The melREDCA Operon Encodes a Utilization System for theRaffinose Family of Oligosaccharides in Bacillus subtilis

Kambiz Morabbi Heravi,a Hildegard Watzlawick,a Josef Altenbuchnera

aInstitut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany

ABSTRACT Bacillus subtilis is a heterotrophic soil bacterium that hydrolyzes differ-ent polysaccharides mainly found in the decomposed plants. These carbohydratesare mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides(RFOs). RFOs are soluble �-galactosides, such as raffinose, stachyose, and verbascose,that rank second only after sucrose in abundance. Genome sequencing and tran-scriptome analysis of B. subtilis indicated the presence of a putative �-galactosidase-encoding gene (melA) located in the msmRE-amyDC-melA operon. Characterization ofthe MelA protein showed that it is a strictly Mn2�- and NAD�-dependent �-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription ofthe msmER-amyDC-melA operon is under control of a �A-type promoter located up-stream of msmR (PmsmR), which is negatively regulated by MsmR. The activity ofPmsmR was induced in the presence of melibiose and raffinose. MsmR is a transcrip-tional repressor that binds to two binding sites at PmsmR located upstream of the�35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms anATP-binding cassette (ABC) transporter that probably transports melibiose into thecell. Since msmRE-amyDC-melA is a melibiose utilization system, we renamed theoperon melREDCA.

IMPORTANCE Bacillus subtilis utilizes different polysaccharides produced by plants.These carbohydrates are primarily degraded by extracellular hydrolases, and the re-sulting oligo-, di-, and monosaccharides are transported into the cytosol viaphosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitatorsuperfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbo-hydrate utilization system of B. subtilis responsible for the utilization of �-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOsare synthesized from sucrose in plants and are mainly found in the storage organsof plant leaves. Our results revealed the modus operandi of a new carbohydrate uti-lization system in B. subtilis.

KEYWORDS �-galactosidase, �-galactosides, ABC transporter, carbohydrate,melibiase, melibiose, repressor, sugar-binding lipoprotein

Bacillus subtilis is a soil bacterium found in the rhizosphere of many plants. Becauseof such a habitat, B. subtilis can utilize a variety of poly- and oligosaccharides

present in the rhizosphere as a major carbon source and therefore has different systemsfor the utilization of poly-, oligo-, and monosaccharides synthesized by plants. Thepolysaccharides are primarily degraded to oligosaccharides with shorter chain lengthsand to monosaccharides by extracellular enzymes such as �-amylase, pullulanase, orxylanase. The generated oligosaccharides and monosaccharides can be then taken upvia the carbohydrate-specific transporters of the phosphoenolpyruvate-dependentphosphotransferase system (PTS), ATP-binding cassette (ABC) transporters, and themajor facilitator superfamily (uniporters, symporters, and antiporters) (1, 2). Dependingon the transport system, the carbohydrates are phosphorylated during (for PTS) and

Citation Morabbi Heravi K, Watzlawick H,Altenbuchner J. 2019. The melREDCA operonencodes a utilization system for the raffinosefamily of oligosaccharides in Bacillus subtilis. JBacteriol 201:e00109-19. https://doi.org/10.1128/JB.00109-19.

Editor Anke Becker, Philipps-UniversitätMarburg

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Kambiz MorabbiHeravi, kambiz.morabbi@iig.uni-stuttgart.de.

K.M.H. and H.W. contributed equally to thiswork.

Received 1 February 2019Accepted 21 May 2019

Accepted manuscript posted online 28 May2019Published

RESEARCH ARTICLE

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after (for non-PTS) the transport and usually converted to one of the intermediates ofthe glycolysis or pentose phosphate pathway (3, 4). Carbohydrate utilization genes canform operons consisting of genes encoding extracellular and/or intracellular degrada-tion enzymes, the transporter components, and a specific regulator. The function ofthese specific regulators can be altered due to the binding of sugar ligands as seen inthe ABC transport systems, such as it is in the maltodextrin and galactan utilizationsystems (5, 6), or as a result of the phosphorylation state of specific regulators in aninteraction with the specific transporter as for the PTSs (5–7). All these systems arecatabolically repressed in the presence of glucose (or other PTS sugars) via the globalCcpA-dependent pathway (CcpA is the carbon catabolite protein) or via more specificpathways, such as inducer exclusion (8, 9).

Among all carbohydrates, oligosaccharides of the raffinose family (RFOs) are ubiq-uitous among the plants. These soluble carbohydrates rank second in abundance, onlyafter sucrose (10). RFOs, such as raffinose, stachyose, and verbascose (Fig. 1A), are infact the �-galactosyl derivatives of sucrose (the major product of photosynthesis) andare synthesized in a reaction catalyzed by galactosyl transferase using sucrose andgalactinol (galactose bound to myo-inositol) as the substrate (11). Therefore, RFOsand melibiose contain terminal �-galactosyl residues that are cleaved by �-galactosidases. Several bacteria, such as Escherichia coli (12, 13), Erwinia chrysanthemi(14), and different lactobacilli (15), are able to utilize RFOs or melibiose as their carbonsource. The genome of B. subtilis also contains a gene for an �-D-galactoside galacto-hydrolase, also known as melibiase (16, 17), which is possibly involved in degradationof the RFOs, such as raffinose and melibiose (18). The melA gene is the last gene of themsmRE-amyDC-melA operon (or melibiose operon) (Fig. 1B) (19). In this study, wecharacterized the functions of the components of the melibiose utilization system of B.subtilis. MelA was shown to have �-galactosidase activity on the substrates transportedby the ABC transport system. Also, the regulation of the system by a specific regulator(MsmR) was addressed, and the promoter region of the operon was characterized.

