Computational Analysis of Cysteine and Methionine Metabolism andIts Regulation in Dairy Starter and Related Bacteria

Mengjin Liu,a,b* Celine Prakash,b* Arjen Nauta,a Roland J. Siezen,b,c,d,e,f and Christof Franckeb,d,e,f

FrieslandCampina Research, Deventer, The Netherlandsa; Center for Molecular and Biomolecular Informatics (260), NCMLS, Radboud University Nijmegen Medical Center,Nijmegen, The Netherlandsb; NIZO Food Research, Ede, The Netherlandsc; Kluyver Center for Genomics of Industrial Fermentation, Delft, The Netherlandsd; NetherlandsBioinformatics Center, Nijmegen, The Netherlandse; and TI Food and Nutrition, Wageningen, The Netherlandsf

Sulfuric volatile compounds derived from cysteine and methionine provide many dairy products with a characteristic odor andtaste. To better understand and control the environmental dependencies of sulfuric volatile compound formation by the dairystarter bacteria, we have used the available genome sequence and experimental information to systematically evaluate the pres-ence of the key enzymes and to reconstruct the general modes of transcription regulation for the corresponding genes. Thegenomic organization of the key genes is suggestive of a subdivision of the reaction network into five modules, where we ob-served distinct differences in the modular composition between the families Lactobacillaceae, Enterococcaceae, and Leuconosto-caceae, on the one hand, and the family Streptococcaceae, on the other. These differences are mirrored by the way in which tran-scription regulation of the genes is structured in these families. In the Lactobacillaceae, Enterococcaceae, and Leuconostocaceae,the main shared mode of transcription regulation is methionine (Met) T-box-mediated regulation. In addition, the gene metK,encoding S-adenosylmethionine (SAM) synthetase, is controlled via the SMK box (SAM). The SMK box is also found upstream ofmetK in species of the family Streptococcaceae. However, the transcription control of the other modules is mediated via threedifferent LysR-family regulators, MetR/MtaR (methionine), CmbR (O-acetyl[homo]serine), and HomR (O-acetylhomoserine).Redefinition of the associated DNA-binding motifs helped to identify/disentangle the related regulons, which appeared to per-fectly match the proposed subdivision of the reaction network.

Many of the characteristic flavors in fermented dairy productssuch as cheese and yoghurt are the result of metabolic reac-

tions involving sulfur-containing amino acids. The microorgan-isms applied in these products degrade cysteine and methionine,resulting in the production of flavor components such as meth-anethiol, dimethyl sulfide (DMS), dimethyl disulfide (DMDS),and dimethyl trisulfide (DMTS). Insight into the regulatory sig-nals and pathways that control the corresponding metabolicfluxes involved in the formation of these flavor compounds andtheir precursors is essential to rationally control and steer the fla-vor profiles of said dairy products.

The microorganisms used to produce fermented dairy prod-ucts belong to the taxonomic order Lactobacillales, which includesthe families Enterococcaceae, Lactobacillaceae, Leuconostocaceae,and Streptococcaceae. Many of the respective species are character-ized by the fact that they produce lactic acid and are thereforeknown as the lactic acid bacteria (LAB). The transcription of genesencoding the proteins that are involved in cysteine and methio-nine metabolism in lactic acid bacteria and other Lactobacillales iscontrolled by both regulator-binding and RNA structuralswitches. In various Streptococcaceae, the LysR-family transcrip-tion regulators MtaR and CmbR have been shown to be involvedin activation as well as repression of genes such as cysD, cysK,metA, metC, metE, and metF (e.g., for Lactococcus lactis [25, 70]and Streptococcus mutans [36, 68]). The transcription regulatorHomR was reported to control the expression of metB in S. mutansand Streptococcus thermophilus (69). In addition, three types ofRNA structural switches for the regulation of cysteine and methi-onine metabolism have been reported for low-GC-content Gram-positive bacteria: the T box, the S box, and the SMK box (14, 22, 59,77, 80). These sequence elements at the 5= untranslated region ofan mRNA molecule can change conformation, depending on the

binding of an effector molecule. The conformational change canterminate transcription (when forming a terminator structure) orallow readthrough (when forming an antiterminator structure)(75, 78). In the case of the T box, a terminator structure is formedshortly after transcription initiation, unless an uncharged tRNArelated to a specific amino acid binds to the specifier codon pres-ent in the T-box element, whereupon the antiterminator structureis formed (see reference 29). In the case of the S box and the SMK

box, the terminator structure is formed in the presence of S-ad-enosylmethionine (SAM), whereas in the absence of this mole-cule, transcription continues (22, 27, 47).

Several studies have described the regulation of sulfur-contain-ing amino acid metabolism for specific LAB and other closelyrelated Gram-positive bacteria. For instance, Hullo et al. (30) re-ported on the regulatory mechanisms related to cysteine and me-thionine conversions in Bacillus subtilis. Sperandio et al. describedthese relations for Lactococcus lactis (70) and Streptococcus mutans(68, 69). Rodionov et al. (59) and Kovaleva and Gelfand (36)performed a comprehensive comparative in silico study for the

Received 2 January 2012 Accepted 4 April 2012

Published ahead of print 20 April 2012

Address correspondence to Christof Francke, c.francke@cmbi.ru.nl.

* Present address: Mengjin Liu, Hero-Huishan Nutrition Co., Ltd., Shangri-La,Shenbei, Shenyang, China; Celine Prakash, Laboratory of Gene Regulation andInflammation, Singapore Immunology Network (SIgN), Agency for Science,Technology and Research (A*STAR), Biopolis, Singapore.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.06816-11

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transcriptional regulators CmbR and MtaR within Gram-positivebacteria. However, the availability of additional experimental andsequence data now allows an overview of the transcriptional con-trol of the key enzymes involved in cysteine and methionine me-tabolism at a higher resolution. We therefore decided to extendthe latter studies and to focus our efforts on the LAB and otherLactobacillales.

In a previous study, we improved the annotation of key en-zymes involved in the metabolism of cysteine and methionine inLAB using genome-wide comparative analyses (41). Here, we ex-tend the list of enzymes on the basis of the pathway informationpresent in the KEGG database (32). Redefinition of the bindingmotifs for CmbR, MetR/MtaR, and HomR in lactococci andstreptococci allowed the identification of transcription factor-spe-cific binding sites for these regulators. Also, Met-specific T boxesand SMK boxes were identified in recently sequenced and pub-lished genomes of, e.g., Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus reuteri, and Lactobacillus casei. The absence of Sboxes (SAM-I) in the Lactobacillales observed hitherto was con-firmed. Potential structure-forming elements associated with thecysK gene and the hom-thrBC operon in various LAB were re-vealed, as presented below.

