions in Com leading to alteration of the prenyl- chain length of the ComX pheromone, which might have been actualized in nature Acknowledgments ‘We are grateful to Ayaka Tamano, Naoki Matsumura, and Hiroyoshi Komatsu for their help with the plasmid con: struction and the in vive bioassay. Author contributions SS performed the plasmid construction for the co-expres: sion in E.coli and the bioassay experiments. KH constructed the B, subilis strains. MO prepared the recombinant ‘ComQs and performed the in vitro analysis. KH and MO drafted the research plan and wrote the manuscript with SS. Disclosure statement ‘No potential conflict of interest was reported by the authors. ORCID Kazutake Hirooka @ _hitp/forcid.org/0000-0001-5879. 936K References {i} Rutherford ST, Bassler BL. 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