RESULTSMelA is an �-galactosidase able to hydrolyze melibiose and RFOs. The melibiose

operon was predicted to encode the components of an �-galactosidic oligosaccharide

FIG 1 (A) Chemical structure of melibiose and the raffinose family of oligosaccharides (RFOs). Galactose(Gal) moieties are �-1,6 linked to Gal or glucose (Glc). Sucrose consists of Glc that is �-1,2 linked tofructose (Fru). (B) The genetic map of the utilization system of melibiose and RFOs in B. subtilis.

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utilization system in B. subtilis (1). Therefore, the first step toward understandingthe utilization of �-galactosidic oligosaccharides by B. subtilis was to show the�-galactosidase activity of MelA. MelA belongs to the glycosyl hydrolase 4 (GH4) family,the members of which are known to require NAD�, Mn2�, and reducing agents for theiroptimal activity (20, 21). In order to measure the MelA activity, melA coding for a proteinwith an N-terminal Strep-tag was overexpressed by using the L-rhamnose-induciblepromoter (rhaPBAD) in Escherichia coli (strain JM109/pHWG1118). The preliminary stud-ies using 4-nitrophenyl-�-D-galactopyranoside (pNP-�-Gal) as the substrate indicatedthat the highest �-galactosidase activity was obtained with Mn2� (3 mM), NAD�

(10 mM), and Tris(2-carboxyethyl)phosphine (TCEP) (1 mM) at pH 8 and 55°C (see TableS4 in the supplemental material). Under these optimal assay conditions, MelA was ableto hydrolyze melibiose, raffinose, and stachyose (Table 1), whereas locust bean gum (apolymeric galactomannan) was not hydrolyzed (data not shown). Among all substrates,the highest specific activity of MelA was obtained with melibiose (Table 1). The Km

values for the hydrolysis of melibiose and raffinose were 10 and 25 mM, respectively(Table 1). Altogether, this result confirmed the connection between the msmRE-amyDC-melA operon and �-galactoside utilization. To better understand this utilization system,the regulation of the operon was further studied.

MsmR represses the melibiose operon in the absence of melibiose or raffinose.Transcriptome analysis of B. subtilis under different conditions (19) indicated that themelibiose operon has two transcription start sites, one located upstream of msmR(promoter PmsmR) and the other one located upstream of msmE (PmsmE). To verify thepresence of two promoters controlling the expression of melibiose operon, the lacZgene was used as a reporter. In detail, the PmsmR-lacZ and PmsmE-lacZ cassettes wereconstructed and integrated into the amyE locus of the wild-type B. subtilis KM0. The�-galactosidase activity of each strain was then measured in LB after the addition ofmelibiose, raffinose, and stachyose as the possible inducers. No or negligible�-galactosidase activity was detected with the PmsmE-lacZ fusion under all conditions(data not shown). This indicated that there is not a promoter in the intergenic regionbetween msmR and msmE. This result contradicted the upshift of the msmE transcrip-tion reported by Nicolas et al. (19). Perhaps, another mechanism, such as mRNAendonucleolytic cleavage, as observed in the regulation of the cggR-gapA operon, isinvolved (22, 23). In contrast to PmsmE, melibiose and raffinose strongly induced PmsmR

activity in the wild-type strain, while stachyose had no effect on PmsmR activity com-pared to the uninduced control (Fig. 2A). Likewise, the addition of glucose repressedthe activity of PmsmR even in the presence of melibiose (Fig. 2A). This showed that themelibiose operon is transcribed under the control of PmsmR and subject to carboncatabolite repression.

Given that melibiose and raffinose induced the activity of PmsmR, a specific regula-tion of PmsmR was further considered. The first gene of the melibiose operon encodesa putative transcriptional regulator, MsmR, which belongs to the LacI family of tran-scriptional regulators (24). The deletion of msmR in the wild-type strain resulted inKM911 and revealed a constitutive high activity of PmsmR under all conditions, showingthat msmR had a negative regulatory effect on the expression of the melibiose operon(Fig. 2B). Next to the in vivo experiments with msmR-deficient strains, in vitro experi-

TABLE 1 �-Galactosidase activity of purified MelA with different substratesa

Substrate Sp act (U/mg) Km (mM)b

pNP-�Gal 2.9 NDMelibiose 4.8 10Raffinose 3.3 25Stachyose 1.5 NDa�-Galactosidase activity was measured in a buffer containing 0.1 M Tris-HCl (pH 8), 1 mM NAD�, 3 mMMn2�, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). The reactions were carried out at 37°C and pH 8, asdescribed in Materials and Methods.

bND, not determined.