MATERIALS AND METHODSGenomic information, tools, and data. Genomic information was re-trieved from the ERGO resource (as of December 2009) (53) and from theNCBI microbial genome database (as of September 2011) (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). BLAST searches were performedas described elsewhere (1). Multiple-sequence alignments and neighbor-joining (NJ) trees (corrected for multiple substitutions) were generatedusing the ClustalX program (37). BioEdit software was used to manipulatethe alignments and to toggle between translated protein and nucleotidesequences (version 7.0.9; http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Hidden Markov models (HMMs) were made, and genome-wideHMM searches were performed using the HMMER package (http://hmmer.janelia.org/) (13). Genome context was visualized and upstreamsequence data were collected using Microbial Genome Viewer 2 (version 1[34] and version 2 [http://mgv2.cmbi.ru.nl/genome/index.html]; L.Overmars, unpublished data). The original data supporting the analysespresented in this paper can be found at www.cmbi.ru.nl/bamics/supplementary/Liuetal_2012_CysMetregulation.

Collection of genes related to cysteine and methionine metabolism.The species and strains that were analyzed included all members of theorder Lactobacillales with a completed genome published before June2011 and present within the NCBI database. The KEGG map cysteine andmethionine metabolism (map 00270) was used to define a core set ofenzyme activities. The set extends the set of enzymes that we previouslydefined (41). The protein sequences of experimentally verified membersof the set (see File S1 in the supplemental material) were used to searchorthologs/functional equivalents in other species using BLAST. An or-thologous relationship and/or functional equivalency was defined on thebasis of our earlier analyses (41), BLAST E values, and, in some cases,multiple-sequence alignments, followed by clustering on the basis ofneighbor joining (as described in reference 73). The complete list of en-zymes, their function annotation, and the relevant experimental literatureare given below. The annotation data were taken from the NCBI (COGnumbers below) (57), KEGG (K numbers below) (32), and PFAM (PFnumbers below) (18) reference databases. The enzymes have beengrouped into five clusters on the basis of the composition of the relatedoperons and shared enzyme nomenclature (EC) numbers.

(i) Enzyme group 1. Enzyme group 1 consisted of homoserine dehy-drogenase (hom; EC 1.1.1.3; COG0460E, K00003, PF03447, and PF00742[7, 44, 54]), homoserine kinase (thrB; EC 2.7.1.39; COG0083E, K00872,

PF08544, and PF00288 [44]), aspartate kinase III (thrA [Bsubtilis_yclM];EC 2.7.2.4; COG0527E, K00928, PF01842, and PF00696 [7, 35]), andthreonine synthase (thrC; EC 4.2.3.1; COG0498E, K01733, and PF00291[45, 63, 66, 72]).

(ii) Enzyme group 2. Enzyme group 2 consisted of serine acetyltrans-ferase (cysE; EC 2.3.1.30; COG1045E, K00640, and PF06426 [23, 30, 70]),homoserine O-acetyltransferase (metA; EC 2.3.1.31; COG1897E, K00651,and PF04204 [83]), cysteine synthase A and cysteine synthase-like protein(Bsubtilis_cysK and Bsubtilis _ytkP; EC 2.5.1.47; COG0031E, K01738, andPF00291 [25, 30, 76]), cystathionine gamma-synthase and O-acetylho-moserine (thiol)-lyase (Bsubtilis_yjcL; EC 2.5.1.48; COG0626E, K01739,and PF01053 [3, 33]), O-acetyl-L-homoserine sulfhydrolase and O-acetyl-L-serine sulfhydrolase (cysD; EC 2.5.1.49; COG2873E, K01740, andPF01053); cystathionine beta-synthase for the reverse transsulfurase path-way (Bsubtilis_yrhA; EC 4.2.1.22; COG0031E, K01738, and PF00291[30]), cystathionine beta/gamma-lyase and homocysteine gamma-lyase(Bsubtilis_yrhB Ecoli_metB; EC 2.5.1.48 and EC 4.4.1.8; COG0626E,K01760, and PF01053 [12, 17, 30, 31]), cystathionine beta/gamma-lyase(Bsubtilis_yjcJ; EC 4.4.1.8 and EC 4.4.1.1; COG0626E, K01760, andPF01053 [3]), and pyridoxal-phosphate (PLP)-dependent C-S lyase(Bsubtilis_patB Llactis_ytjE Ecoli_malY; EC 4.4.1.8 and EC 4.4.1.1;COG1168E, K14155, and PF00155 [2, 31, 46]).

(iii) Enzyme group 3. Enzyme group 3 consisted of 5,10-methylenet-edrahdrofolate reductase (metF; EC 1.5.1.20; ?, K00297, and PF02219[64]), bifunctional homocysteine S-methyltransferase 5,10-methylenetet-rahydrofolate reductase protein (Bsubtilis_yitJ; EC 2.1.1.10 and EC1.5.1.20; COG0646E [cobalamin dependent], K00547, PF02219, andPF02574 [42]), homocysteine S-methyltransferase (mmuM; EC 2.1.1.10;COG2040E, K00547, and PF02574 [74]), MmuM-associated amino acidpermease (mmuP; COG0833E, K03293, and PF00324), methyltransferase(Bsubtilis_yxjG and Bsubtilis_yxjH Llactis_yhcE; EC 2.1.1.14[?];COG0620E [cobalamin independent], K00548, and PF01717 [9, 38]),5-methyltetrahydropteroyltriglutamate– homocysteine S-methyltrans-ferase (metE; EC 2.1.1.14; COG0620E [cobalamin independent], K00549,PF08267, and PF01717 [21, 26]), and S-ribosylhomocysteinase (luxSLlactis_ycgE; EC 4.4.1.21; COG1854T, K07173, and PF02664 [38, 58).

(iv) Enzyme group 4. Enzyme group 4 consisted of C-5 cytosine-specific DNA methylase and SP-beta prophage DNA (cytosine-5-)-methyltransferase (Bsubtilis_ydiO Bsubtilis _ydiP Bsubtilis_mtbP; EC2.1.1.37; COG0270L, K00558, and PF00145 [51, 81]), and 5=-methyl-thioadenosine nucleosidase and S-adenosylhomocysteine nucleosid-ase (mtn Streptococci_pfs; EC 3.2.2.16 and EC 3.2.2.9; COG0775F,K01243, and PF1048 [10, 55]).

(v) Enzyme group 5. Enzyme group 5 consisted of S-adenosylmethio-nine synthetase (metK; EC 2.5.1.6; COG0192H, K00789, PF02773, andPF00438 [22, 48]).