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ments were carried out with purified MsmR. In practice, the MsmR protein wasproduced by E. coli JM109 with plasmid pHWG1169, as described in Materials andMethods, and purified MsmR–Strep-tag protein was used for in vitro studies. In order tostudy the interaction between PmsmR and MsmR, the promoter elements of PmsmR wereprimarily identified (Fig. 3A). The transcription of msmR starts at a thymidine residuelocated 51 bp upstream of the start codon of MsmR (Fig. 3B). Accordingly, the �10(TAATAT) and �35 (TTGTAA) boxes were found showing a �A-type (housekeepingsigma factor) promoter structure (Fig. 3A). Likewise, the presence of a cataboliteresponsive element (cre) site (25) within the spacer of PmsmR indicated the possibility ofCcpA-dependent carbon catabolite repression of the melibiose operon similar to thatin other carbohydrate utilization systems in B. subtilis (8, 26). The DNA fragmentcomprising the PmsmR region specifically interacted with purified MsmR compared tothe control DNA fragment containing the PglcR region (Fig. 3C). DNase I footprintingrevealed two MsmR binding sites at PmsmR (Fig. 3D) with the inverted repeat ofATTTACTAAAT (Fig. 3A). One of these MsmR binding sites is located upstream of the�35 box (FP1), whereas the other one was found downstream of the transcriptionalstart site (FP2) (Fig. 3A). An electrophoretic mobility shift assay confirmed the bindingof MsmR to DNA fragments of the FP1 or FP2 inverted repeats (green boxes in Fig. 3C).To find the specific effector of MsmR, a thermal shift assay was carried out usingpurified MsmR and tested with different effectors, i.e., galactose, melibiose, and raffin-ose. The melting temperature of MsmR was elevated only in the presence of melibioseor raffinose (Fig. 4A). MsmR was, however, not dissociated from PmsmR DNA in thepresence of raffinose or melibiose, as shown by the electrophoretic mobility shift assay(Fig. S1). This was similar to regulators such as MerR (27, 28) and GalR (29) in E. coliwhich remain bound to DNA even in the presence of their inducer. Altogether, theseresults indicated that MsmR represses the melibiose operon when there is no melibioseor raffinose as the inducer.

MsmEX-AmyCD forms the probable transporter of melibiose. The melibioseoperon encodes the components of a predicted ABC transport system, i.e., AmyC,AmyD, and MsmE, in addition to the intracellular �-galactosidase MelA. Among theseproteins, MsmE was predicted to be a sugar-binding lipoprotein, which probably formsan ABC transporter together with the transmembrane proteins AmyC and AmyD (4). Toconfirm the function of these proteins, in vivo experiments with the different mutantsof B. subtilis strains lacking msmE, amyC, or amyD were carried out, and �-galactosidasemeasurements were performed to investigate the inducibility of the PmsmR-lacZ cas-

FIG 2 (A) Regulation of the melibiose operon in B. subtilis was measured by integration of the PmsmR-lacZ cassette(pKAM384) into the amyE locus of the wild-type strain (KM0). B. subtilis KM845 (wt) was cultured in LB, and the�-galactosidase activity was measured 1 h after the addition of carbohydrates (0.2%), melibiose (Mel), raffinose(Raf), stachyose (Sta), and glucose (Glc). (B) �-Galactosidase activity of B. subtilis strains KM845 (wt), KM861(ΔmsmX), KM911 (ΔmsmR), KM912 (ΔamyD), KM913 (ΔamyC), and KM914 (ΔmelA) carrying the PmsmR-lacZ cassettewas measured in minimal medium (MM) with succinate and glutamate as the basal carbon source. Melibiose,raffinose, and stachyose were added with a final concentration of 0.2% to the cultures, and the �-galactosidaseactivity was measured 4 h after the addition of carbohydrates.

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sette in the mutants lacking amyC, amyD, or msmE genes. The deletion of all thesegenes rendered PmsmR uninducible in the presence of melibiose and raffinose, confirm-ing their probable function as an �-galactoside transport system (Fig. 2B). Nevertheless,an important missing piece of the melibiose transport puzzle was the nucleotide-binding domain. Previous studies of the carbohydrate transport systems showed thatMsmX acts as the nucleotide binding domain of different carbohydrate ABC transport-ers (6, 30). Also here, the deletion of msmX resulted in the loss of inducibility of PmsmR

by melibiose, showing that MsmX hydrolyzes ATP as the nucleotide binding domain forthe transport of �-galactosides (Fig. 2B). The substrate specificity of the MsmEX-AmyCDtransporter was also considered and investigated by in vitro experiments. Since thesubstrate specificity of ABC transporters depends on their substrate binding lipopro-tein, MsmE was further studied in order to find its effector specificity. Analysis of theamino acid sequence of MsmE with the SignalP program (31) indicated a signal peptidemotif with a possible cleavage site between amino acid positions 20 and 21. Therefore,the signal peptide sequence was replaced with the His6 tag, and the His6-msmEtranscriptional fusion was overexpressed in E. coli (strain JM109/pHWG1149). Thepurified His6-MsmE (Fig. S2) was used for the thermal shift assay to examine galactose,