Identification of putative regulatory elements and their regulons.cis-Regulatory elements were defined according to the specific footprint-ing method set out by Francke et al. (20). The method relies on the defi-nition of groups of orthologous functional equivalents (GOOFEs) on thebasis of orthology and conserved genomic context. The comparative lin-ear genome maps generated by the Microbial Genome Viewer were usedto visualize and inspect the context. For every GOOFE, the upstreamregions (normally �200 nucleotides) were collected, and conserved se-quence elements were searched for by eye from a multiple-sequence align-ment and by using the MEME tool (4). The highest-scoring motifs result-ing from MEME and the conserved elements in the multiple-sequencealignment were compared, and potential regulatory regions were identi-fied. In case the conserved elements resembled transcription factor bind-ing motifs reported in literature, experimental data on regulators of thesame protein family were searched for directly via PubMed (61) or in thereference databases Regulon DB (24) and DBTBS (65). Because membersof the same regulator-protein family will, in general, adopt the same fold,the DNA-binding motif should be similar (i.e., have similar compositionsand the same size and spacing). Therefore, established binding motifs of

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regulator-protein family members were taken into account to define theactual binding motif. In addition, we defined the motifs such that theyobey general constraints imposed by the molecular nature of the bindingprocess and the helical nature of the DNA molecule. Since most regulatorproteins bind to the DNA by virtue of a helix-turn-helix (HTH) domainand as a dimer, a binding site will, in general, be made up of two monomerbinding sites and will have to be either palindromic or represent a directrepeat. Moreover, since the DNA is helical, the actual monomer bindingsite in general has to be shorter than 7 nucleotides and the two sites thatmake up the dimer binding site have to be interspaced by a fixed numberof nucleotides.

The defined motifs (given in File S2 in the supplemental material)were converted to a position frequency matrix, which was used directly toscore potential transcription factor binding sites and other regulatory el-ements of fixed composition and size. In this way, the score of any DNAsequence related directly to its similarity to the input motif. In general, wescore the “relative similarity,” which we define as the percentage of themaximally attainable score given the input motif. We have validated andused this approach with success to identify potential binding sites of CcpAand Spo0A in low-GC-content Gram-positive organisms and the sigma 54promoter in all organisms (19). A cutoff score of �83% relative similarityand a positioning of a maximum of about 200 nucleotides upstream (withsome exceptions) of the translation start were used to select potentialbinding sites for the various regulators. The uniform cutoff score waschosen such that the number of false-positive assignments should be lim-ited, i.e., such that experimentally validated sites were included and thatthe number of correctly positioned sites was high (position in terms ofdistance and orientation with respect to translation start of the genedownstream). The identified regulatory elements were related to all genespresent in the downstream operon, where an operon was defined as thosegenes on the same strand that are separated by an intergenic region of lessthan 250 nucleotides without a termination signal. The locations of (rho-independent) transcription terminators were determined with the Tran-sterm system (15). The analyzed results of the motif searches are given inFile S3 in the supplemental material.

Identification of riboswitches and other structural elements. Hid-den Markov models were constructed for the T-box motif and for theS-box motif (SAM-I) on the basis of the available literature (59, 77, 80).Both HMMs were used to scan the selected genomes (cutoff E value, 1[38]), and the locations of putative boxes were identified. The amino acidspecificity of the detected T boxes was established on the basis of thespecifier codon, as described by Wels et al. (80) and exemplified in Fig. S1in the supplemental material. Two characteristic SMK-box sequences weredefined on the basis of information presented elsewhere (22), as given inFig. 2A, and these were used to scan the selected genomes using the similarmotif search procedure (results are presented in File S3 in the supplemen-tal material). Only in case both motifs were found directly upstream of agene and they were complementary did we consider the site to be a puta-tive SMK box.

RESULTSComparative analysis of the enzymes involved in central cys-teine and methionine metabolism. The set of genes related tocentral cysteine and methionine metabolism was identified on thebasis of KEGG map 00270 (32) and earlier work by others (36, 59)and us (41). Orthologs and homologs of these genes were collectedfrom the genomes of all sequenced species/strains of the LAB andother Lactobacillales, as described in Materials and Methods. Therelated reaction network is given in Fig. 1, and the results of thesearch and analysis procedure are presented in Tables 1 and 2 andgiven in File S1 in the supplemental material. Remarkably, the setof genes (and corresponding proteins) can be divided into fiveseparate groups on the basis of genomic organization, the identityof the EC numbers, and the position in the reaction network

(Fig. 1). Small differences in operon organization between thefamilies Lactobacillaceae, Enterococcaceae, and Leuconostocaceae(Table 1) and the family Streptococcaceae (Table 2) were observed,implying a difference in pathway modularization between thefamilies.

The presence-absence list of genes is in agreement with theresults obtained in earlier analyses (36, 41, 59). Nevertheless, weobserved a few differences. For instance, we found that O-acetyl-homoserine sulfhydrolase (gene cysD) is absent in various Strep-tococcus pneumoniae strains, including strain TIGR4, and that vi-tamin B12-dependent methionine synthase (gene yxjH) is presentin Streptococcus gordonii. We also found that the enzyme cystathi-onine beta/gamma-lyase of Streptococcus pyogenes is more similarto that of B. subtilis (gene yjcJ) than to that of Lactococcus lactis(gene ytjE). A multiple-sequence alignment of MetA showed thatthe protein in all Lactobacillales carries the specific Glu residue atposition 111 that renders the MetA protein of Bacillus cereus anacetyltransferase instead of a succinyltransferase (as shown else-where [83]) (see File S4 in the supplemental material).

Because we could include a large number of species and strains(i.e., 96 genomes), the general trends in the presence or absence ofcertain enzymes became more apparent than in the earlier com-parative analyses (36, 41, 59). Owing to the larger number of ge-nomes, cases of putative horizontal gene transfer could be recog-nized. As an example of the latter, orthologs of cystathioninesynthase and cystathionine beta/gamma-lyase, represented bygenes yrhA and yrhB, respectively, in B. subtilis, were not foundamong the streptococci, except for the sequenced S. thermophilusstrains. We observed that the upstream region associated with theyrhA gene in S. thermophilus was identical (besides a few singlenucleotide polymorphisms) to that of yrhA in Lactobacillus helve-ticus and L. delbrueckii subsp. bulgaricus and that of yrhA on plas-mid pLC1 of Lactobacillus rhamnosus Lc 705w (see Fig. S2 in thesupplemental material), suggesting a recent plasmid-mediatedtransfer of the genes and upstream sequences to all the differentstrains individually or an extreme stability of this particular se-quence.