FIG 3 Characterization of PmsmR. (A) The DNA sequence between msmR and ytaP start codons is shown. The open arrows show the start codons of ytaP andmsmR. The core elements of PmsmR (�35 and �10 boxes) and the transcription start site of msmR are indicated with boxes. The protected regions of PmsmR DNAby MsmR from DNase I digestion (FP1 and FP2) are in red letters. The inverted repeats within the MsmR binding site are indicated by solid arrows. The putativecre site (gray highlighted) is also demonstrated. The green boxes show the DNA regions used for the electrophoretic mobility shift assay. (B) Identification ofthe transcription start site (TSS) of msmR was performed by primer extension. The migration of the generated cDNA fragment (orange) was compared with thesequencing reaction. (C) An electrophoretic mobility shift assay was carried out using 5=-end Cy5-labeled DNA fragments of PmsmR, the FP1 inverted repeat, andthe FP2 inverted repeat. The amplified DNA fragment from the GlcR binding site was used as a negative control. The migration of the DNA fragment wasinvestigated in the absence (�) or presence (�) of MsmR. (D) The chromatographs of the DNA footprinting and DNA sequencing reactions are separately shown.The 6-FAM-labeled PmsmR DNA was digested with DNase I in the absence (orange) or presence (blue) of 0.27 mM MsmR. The identified DNA footprints, FP1 andFP2, were then compared with the DNA sequencing reaction utilizing ddATP (green), ddGTP (black), ddCTP (blue), and ddTTP (red).

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melibiose, raffinose, and stachyose as the possible effectors for MsmE (Fig. 4B). Amongthese substrates, stachyose, raffinose, and melibiose were able to increase the meltingtemperature of MsmE. Surprisingly, only melibiose could support the growth of the B.subtilis cells in minimal medium as the main carbon source (Fig. S3). All strains lackingone of the components of the melibiose utilization system were unable to grow withmelibiose (Fig. S3). Altogether, it seems that MsmE-AmyCD together with MsmX formsan ABC transporter for melibiose and, probably, raffinose and stachyose.

DISCUSSION

Most of the carbohydrates in the soil are released after decomposition of planttissues and are rich in RFOs and �-galactosides. In this study, we showed that B. subtilishas an �-galactosidase, called MelA, that belongs to the rare GH4 family of�-galactosidase for the degradation of melibiose and RFOs. So far, there is littleinformation on the enzymes of the GH4 family. By determination of the biochemicalproperties of MelA, another member of the rare GH4 family of �-galactosidase is nowwell known. MelA is a strictly Mn2�- and NAD�-dependent enzyme capable of hydro-lyzing melibiose, raffinose, and stachyose, whereas it is unable to act on polymericsubstrates, such as locust bean gum. Locust bean gum is a galactomannan consistingof a linear polymer of mannose with branching of an �-galactose after each fourthmannose. The substrate specificity and mode of action of MelA are highly similar tothose of MelA of E. coli K-12 (32) and recombinant Mel4A (rMel4A) of Bacillus halodurans(33). Compared to MelA from E. coli and B. subtilis, recombinant Mrl4A (rMrl4A) hashigher affinity toward raffinose than toward melibiose (33). Interestingly, B. haloduranshas two extra putative �-galactosidase genes encoding enzymes of the glycosidehydrolase families GH27 and GH36 (33). In addition to melA, there is a putativeintracellular hydrolase-encoding gene (ytaP) located upstream of melREDCA; however,the deletion of ytaP had no influence on the regulation of the melREDCA. Also, PytaP wasnot inducible with melibiose or raffinose (data not shown). Likewise, YtaP showed nohydrolase activity with sucrose as the substrate similar to an invertase (data not shown).Clearly, further studies are necessary to find the substrate specificity of YtaP hydrolase.

The melibiose utilization system of B. subtilis was inducible with both melibiose andraffinose. In. B. halodurans, however, the rMel4A activity was induced only after theaddition of raffinose and not melibiose or stachyose (33). More-complex substrates,such as guar gum or locust bean gum, could not induce the system as observed in B.megaterium VHM1 (34). The regulator of the melibiose operon, MelR, is one of the 12transcriptional regulators of the LacI family in B. subtilis, all of which are involved incarbon catabolic pathways. All of the well-studied LacI regulators in B. subtilis, includingCcpA (25), AraR (35), GanR (36), KdgR (37), ExuR (38), and IolQ (39), recognize invertedrepeats. The LacI family of regulators can have more than a single binding site leadingto DNA bending or looping (40, 41). The presence of two binding sites at PmsmR mayalso result in bending of the DNA causing steric hindrance for the RNA polymerase. This

FIG 4 Determination of substrate specificity of MsmR and MsmE by thermal shift assay. (A) Alteration ofthe melting temperature of purified MsmR–Strep-tag (concentration) in the absence of effectors wascompared to the presence of galactose, melibiose, or raffinose with the final concentration of 10 mM. (B)Purified His6-MsmE was mixed with different effectors (10 mM), i.e., galactose, melibiose, raffinose, andstachyose, and its melting temperature was measured.

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mode of action is different from that of E. coli, in which MelR is a dual regulator, atranscriptional activator for melAB, and a repressor for melR that binds to five bindingsites at the intergenic region of melR and melA in the absence of melibiose (42).