We found the enzymes 5=-methylthioadenosine nucleosidase/S-adenosylhomocysteine nucleosidase (the corresponding gene isdesignated mtn in B. subtilis and Escherichia coli or pfs in Strepto-coccaceae) and S-adenosylmethionine synthetase (gene metK) tobe present in all analyzed genomes and thus potentially essential.Indeed, the metK gene product was shown to be essential to thegrowth of E. coli K-12 (79), and the mtn (i.e., pfs) gene product wasrecently put forward as a critical enzyme for bacterial metabolism(55). The enzyme S-ribosylhomocysteinase (gene luxS) was foundto be absent only in Lactobacillus sakei. Besides, serine acetyltrans-ferase (gene cysE) and cysteine synthase A (gene cysK) are presentin all Streptococcaceae and N5-methyltetrahydrofolate methyl-transferase (gene yxjH or metE2) is present in all Lactobacillaceaeexcept for L. sakei and L. helveticus. The remaining makeup ofcysteine and methionine metabolism appeared to be more vari-able between species. L. sakei (Lactobacillaceae) and Streptococcusequi (Streptococcaceae) have the least extensive enzyme repertoire,with 2 and 7 enzymes, respectively.

Identification of riboswitches. Recently, three comprehensivestudies described the occurrence and evolution of T boxes amongprokaryotes (29, 77, 80). We have used the T-box HMMs de-scribed previously (80) to search recently acquired genome se-quences of, e.g., L. delbrueckii subsp. bulgaricus, Lactobacillus bre-

Liu et al.

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vis, and L. reuteri. We identified many new T boxes associated withgenes/operons involved in cysteine and methionine metabolismin these genomes. As a control, we also scanned the other genomesincluded in previous studies (77, 80). The conservation of theATG specifier codon in the multiple-sequence alignment of thenewly recovered T boxes (see Fig. S1 in the supplemental material)implies that they all respond to the absence of methionine. Wefound that genes/operons metB (BS_yjcL), metE-metH (BS_metE-yitJ), and hom1-metA-cysD from Lactobacillus plantarum WCFS1and genes/operons LEUM_1806-LEUM_1803 (BS_metA-yjcL-yjcJ-yxjG), LEUM_1802 (luxS), and LEUM_1795-LEUM_1794(metE-metF) from Leuconostoc mesenteroides are regulated by aMet T box, in agreement with previously published findings (77,80). cysE, encoding a serine acetyltransferase, was also found to bepreceded by a Met T box in B. subtilis, as reported previously (56).The association with a Met T box appeared to be almost fullyconserved within the Lactobacillus species for the genes that en-code the proteins responsible for the synthesis of methionine fromhomocysteine, i.e., metEF, yxjH-yxjG, and luxS, and for the genesencoding a methionine ABC import system, where metQ encodesthe substrate binding protein.

S-Adenosylmethionine-sensitive SAM-I riboswitches are oftenfound upstream of genes involved in sulfur metabolism and trans-

port in bacilli and clostridia (3, 59), but they have not been re-ported in LAB. In accordance, we did not detect SAM-I ribo-switches upstream of genes involved in cysteine and methioninemetabolism in the genomes that were analyzed. However, anotherSAM-responsive element was reported (22) upstream of the metKgene in lactobacilli and streptococci and was named the SMK box.We used two conserved structures forming stretches of about 6nucleotides from the reported box (given in Fig. 2A) to search forpotential SMK boxes. We found the two motifs in the correct orderupstream of the metK gene in all analyzed genomes but no addi-tional hits (see File S3 in the supplemental material). For all Lac-tobacillaceae and many Streptococcaceae, the distance was about 50nucleotides, whereas in other streptococci, like Streptococcus dys-galactiae, Streptococcus gallolyticus, Streptococcus mitis, S. mutans,Streptococcus pasteurianus, and S. pyogenes, this distance wasmuch larger, at about 350 nucleotides. This huge variability inspacing that was also observed previously (22) raises interestingquestions related to the way in which sequences of such differentlengths can form similar three-dimensional structures to accom-modate SAM binding.

LysR-family regulators related to cysteine and methioninemetabolism. MetR from S. mutans, MtaR from Streptococcus aga-lactiae, CmbR from L. lactis, and HomR from S. mutans have been

FIG 1 Generalized cysteine and methionine metabolism in the Lactobacillales. For most of the studied species, only part of the depicted reactions can take place,as can be concluded from Tables 1 and 2. The map is divided into differently colored boxes on the basis of the operon composition and EC numbers in line withthe color scheme in Tables 1 and 2. Abbreviations: DMS, dimethyl sulfide; DMDS, dimethyl disulfide; DMTS, dimethyl trisulfide; CoA, coenzyme A.

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identified as being important regulators of the genes related tocysteine and methionine metabolism in streptococci (25, 68, 69).They belong to the LysR family of transcription regulators. Aneighbor-joining tree of LysR-family proteins was constructed onthe basis of a comprehensive BLAST search for homologs (cutoff Evalue, 1e5). There was a clear division of protein sequences inthree subclusters, corresponding to MetR/MtaR, CmbR, andHomR, within the resulting NJ tree, as was also observed previ-ously (36). The tree (Fig. 3) clearly shows that MetR of S. mutansand MtaR of S. agalactiae are orthologous and that CmbR andHomR are their closest relatives. The phylogenetic analysis furtherimplies that CysR from S. mutans is orthologous to CmbR from L.lactis, although CysR was proposed to be a new regulator sepa-rated from the CmbR cluster (69). Our assessment of orthologybetween the two transcription factors is supported by the similar-ity of their cognate DNA-binding motif (see below). For the otherspecies, only Lactobacillus delbrueckii subsp. bulgaricus, L. planta-rum, and Enterococcus faecalis possessed a homolog. The first twoare clearly orthologous to MetR/MtaR, whereas the enterococcalregulator seems to be the most related to CmbR.

The LysR-family proteins have a domain architecture that iscommon for transcriptional regulators in prokaryotes. The mem-ber proteins consist of a signal molecule binding domain, followedby an HTH DNA-binding domain. The family contains a numberof well-studied proteins, including AlsR, CcpC, CitR, GltC, YwfK,and CmbR (43). We have compared the results of DNA-bindingstudies for LysR-family members for a number of Firmicutes (seethe data in Table S1 in the supplemental material). A straightfor-ward alignment and comparison of the reported binding sites re-veal a common motif structure of the cis elements, namely, ATNNNN---NNNNAT. The motif displays a clear dyad symmetry anda conserved spacing of 3 nucleotides. This architecture agrees wellwith the observation made by Schell (62) that LysR-family mem-bers generally recognize a box with a conserved sequence,T-N11-A, located 50 to 80 bp upstream of the transcriptional startsite. In fact, LysR-family members in general assemble as tetram-ers (dimer of dimers), and thus, their binding locus is most oftencomposed of two adjacent dimer binding sites where the spacingbetween the two sites may vary (43). The motifs for LysR bindingthat have been defined more recently in some cases deviate from