E. coli naturally takes up only melibiose via a melibiose:H�/Na�/Li� symporter(MelB) (43), and raffinose can be transported via a plasmid-based system (44, 45), UnlikeE. coli, RFOs and melibiose are mainly transported by the ABC transport systems in B.subtilis (1, 2). Interestingly, most of the carbohydrate ABC transporters, such as malto-dextrin (5), galactan (6, 36, 46), and arabinan (47), optimally transport oligosaccharideswith 3 to 4 degrees of polymerization. It seems that ABC transporters of B. subtilis aremainly involved in the uptake of oligosaccharides compared to the PTS transporters,which are mainly involved in the transport of mono- and disaccharides (48). Anothermissing piece of the �-galactoside ABC transport systems was the nucleotide bindingdomain. Interestingly, MsmX was shown to be the nucleotide binding domain of theMelECD ABC transporter, similar to the GanSPQ, AraNPQ, YtcQP-YteP, and MdxEFG ABCtransporters (5, 6, 30). Despite the results from thermal shift assay and the inducibilityof PmsmR with raffinose and melibiose, B. subtilis weakly grew only with melibiose. Sucha weak growth could be due to weak promoter activity (approximately 250 Miller units)of PmsmR compared to other sugar-inducible promoters, such as PmtlA (49) or PmanP (50),or due to the low efficiency of the transport system. Indeed, further studies arenecessary to clarify the transport efficiency of �-galactosides by the MelECD-MsmXtransporter.

In conclusion, based on the results of this study, a model for the function ofmelibiose utilization system is demonstrated in Fig. 5 Briefly, B. subtilis takes upstachyose, raffinose, and melibiose via the MsmE-AmyCD-MsmX ABC transport system.These carbohydrates are then converted to glucose, galactose, and sucrose by theaction of the �-galactosidase MelA. The melibiose operon is regulated by MsmR. MsmRacts as a transcriptional repressor for the melibiose operon by binding to FP1 and FP2regions at PmsmR and inhibits the transcription of the mel operon.

FIG 5 Model of the RFOs degradation by B. subtilis. After degradation of polysaccharide complexesproduced by plants, the released �-galactosides are consumed by B. subtilis. MsmE as the substrate-binding lipoprotein binds melibiose, stachyose, and raffinose. The transport takes place via the channelformed by transmembrane proteins MelCD. The energy of the transport is provided by MsmX. Raffinoseand melibiose induce the derepression of melibiose operon by interaction with MelR. The carbohydratesare then converted to glucose, galactose, and sucrose via the action of the �-galactosidase activity ofMelA. Raffinose and melibiose are the effectors of MsmR. CM, cytoplasmic membrane.

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MATERIALS AND METHODSStrains, media, and growth conditions. All strains used in this study are listed in Table S1 in the

supplemental material. To propagate plasmids or express the desired genes, Escherichia coli JM109 wasused as a host. The transformants of E. coli were selected on LB agar supplemented with ampicillin(100 �g/ml) or spectinomycin (100 �g/ml), depending on the plasmid selection marker. To overexpressthe desired gene, E. coli JM109 carrying the expression plasmid was inoculated into LB medium withampicillin (100 �g/ml) with a starting optical density at 600 nm (OD600) of 0.05. After 2 h of incubationat 37°C with 200 rpm shaking intensity, L-rhamnose (0.2% [wt/vol]) was added to the bacterial culture,and the cells were harvested by centrifugation after 4 h of incubation at 30°C. The harvested cell pelletwas kept at �20°C for further analysis.

Bacillus subtilis KM0, a tryptophan prototroph derivative of strain 168, was used as the wild-typestrain in this study. B. subtilis knockout erythromycin (BKE) strains (51) were also obtained from theBacillus Genetic Stock Center (BGSC, OH). B. subtilis transformants were selected on LB plates containingspectinomycin (100 �g/ml) or erythromycin (5 �g/ml). The tryptophan auxotroph BKE strains werecultured in Spizizen’s minimal medium (52) supplemented with tryptophan (50 �g/ml). To find theinducer of the melibiose utilization system, LB medium was inoculated with the desired strains with astarting OD600 of 0.05. After 2 h of incubation at 37°C with 200 rpm shaking intensity, raffinose, melibiose,stachyose, or glucose was added to the bacterial cultures at a final concentration of 0.2% (wt/vol). Eachbacterial culture was harvested after 1 h to measure the �-galactosidase activity.

To study the regulation of the melibiose utilization genes and isolation of total RNA, B. subtiliswild-type and mutant strains were cultured in Spizizen’s minimal medium [(NH4)2SO4 (2 g/liter), K2HPO4

(14 g/liter), KH2PO4 (6 g/liter), Na3 citrate·2H2O (1 g/liter), MgSO4·7H2O (0.2 g/liter)] supplemented with0.6% (wt/vol) succinate, 0.8% (wt/vol) glutamate, trace elements (CaCl2·2H2O [0.5 mg/liter], FeCl3·6H2O[16.7 mg/liter], Na2-EDTA [20.1 mg/liter], ZnSO4·7H2O [0.18 mg/liter], MnSO4·H2O [0.1 mg/liter],CuSO4·5H2O [0.16 mg/liter], CoCl2·6H2O [0.18 mg/liter]), and 0.02% (wt/vol) Casamino Acids. Fifty millili-ters of the minimal medium was inoculated with a starting OD600 of 0.1 and incubated at 37°C with200 rpm shaking intensity. After 3 h and 45 min of incubation, 0.2% (wt/vol) of raffinose, melibiose, orstachyose was added to the 8-ml aliquots of bacterial culture. After 4 h of incubation, the bacterial culturewas harvested and used for the measurement of �-galactosidase activity. All experiments were repeatedtwice, and the mean values were used for further analysis.

DNA manipulation and plasmid construction. All plasmids used in this study are listed in Table S2.Standard molecular techniques were carried out as described by Sambrook and Russell (53). DNAfragments were amplified by PCR using Phusion high-fidelity (HF) DNA polymerase (catalog no. M530S;New England BioLabs, Frankfurt am Main, Germany) on a LifeECO thermal cycler (Hangzhou BioerTechnology Co. Ltd., China). Unless otherwise specified, the chromosomal DNA of B. subtilis KM0 wasused as the template in all PCRs. Chromosomal DNA of B. subtilis was isolated using the innuPREPbacterial DNA kit (Analytik Jena AG, Jena, Germany), according to the manufacturer’s instructions. Alloligonucleotides used were synthesized by Eurofins MWG Operons (Ebersberg, Germany) (Table S3).Restriction enzymes purchased from New England BioLabs were used for the digestion of DNA frag-ments. PCR or digested DNA fragments cut from agarose gel were isolated using the NucleoSpin gel andPCR cleanup kit (Macherey-Nagel GmbH, Düren, Germany). Ligation of the desired DNA fragments wascatalyzed by T4 DNA ligase (Thermo Fisher Scientific, Inc., Karlsruhe, Germany). The innuPREP plasmidminikit (Analytik Jena AG) was purchased for plasmid extraction. All newly constructed plasmids weresequenced by GATC Biotech AG (Constance, Germany). The construction of each plasmid is thoroughlyexplained in Table S2.

Construction of the B. subtilis strains. Natural transformation of B. subtilis strains was performedaccording to the Paris method (54). All of the mutants used in this study were derivatives of the Bacillusknockout erythromycin (BKE) strains constructed by Koo et al. (51). To remove the erythromycinresistance gene, each strain was transformed with the unstable plasmid pJOE6732.1 (6) expressing Crerecombinase. After selection on LB with spectinomycin, a single colony was further cultured in LB for 24 hat 37°C, and a 10�6 dilution was plated on LB. Approximately 50 colonies were checked for the loss ofspectinomycin and erythromycin. Finally, each deletion was verified with appropriate oligonucleotides ina PCR. Since all BKE strains and their derivatives were tryptophan auxotrophs, the trpC2 mutation wasrepaired using plasmid pKAM041 (55). After transformation of the strains, selection was performed onminimal medium without tryptophan. To investigate the regulation of the promoter regions, thederivatives of pKAM263 (48), i.e., pKAM384 (PmsmR) and pKAM385 (PmsmE), containing the promoter-lacZfusion were integrated into the amyE locus of the desired strain. The transformants were selected on LBcontaining spectinomycin, and the sensitivity to erythromycin (single-crossover integration) and loss of�-amylase activity (LB containing 1% starch) verified the integration of the promoter-lacZ via doublecrossover.

Gene expression and protein purification. For expression in E. coli, the coding regions of melA,msmE, and msmR were amplified by PCR, with genomic DNA from Bacillus subtilis 168 serving as thetemplate. The specific primers additionally introduced the restriction sites necessary for the cloning ofthe desired genes (see Table S2) and resulted in the expression plasmids pHWG1118 for the Strep-tag–melA transcriptional fusion, pHWG1149 for the His6-msmE transcriptional fusion, and pHWG1169 for themsmR–Strep-tag transcriptional fusion, respectively. The growth conditions of the strains for overpro-duction of proteins have previously been described. The cell pellet of E. coli strain JM109/pHWG1118(Strep-tag–MelA) was dissolved in 12 ml of 0.1 M Tris-HCl (pH 8.0), while JM109/pHWG1169 (for MsmR–Strep-tag) was dissolved in 12 ml of 0.1 M Tris-HCl (pH 8.0), 0.3 MnSO4, and 1 mM Tris(2-carboxylethyl)phosphine (TCEP). The cells were disrupted by passing the cell suspension through

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pouring into the ice-cooled chamber of an EmulsiFlex-C5 high-pressure homogenizer (Avestin, Mann-heim, Germany) at 15,000 lb/in2. The bacterial suspension was completely ruptured after 2 to 3 min. Thebacterial lysate was centrifuged for 30 min at 12,000 � g. The cleared bacterial lysate was then passedthrough 1 ml of Strep-Tactin resin for the purification of streptavidin-tagged proteins. The purificationsteps were carried out according to the manufacturer’s instructions. Purification of MsmE without itssignal peptide was performed using strain JM109/pHWG1149. The cell disruption steps were accom-plished as mentioned above, and the cleared cell lysate was passed through 1 ml of Talon metal affinityresin (Clontech Laboratories, Inc., Mountain View, CA) to purify His6-tagged MsmE.