TABLE 1 Presence and regulation of genes encoding central cysteine and methionine metabolism in Enterococcus, Lactobacillus, and Leuconostocgenomesa

a The genes present in the same operon are indicated by a similar coloring of the cells and/or by †. In case more than one closely related sequence was present, the total number isgiven. The number is in parentheses in case not all are present in the same operon and/or preceded by a similar putative binding site. In case the gene might be present but was notcalled, it is indicated by “0?.” The genes have been grouped into five clusters on the basis of the composition of the operons and in case of shared EC numbers. Putativeregulator-binding sites upstream of the indicated gene are given in capital letters in superscript: C, CmbR motif; M, MetR motif; N, CodY motif; S, SMK box; T, T box.Low-scoring putative binding sites are indicated by lowercase letters and a question mark. In several cases we found putative binding sites for two regulators. Species nameabbreviations: Lb., Lactobacillus; L., Leuconostoc; P., Pediococcus. The related data and NCBI gi codes can be found in File S1 in the supplemental material, and the analysis ofupstream regions can be found in File S3 in the supplemental material. The original data are provided at www.cmbi.ru.nl/bamics/supplementary/Liuetal_2012_CysMetregulation.*, for most enzymes, the related gene names are provided. In most cases these represent names common in all lactobacilli. In cases with little uniformity, the names are derived fromthe orthologs found in the Bacillus subtilis genome (also see Materials and Methods). Abbreviations in enzyme names: methylenethf, methylenetedrahdrofolate; synth. rev. pathway,synthase for reverse pathway. The E. faecalis V583 genome has a copy of the cmbR gene (indicated by #), and the Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus planta-rum genomes have a copy of the metR gene (indicated by ##). In Lactobacillus plantarum, the second copy of hom is associated with metA and cysD in an operon.

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this family consensus, and we have therefore redefined them, asdescribed below, taking the mechanistic/molecular characteristicsof the binding into account. The motifs were then used to searchthe lactobacillus genomes for putative binding sites, and the re-sults are given in Tables 1, 2, and 3.

MetR/MtaR. A 17-bp palindromic conserved sequence, TATAGTTTNAAACTATA, was identified upstream of metY, metA,metQ, metI, and the metEF operon in streptococci and upstreamof the metEF operon in L. lactis (36, 59). However, at that time, theregulatory protein, which should bind to the so-called MET box,had not yet been identified. Rodionov et al. (59) initially proposedthat the transcriptional regulator MtaR, known to be involved inmethionine uptake in S. agalactiae, was a good candidate. Indeed,it was shown soon after that MetR is the regulator protein thatbinds to the MET box in S. mutans (68). In fact, MetR and MtaRcan be inferred to fulfill the same role, as they are orthologs (Fig.3). The MET-box motif was identified in the upstream regions ofatmB, metE, cysD, metA, and smu.1487 in S. mutans, and bindingof MetR to these MET boxes was confirmed using gel mobilityshift assays and base substitutions in the MET boxes (68).

We have analyzed the upstream regions of the genes ortholo-

gous to metE and metH (mmuM) in the selected genomes for thepresence of a sequence similar to that of the MET box. Like others,we observed two MetR/MtaR-binding sites in the upstream regionof the metH (mmuM) and metE genes in the L. lactis and S. ther-mophilus genomes. The first site is often located about 60 to 70 bpfrom the transcriptional start and displays an activating role (68).The second putative binding site, which is closer to the transcrip-tional start, is far less conserved. The observed organization of thebinding sites appears to be general for LysR-family members andrelates to a mechanism that requires tetramer formation of thetranscription factor (43). It has clear implications for the dynam-ics of LysR-family-mediated transcriptional regulation, as de-scribed for the enterobacterial nitrogen assimilation control pro-tein Nac (60).

The dissected sites were used to redefine the binding motif forMetR/MtaR in both lactococcal and streptococcal strains (Fig. 2B;see File S2 in the supplemental material). We used the experimen-tally identified binding sites of all LysR-family regulators to guidethe definition (see above and Materials and Methods). The puta-tive binding sites of MetR/MtaR showed an overall palindromicstructure, ATA-N9-TAT, which is typical for the LysR family. The

TABLE 2 Presence and regulation of the genes encoding central cysteine and methionine metabolism in streptococcal genomesa

a The genes present in the same operon are indicated by a similar coloring of the cells. In case more than one closely related sequence was present, the total number is given. Thenumber is in parentheses in case not all are present in the same operon and/or preceded by a similar putative binding site. In case the gene might be present but was not called, it isindicated by “0?.” The genes have been grouped into five clusters on the basis of the composition of the operons and in case of shared EC numbers. Putative regulator-binding sitesupstream of the indicated gene are given in capital letters in superscript: C, CmbR motif; M, MetR motif; N, CodY motif; S, SMK box; T, T box. Low-scoring putative binding sitesare indicated by lowercase letters and a question mark. In several cases, we found putative binding sites for two regulators. Species name abbreviation: Lc., Lactococcus. The relateddata and NCBI gi codes can be found in File S1 in the supplemental material, and the analysis of upstream regions can be found in File S3 in the supplemental material. The originaldata are provided at www.cmbi.ru.nl/bamics/supplementary/Liuetal_2012_CysMetregulation. *, for most enzymes the related gene names are provided. In most cases these repre-sent names common in all lactobacilli. In cases with little uniformity, the names are derived from the orthologs found in the Bacillus subtilis genome (also see Materials and Meth-ods). Abbreviations in enzyme names: methylenethf, methylenetedrahdrofolate; synth. rev. pathway, synthase for reverse pathway.

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most conserved element, ATAGTT , is located upstream, whereasthe downstream element, N3-XXCTAT, shows somewhat lessconservation. The complete MetR/MtaR dimer-binding motifthat we thus define is ATAGTT-N3-XXCTAT. The newly definedMetR/MtaR-binding motif is completely covered by the earlierdefined MET box, yet it is 2 nucleotides shorter so that it agreeswith the LysR-family characteristic. The recovery of putativebinding sites in genome-wide searches is very sensitive with re-spect to the precise composition of the search motif, and there-fore, we used both the newly defined motif and the extended motif(1 nucleotide at each side) reported previously (59) to identifyMetR/MtaR-binding sites. We observed only a few differences be-tween the two searches (not shown), although the latter searchseemed to be more discriminative, and the results given in Tables2 and 3 and File S3 in the supplemental material therefore relate tothe second search. The observed positive effect of the addition ofthe flanking nucleotides in the search and their conservation couldwell be related to potential effects of the flanking nucleotides onthe molecular structure of the binding site (where the physicalbinding occurs) and therefore on binding affinity.