DNA sequencing. The sequencing reaction of pKAM0138 carrying the msmR promoter region wasperformed with 5=,6-carboxyfluorescein (5=,6-FAM)-labeled oligonucleotide s12050 using the ThermoSequenase cycle sequencing kit (Affymetrix, High Wycombe, UK). The master mix for sequencing wasprepared by mixing 2 �l of pKAM0138 (30 fmol/�l) with 2 �l of the reaction buffer, 1 �l of the desiredoligonucleotide (4 pmol/�l), 1 �l dimethyl sulfoxide (DMSO), 2 �l DNA polymerase, and 9.5 �l double-distilled water (ddH2O). Four microliters of the sequencing master mix was then added to 4-�l aliquotsof ddGTP, ddATP, ddTTP, and ddCTP. The sequencing reaction was accomplished in a LifeECO thermalcycler (Hangzhou Bioer Technology Co. Ltd., China). The amplification program included initial denatur-ation for 2 min at 95°C, 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and a final extensionfor 1 min at 72°C. Finally, amplified DNA in each reaction mixture was precipitated with 3 M sodiumacetate (pH 6.3) and isopropanol. The DNA was then dissolved in 6 �l ddH2O and 34 �l Hi-Di formamide(catalog no. 4401457; Applied Biosystems by Thermo Fisher Scientific, USA). After the addition of 0.5 �lGeneScan 600 LIZ size standard v2.0 from the GeneScan installation kit DS-33 (catalog no. 4376911;Applied Biosystems by Thermo Fisher Scientific, USA), the samples were run on a SeqStudio geneticanalyzer (Applied Biosystems by Thermo Fisher Scientific), and the results were analyzed using theGeneMapper software 5 (Applied Biosystems by Thermo Fisher Scientific).

Primer extension. To find the transcription start site (TSS) of msmR, strain KM845 containing theintegrated PmsmR-lacZ cassette was used. Strain KM845 was inoculated to 20 ml minimal mediumcontaining 0.2% melibiose and incubated overnight at 37°C. The total mRNA was isolated using theQiagen RNeasy minikit (Hilden, Germany), as instructed by the manufacturer. Approximately 30 �g oftotal RNA was precipitated using 3 M sodium acetate (pH 6.3) and ethanol. The precipitated RNA wasdissolved in 5 �l of RNase-free ddH2O and 0.5 �l of RNasin RNase inhibitor (40 U/�l; Promega, Mannheim,Germany). After incubation at 65°C for 3 min, 0.5 �l of the 5=,6-FAM-labeled s11996 or s12048 oligonu-cleotide (10 pmol/�l) and 2 �l of 5� avian myeloblastosis virus reverse transcriptase (AMV-RT) reactionbuffer (New England BioLabs GmbH, Frankfurt am Main, Germany) were added. The mixture wasincubated for 20 min at 51°C, followed by incubation for 5 min at room temperature. Afterwards, 1 �ldinucleoside triphosphate (dNTP) (10 mM) and 1 �l AMV-RT (20 U/�l; New England BioLabs GmbH) wereadded to start the reverse transcription, and the reaction mixture was incubated for 1 h at 42°C. Finally,the generated cDNA was purified using DNA Clean & Concentrator-5 kit (Zymo Research GmbH, Freiburg,Germany) and eluted in 6 �l ddH2O. After the addition of 34 �l Hi-Di formamide (catalog no. 4401457;Applied Biosystems by Thermo Fisher Scientific, USA) and of 0.5 �l GeneScan 600 LIZ size standard v2.0from the GeneScan installation kit DS-33 with 600 LIZ size standard v2.0 (catalog no. 4376911; AppliedBiosystems by Thermo Fisher Scientific), the samples were run on a SeqStudio genetic analyzer (AppliedBiosystems by Thermo Fisher Scientific). The GeneMapper software 5 (Applied Biosystems by ThermoFisher Scientific) was used to compare the primer extension peak with the DNA sequencing results ofpKAM384 (using the same oligonucleotides) to find the TSS.

Electrophoretic mobility shift assay. 5=-end Cy5-labeled oligonucleotides were used to createCy5-labeled DNA fragments by PCR or hybridization. The Cy5-PmsmR DNA fragment was amplified frompKAM384 using the s11321 and s5960 oligonucleotides. The footprinting regions at PmsmR, i.e., FP1 andFP2, were also labeled by DNA hybridization of the s12280 and s12281 oligonucleotides for FP1 and thes12282 and s12283 oligonucleotides for FP2. As the negative control, the DNA fragment containing theGlcR binding site of PglcR was labeled after hybridization of s12284 and s12285. All electrophoreticmobility shift assays were carried out in a total volume of 20 �l containing 2 �l of Cy5-labeled DNAfragment (50 fmol/�l) and 4 �l of 5� shift buffer (50 mM Tris-HCl [pH 7.5], 250 mM KCl, 10 mMdithiothreitol [DTT], 25% [vol/vol] glycerol, 250 �g/ml bovine serum albumin [BSA], 25 �g/ml herringsperm DNA). Fourteen microliters of purified MsmR–Strep-tag (0.015 mg/ml) was added to the reactionmixture, and the reaction mixture was incubated on ice for at least 15 min. Finally, 10 �l of the reactionmixture was loaded onto a 6% (wt/vol) native polyacrylamide gel to separate the free DNA andDNA-protein complexes. The migration of the bands of free DNA and the DNA-protein complexes wasvisualized using the Storm 860 PhosphorImager (Molecular Dynamics).

DNase I footprinting. Fluorescence labeling of the noncoding strand of PmsmR DNA was carried outby PCR using T7 and 5=,6-FAM-labeled s12050 oligonucleotide from pKAM0138. DNase I digestion wasperformed by mixing of 20 �l of the 6-FAM-labeled PmsmR DNA fragment (50 fmol/�l) with 48 �l of 5�shift buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM KCl, 10 mM DTT, 25% (vol/vol) glycerol,250 �g/ml BSA, and 25 �g/ml herring sperm DNA to prepare the master mix. Seventeen microliters of themaster mix was then mixed with 43 �l of purified MsmR–Strep-tag (0.015 mg/ml). As the negativecontrol, purified MsmR was similarly added to the master mix after denaturation by incubation for 10 minat 99°C. After 15 min of incubation on ice, the DNA-protein mixture was preheated for 1 min at 25°C.Next, 10 �l of the DNase I master mix containing 7 �l of 10� DNase I buffer, 2.75 �l ddH2O, and 0.25 �lDNase I (2,000 U/ml; New England BioLabs GmbH) was added. The reaction was stopped after 1 min ofincubation at 25°C with the addition of stop solution (50 mM EDTA [pH 8.0], 15 �g/ml calf thymus DNA).Phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol/vol]) extraction, followed by washing of the DNA

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with ethanol, was carried out to remove the protein. The DNA was then dissolved in 6 �l ddH2O and 34 �lHi-Di formamide (catalog no. 4401457; Applied Biosystems by Thermo Fisher Scientific, USA). After theaddition of 0.5 �l GeneScan 600 LIZ size standard v2.0 from the GeneScan installation kit DS-33 with 600 LIZsize standard v2.0 (catalog no. 4376911; Applied Biosystems by Thermo Fisher Scientific), the samples wererun on a SeqStudio genetic analyzer (Applied Biosystems by Thermo Fisher Scientific), and the results wereanalyzed using the GeneMapper software 5 (Applied Biosystems by Thermo Fisher Scientific).

Thermal shift assay. To find the possible effectors of MsmR and MsmE, a thermal shift assay (alsoknown as differential scanning fluorimetry) was performed with a Mastercycler ep realplex (Eppendorf,Hamburg, Germany) utilizing the melting capability. Each reaction was carried out in a total volume of50 �l by mixing purified MsmR–Strep-tag (12 �g), purified His6-MsmE (20 �g) with or without theeffectors, galactose, melibiose, raffinose, and stachyose at a final concentration of 1 mM, with SYPROOrange (5 �l of the 50� stock solution in DMSO; Sigma, Munich, Germany). The fluorescence intensityprofile (or melting curve) of the SYPRO Orange-protein complex was measured at 520 nm with intervalsof 0.2°C from 20°C to 90°C. The thermal stability (melting temperature) was recorded from the formulaΔfluorescence/Δtemperature.

Measurement of �-galactosidase activity. The �-galactosidase activity was determined by mea-suring the rate of para-nitrophenyl-�-galactopyranoside (pNPG) hydrolysis, as described previously (56),in 0.1 M Tris-HCl (pH 8.0) at 37°C. The effects of Mn2� (3 mM), NAD� (1 to 10 mM), and the reducingagents, such as mercaptoethanol (100 mM) and Tris(2-carboxylethyl)phosphine (TCEP) (1 mM), werestudied in a 50-�l reaction mixture. The standard melibiase activity test was performed in the presenceof 3 mM MnCl2, 10 mM NAD�, and 1 mM TCEP. One unit of the enzyme activity was defined as theamount of enzyme required to hydrolyze 1 �M pNPG per minute. The protein concentration wasdetermined by the method of Bradford (57) using bovine serum albumin (BSA) as a standard.

The rate of melibiose hydrolysis was determined by assessing the amount of released glucose, whichwas determined by a glucose-hexokinase test (DiaSys GmbH, Holzheim, Germany) and quantifiedaccording to a glucose standard curve. The rates of raffinose and stachyose hydrolysis were measured byassessing the release of galactose after separation of the reaction mixture by high-performance liquidchromatography (HPLC), as described before (56). The hydrolysis of the mentioned sugars was measuredfrom a final concentration of 100 mM under standard assay conditions. Kinetic parameters (apparentMichaelis constant [Km] were determined for the substrates (melibiose and raffinose) and were obtainedby curve fitting analysis using the KaleidaGraph software (Synergy Software, USA).

Measurement of �-galactosidase activity. The �-galactosidase activity was measured usingp-nitrophenyl-�-D-galactopyranoside (pNP-�-Gal), according to the Miller assay (58).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/JB

.00109-19.SUPPLEMENTAL FILE 1, PDF file, 0.3 MB.

ACKNOWLEDGMENTSWe thank Gisela Wajant, Gisela Kwiatkowski, Silke Weber, and Annette Schneck for

their technical assistance during this study.

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