A clear MetR/MtaR-binding motif was discerned in the up-stream region of metA, yjcL patB, metEF or metE yitJ, mmuM(metH), and yxjH and the operon related to ABC-mediated trans-port of methionine in most streptococcal genomes (Tables 2 and3; see File S3 in the supplemental material), as reported earlier(59). These regulatory relations seem to be conserved (with a fewexceptions, as described below); i.e., when the genes are present,

the MetR/MtaR-binding site is also present. However, some sitesscore relatively low, which could be indicative of lost function butcould also be very well related to slightly altered binding prefer-ences between species. In the case of S. gordonii, Streptococcusparasanguinis, and Streptococcus sanguinis, the SMK box upstreamof metK is preceded by a MetR/MtaR-binding site. As S. equi, S.dysgalactiae, Streptococcus parauberis, and Streptococcus uberis lackthe related genes, MetR/MtaR-mediated regulation seems to berestricted to methionine import via the ABC transport system.Another regulatory connection that was conserved in at least threespecies was observed in S. mitis, Streptococcus oralis, and S. pneu-moniae for the genes fhs and folD, encoding formate-tetrahydro-folate ligase and a bifunctional methylenetetrahydrofolate dehy-drogenase (NADP�)/methenyltetrahydrofolate cyclohydrolase,respectively, which are important in the biosynthesis of the cofac-tor tetrahydrofolate.

In L. lactis, MetR/MtaR-mediated regulation seems to be re-stricted to metEF, although most of the other genes are present inthe L. lactis genomes. In the lactobacilli L. delbrueckii subsp. bul-garicus and L. plantarum, only one gene seems to be connected toMetR/MtaR. In L. delbrueckii subsp. bulgaricus, the gene metE2 ispreceded by an obvious binding site. Remarkably, in other lacto-bacilli, the gene is preceded by a T box. As the gene is adjacent to

FIG 2 Binding motifs of various cysteine and methionine metabolism-relatedregulators in the Lactobacillales. (A) The characteristic SMK box motifs up-stream of the metK gene in lactobacilli and streptococci as reported previously(22). (B) Redefined MetR, CmbR, and HomR dimer binding motifs in lacto-bacilli and streptococci. See the main text for details. (C) CodY binding motifin lactobacilli and streptococci. The motif was created on the basis of the dataprovided elsewhere (11). The original data can be found in File S2 in thesupplemental material. The motifs were created with WebLogo (frequencyrepresentation; no correction applied; http://weblogo.berkeley.edu).

FIG 3 Bootstrapped (n � 1,000) partial neighbor-joining tree of LysR-familytranscription regulators in representative members of the Lactobacillales. Onlythe branches of the CmbR, MetR/MtaR, and HomR subfamilies are shown.NCBI reference sequence (RefSeq) gi codes precede the species/strain names (syn-chronized with Tables 1 and 2). Experimentally validated regulatory proteins areindicated by dots. Abbreviations: L., Lactococcus; Lb., Lactobacillus.

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the gene encoding the regulator, it could well be that this unit hasbeen acquired from some Streptococcus. In fact, the genes metEand metR are also neighbors on the L. plantarum genome. Surpris-ingly, in L. plantarum the MetR/MtaR-binding site appears tohave been lost. However, a binding site was found upstream oftrxB1 (lp_0761), a gene encoding a thioredoxin reductase, moreremotely connected to sulfur metabolism. Similarly, a binding sitewas found upstream of coaE in L. lactis, which encodes dephos-pho-coenzyme A kinase, the enzyme that catalyzes the final step inthe biosynthesis of the thiol compound coenzyme A.

CmbR/CysR. CmbR positively regulates the metC-cysK (i.e.,yrhB-cysK1) operon in L. lactis (25). In fact, the expression of 18genes is affected by a cmbR knockout in this species (70). Thegenes, which are grouped in several transcriptional units, includecysD, cysM (i.e., cysK2), yhcE (i.e., yxjG or yxjH), metC-cysK (i.e.,yrhB-cysK1), and metA-metB1-ytjE (i.e., metA-yjcL-patB) and themethionine (plpABCD ydcBD) and cystine (yjgCDE) ABC trans-port systems (70). It was shown that binding of CmbR to themetB2-cysK (i.e., yrhB-cysK1) promoter is stimulated by O-acetyl-L-serine and by low cysteine and methionine concentrations in L.lactis (16, 25). In vitro promoter binding studies confirmed strongbinding of CmbR to the metB2-cysK (i.e., yrhB-cysK1) promoterand weaker binding to the promoter of cysD and cysM and of themethionine (plpA) and cystine (yjgC) ABC transport system oper-ons. Hardly any binding was observed for metA-metB1-ytjE (i.e.,metA-yjcL-patB) and yhcE (i.e., yxjG or yxjH) (see Fig. 2 in refer-ence 70). In S. mutans, cysK, tcyD (i.e., tcyJ or tcyK), metBC (i.e.,yjcL-patB), and homR expression was shown to be affected upondeletion of cmbR (69).

As CmbR belongs to the LysR family of transcription regula-tors, one would expect its binding site to have a common LysR-family motif. However, all studies dedicated to CmbR binding todate have resulted in a distinctly different CmbR-binding motifdefinition. In the first study, a deletion analysis showed that adirect repeat of ATAAAAAAA is required for metC activation by

CmbR (25). In the second study, the upstream regions of seventranscriptional units found to be regulated by CmbR in L. lactiswere analyzed. A first consensus binding sequence, TWAAAAATTNNTA, centered 46 to 53 bp upstream of the transcriptionalstart, with a second consensus, TWAAAWANNTNNA, located 8to 10 bp upstream, was proposed (70). The approximate locationof CmbR binding in the metB2 and cysD upstream regions wasdetermined by gel-shift experiments (25, 70). A more recent pub-lication by Kovaleva and Gelfand (36) describes an in silico analy-sis of upstream regions of cysteine biosynthesis genes in strepto-coccal species and, to increase the recognition power, defines theCmbR-binding motif to be TGATA-N9-TATCA-N2-4-TGATA.

To resolve the discrepancies resulting from the previous anal-yses, we reanalyzed the reported CmbR-binding sites in L. lactisand Streptococcus species. We identified the consensus bindingsequences in the upstream region of the CmbR-regulated cysKgene and its paralogs/orthologs from lactococcal and streptococ-cal genomes (Fig. 2B; see File S2 in the supplemental material).Since two subclusters with respect to the cysK gene can be distin-guished by phylogeny and gene context in the L. lactis strains, atfirst, a putative CmbR-binding site was identified for each sub-cluster. In case of the so-called cysM1 cluster, CmbR-binding sitesfrom other streptococci such as S. agalactiae and S. gordonii werealso taken into account. Nevertheless, all the putative CmbR-binding sites in both Lactococcus and Streptococcus could be sum-marized by a motif of dyad symmetry ATA-N9-TAT, with a pref-erence for a long stretch of adenines following the starting ATA.This putative CmbR-binding motif has the general LysR-familysignature, and, in addition, all the previous experimental work onCmbR binding supports this assignment. Moreover, like in thecase of MetR/MtaR, a second more degenerate site is found di-rectly downstream. Similarly, we observed a positive effect of theaddition of the flanking nucleotides in the search for putativeCmbR-binding sites, and we have thus used the extended motif inour searches (Fig. 2B).

TABLE 3 Presence and regulation of the cysteine and methionine ABC transport systems in streptococcal genomesa

Con

trol

led

tran

spor

tsy

stem

Lc.l

acti

sIL

1403

S.ag

alac

tiae

2603

V/R

S.dy

sgal

acti

aeG

GS1

24

S.eq

ui40

47

S.ga

lloly

ticu

sA

TC

C_B

AA

2069

S.go

rdon

iiC

hal

lisC

H1

S.m

itis

B6

S.m

utan

sU

A15

9

S.or

alis

Uo5

S.pa

rasa

ngui

nis

AT

CC

1591

2

S.pa

raub

eris

KC

TC

1153

7

S.pa

steu

rian

usA

TC

C43

144

S.pn

eum

onia

eT

IGR

4

S.py

ogen

esM

1-G

AS

S.sa

ngui

nis

SK36

S.su

is05

ZY

H33

S.th

erm

ophi

lus

CN

RZ

1066

S.ub

eris

0140

J

SBP_bac_3 (tcyA, i.e., yxeM) 1 1C 1c? 1c? 1c? 1C 1C 1C 1c? 1c?

BPD_transp_1, ABC_tran(tcyBC, i.e., yxeNO)

1 1C 1c? 1c? 1c? 1C 1C 1C 1C 1C 1c?

SBP_bac_3 (tcyA-, i.e., yxeM-, like) 1 1 1c? 1c? 2C(3) 1c? 2C(3C) 1C 1C 1C 1C 1C 1C(2C) 1c? 1C 1 1C(2C) 1C

Lipoprotein_9 (metQ-like) 4 1M(2M) 2M 1M 1M 1m? 1M 1M 1M 1m? 1M 1 1M 1M 1C 1C 1M(2) 1M

EC 3.5.1.16/EC 3.5.1.18(dapE-like)

1M 1M 1M 1M 1m? 1M 1M 1m? 1M 1 1M 1C 0? 1M

ABC_tran-NIL, BPD_transp_1 1 1M 1M 1M 1M 1m? 1M 1M 1M 1m? 1M 1 1M 1M 1C 1C 1M 1M

a The genes present in the same operon are indicated by similar shading of the cells. In case more than one closely related sequence was present, the total number is given. In casethe gene might be present but was not called, it is indicated by “0?.” The number is in parentheses in case not all are present in the same operon and/or preceded by a similarputative binding site. Putative regulator-binding sites upstream of the indicated gene are given in capital letters in superscript: C, CmbR motif; M, MetR motif. Low-scoringputative binding sites are indicated by lowercase letters and a question mark. In several cases we found putative binding sites for two regulators. Species name abbreviation: Lc.,Lactococcus. The related data and NCBI gi codes can be found in File S1 in the supplemental material, and the analysis of upstream regions can be found in File S3 in thesupplemental material. The original data are provided at www.cmbi.ru.nl/bamics/supplementary/Liuetal_2012_CysMetregulation.

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In the NJ tree, the CmbR subfamily is most closely related tothe MetR/MtaR subfamily (Fig. 2). Therefore, one might expectthat their binding sites resemble each other. Indeed, the motifs areremarkably similar. However, there were some small differences,especially in the conserved flanking residues. As in the case ofMetR/MtaR, we have thus used the extended CmbR motif tosearch for additional binding sites (in this case, 3 nucleotides oneach side). The results are depicted in Tables 1 to 3 (see File S3 inthe supplemental material). We retrieved the known binding sitesin the region upstream of metB2-cysK in the Lactococcus strainsand cysK in the Streptococcus strains. We also identified a novelsite upstream of cysD in the S. thermophilus strains, whereas in, forexample, S. mutans, the regulation of cysD seems to be mediatedby MetR/MtaR. Another conserved relation of CmbR was foundwith homologs of the gene encoding the ABC transport-relatedcystine substrate binding protein (6, 52). In many streptococci,two CmbR-regulated copies of the gene are present; the first isrelated to tcyA or yxeM of B. subtilis, and the second is related totcyJ and tcyK of B. subtilis. The first is associated with the MetR/MtaR-regulated methionine ABC transport-related operon (49,82), and the second is associated with genes encoding the per-mease and ATP-binding subunit of a separate cystine ABC trans-port system (related to tcyLMN in B. subtilis [6]).

Remarkably, the scoring suggests that in S. suis the regulationof metBC, metEF, and the methionine ABC transport-relatedoperon is also CmbR dependent instead of MetR/MtaR depen-dent, like in the other streptococci. For S. mutans, our observa-tions fit the regulon derived on the basis of the cmbR-knockoutexperiments (69). In contrast, the number of putative CmbR-binding sites detected in L. lactis was low compared to the numberof reported putative sites (70). Only the highest-affinity promoterwas retrieved. Apparently, the defined motif is not very discrimi-native in the analysis of the L. lactis genomes. Other regulatoryconnections that were conserved in at least three species were ob-served with genes encoding pyruvate formate lyase, dihydrofolatereductase, a glutamine amidotransferase, a beta-lactamase, glyc-eraldehyde 3-P dehydrogenase, and a specific RNA methyltrans-ferase in various species (Table S2 in the supplemental material).

HomR. HomR is the third transcriptional regulator of the LysRfamily that is closely related to MetR/MtaR and CmbR and whichwas found only in S. gallolyticus, S. mutans, S. pasteurianus, and S.thermophilus. The expression of S. mutans metBC, encoding cys-teine biosynthesis genes, and the tcyDEFGH (i.e., tcyJKLMN) clus-ter, encoding a cysteine transport system, is specifically affected ina homR-knockout mutant (69). We examined the upstream re-gion of metB in the HomR-containing streptococci and couldidentify a clear HomR-binding motif according to the commonstructure of LysR-family DNA elements (as given in Fig. 2B). Thebinding motif resembles that of MetR/MtaR, with the most obvi-ous differences being located in the flanking nucleotides. Similarto MetR/MtaR and CmbR, HomR exerts control over metB ex-pression via a second binding site located downstream with lowersimilarity. We have used the extended HomR-related motif tosearch for additional binding sites (see File S3 in the supplementalmaterial). Besides the conserved relation with regulation of themetBC (i.e., yjcL-patB) operon, we found a HomR-specific siteupstream of folD in S. pasteurianus and panE (encoding a 2-dehy-dropantoate 2-reductase) in S. thermophilus. In addition, the rel-ative scoring is suggestive of some overlap with the regulation via

MetR/MtaR for the metE yitJ operon in S. gallolyticus, S. mutans,and S. pasteurianus and for metA in S. thermophilus.

CodY. A nutritional regulator involved in the global control ofamino acid metabolism in Gram-positive bacteria is CodY (67,71). It has been extensively studied in L. lactis (11, 28) and otherstreptococci (8, 39, 40) and was reported to control the expressionof hom, thrA, thrB, thrC, and cysD in these organisms. We defineda CodY binding motif on the basis of the data provided elsewhere(11). The motif is given in Fig. 2C and is almost identical to theexperimentally defined motif of B. subtilis (5). The motif was usedto search for putative CodY binding sites upstream of the genesrelated to cysteine and methionine metabolism. The results of thesearch are listed in Tables 1 and 2 and File S3 in the supplementalmaterial. We found similar motifs upstream of thrA, thrC, and/orthe hom-thrB operon in many of the streptococcal genomes. How-ever, the upstream regions are relatively dissimilar between thespecies, and as a result, for various other streptococci, it is notparticularly clear whether the CodY binding site is conserved (notshown). We found a potential site upstream of the metA-metB1-ytjE (i.e., metA-yjcL-patB) operon in L. lactis and the metB1-ytjE(i.e., yjcL-patB) operon in S. thermophilus.

Novel elements upstream of cbs and thrB genes. A compara-tive analysis (see Materials and Methods) of the upstream regionsof the other genes involved in cysteine and methionine metabo-lism did yield three additional putative regulatory elements. TheyrhA-yrhB operon in various lactobacillus strains, such as Lacto-bacillus salivarius, L. plantarum, L. delbrueckii subsp. bulgaricus,and Lactobacillus acidophilus, as well as in Oenococcus oeni andLeuconostoc, is preceded by a conserved palindromic motif, AAAGGGCGCGAA-N(11-18)-TTCGCGCCTTTT (Fig. 4A). As de-scribed earlier, all S. thermophilus strains carry an orthologousoperon with a completely conserved upstream region. We couldnot detect identical sites elsewhere on the genome. Consideringthe variable spacing between the complementary stretches, the

FIG 4 Potential regulatory motifs related to the control of Cys and Met me-tabolism. The motifs were identified upstream of the yrhAB operon in LAB(A), in thrB in LAB (B), and in L. lactis (C). The motifs are palindromic andtherefore could relate to structure-forming elements. The matching parts ofthe sequence have been underlined in different shades. The motifs were createdwith WebLogo (frequency representation; no correction applied; http://weblogo.berkeley.edu).

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motif probably represents a structural element. Similarly, a mul-tiple-sequence alignment of the upstream regions of thrB from L.acidophilus and Lactobacillus gasseri revealed another previouslyunidentified conserved motif (Fig. 4B). The putative motif con-sists of inverted repeats separated by 15 bp (ATTGTAAC-N15-GTTACAAT). Interestingly, each part of the inverted repeat comple-ments itself (e.g., in the first part, ATTG is immediately followedby its complement sequence, TAAC). Again, no similar sites werefound elsewhere on the genome. Another potential structural mo-tif was recovered upstream of thrB in L. lactis (Fig. 4C). The 12-nucleotide sequence is found more than 100 times in a seeminglyrandom distribution in all L. lactis genomes and less than 2 timesin all other analyzed genomes (not shown). The motif was discov-ered before and previously called the highly repetitive motif (50).The absolute conservation and occurrence imply functional im-portance to L. lactis, although it has yet to be discovered what theprecise regulatory role might be.

DISCUSSION

We have performed a genome-wide in silico analysis to reveal thetranscription regulatory interactions that control the expressionof the genes encoding various key enzymes involved in cysteineand methionine metabolism in all sequenced species of the orderLactobacillales. The associated regulators that we could identifywere the known regulatory proteins, such as CmbR, MetR/MtaR,HomR, and CodY, as well as known RNA riboswitches. In addi-tion, we found two potential regulatory structure-forming ele-ments. We redefined specific binding motifs for CmbR, MetR/MtaR, and HomR in lactococci and streptococci. All motifs followthe characteristic LysR-family signature (ATA-N9-TAT) and aresubstantiated by the available experimental data. The motifs al-lowed a computational separation of the various binding siteswithin the streptococci. The identified regulator-gene associationsoverlapped well with the subdivision of the reaction network thatwas made on the basis of the operon composition and on the basisof EC numbers (compare Fig. 1 with Tables 1 and 2).

A clear diversity in the transcriptional regulation network be-tween the different families was observed. In various Lactobacil-laceae like L. plantarum, the biosynthesis of methionine, as well asits precursors, i.e., homocysteine and cystathionine, is responsiveto low methionine levels through T-box-mediated regulation.Similarly, the ABC transport of methionine is regulated via a T-box riboswitch. We could not identify new general sites related tothe synthesis of cysteine from homocysteine and the degradationof both cysteine and methionine in the Lactobacillaceae. This mayindicate that most of the relevant regulators are known or thatpotential other elements might be less conserved or species spe-cific. In streptococci, the gene-regulator associations nicely fit theestablished inducer substrates. MetR/MtaR controls the expres-sion of the genes that encode biosynthesis and transport of methi-onine, whereas CmbR relates to the control of cysteine biosynthe-sis and cystine transport. HomR appears to be specificallydedicated to the control of yjcL-patB expression in four strepto-cocci. The latter relationship makes sense, as the genes encode theenzymes responsible for the conversion of acetylhomoserine tocystathionine and further. In S. thermophilus, an additional genecontrolled by HomR encodes the protein that catalyzes the pro-duction of acetylhomoserine. The intracellular levels of homoser-ine seem to be controlled by the global regulator CodY.

The biosynthesis of flavor compounds, e.g., H2S, methane-

thiol, DMS, DMDS, and DMTS, is catalyzed directly by cystathi-onine beta/gamma-lyase, which is encoded in most streptococciby a single gene, the expression of which is controlled by MetR/MtaR (methionine). Of course, changing the levels of the flavorprecursors methionine, cystathionine, and cysteine will also affectthe rate of synthesis of flavor compounds. These levels are deter-mined by the activity of cysteine synthase (cysK) and cystathioninesynthase (yrhA), homocysteine S-methyltransferase (yitJ andmmuM), and S-adenosylmethionine synthetase (metK) and thuscontrolled by CmbR (i.e., O-acetyl[homo]serine), MetR/MtaR(i.e., methionine), and an SMK box (i.e., S-adenosylmethionine),respectively. Inclusion of the above-described reaction networkand the regulatory interconnections into a quantitative metabolicnetwork model may help to rationalize new strategies for control-ling the sulfuric flavor formation in various LAB-fermented foodproducts.

ACKNOWLEDGMENT

This work was supported by grant CSI4017 from the Casimir Program ofthe Ministry of Economic Affairs, The Netherlands.

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