Serine threonine protein kinases of mycobacterial genus: phylogeny to function

doi: 10.1152/physiolgenomics.00221.200629:66-75, 2007. First published 5 December 2006;Physiol. Genomics

Yogendra SinghAzeet Narayan, Preeti Sachdeva, Kirti Sharma, Adesh K. Saini, Anil K. Tyagi andphylogeny to functionSerine threonine protein kinases of mycobacterial genus:

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Serine threonine protein kinases of mycobacterial genus:phylogeny to function

Azeet Narayan,1,2 Preeti Sachdeva,1 Kirti Sharma,1 Adesh K. Saini,1 Anil K. Tyagi,2 and Yogendra Singh1

1Institute of Genomics and Integrative Biology, Delhi; and 2Departmentof Biochemistry, University of Delhi South Campus, New Delhi, India

Submitted 9 October 2006; accepted in final form 29 November 2006

Narayan A, Sachdeva P, Sharma K, Saini AK, Tyagi AK, SinghY. Serine threonine protein kinases of mycobacterial genus: phylogeny tofunction. Physiol Genomics 29: 66–75, 2007. First published December 5,2006; doi:10.1152/physiolgenomics.00221.2006.—Serine/threonine pro-tein kinases (STPKs) are known to act as sensors of environmentalsignals that thereby regulate developmental changes and host patho-gen interactions. In this study, we carried out comparative genomeanalysis of six completely sequenced pathogenic and nonpathogenicmycobacterial species to systematically characterize the STPK com-plement of mycobacterium. Our analysis revealed that while Myco-bacterium tuberculosis strains have 11 conserved kinases, this numbervaries from 4 to 24 in other mycobacterial species. pknA, an essentialSTPK encoding gene, was found to be truncated in the initial analysisof M. avium subsp. paratuberculosis (Map) and M. tuberculosis Cgenomes. However, resequencing of pknA gene in Map confirmed thatthe truncation was due to a sequencing error. The conservation ofdivision and cell wall gene cluster involved in cell envelope biosyn-thesis and cell division, in the vicinity of pknL locus, implicates apossible role of PknL in cell division and envelop biosynthesis. Weidentified a cyclophilin domain as part of a mycobacterial kinase inMap that suggests a plausible regulation of cyclophilins by phosphor-ylation. The co-inheritance of pknA, pknB, pknG, and pknL loci acrossgenomes and some unique repertoire of pathogen-specific kinasessuch as pknI and pknJ of Mtb complex suggest similitude anddivergence between pathogenic and nonpathogenic signaling. Thisstudy would add another dimension toward identification of physio-logical substrates and thereby function, while resolving the existingcomplexities in signaling network between the two domains of life,pathogen and nonpathogen.

kinase; Mycobacterium; synteny; SymBet; comparative genomics

THE GENUS MYCOBACTERIUM INCLUDES acid fast bacilli with morethan 90 species, including slow-growing pathogens of Myco-bacterium tuberculosis complex [M. tuberculosis (Mtb), M.bovis (Mbo), M. africanum, M. microti, and M. canettii], M.avium-intracellulare complex (M. intracellulare and M.avium), M. marinum (Mma), M. leprae (Mle), and nonpatho-genic, fast-growing species such as M. smegmatis (Msm).Human tuberculosis is caused mainly by Mtb; however, theetiological agent of bovine tuberculosis, Mbo can also causehuman disease. Two genetically distinct species within M.avium-intracellulare complex are M. avium, which tends toinfect human immunodeficiency virus-infected patients, and M.intracellulare, more common among immunocompetent indi-viduals (27). Unlike M. intracellulare, which relates to a singlespecies, M. avium is separated into three subspecies; M. avium

ssp. avium, a major opportunistic pathogen leading to a dis-seminated disease among terminal acquired immune deficiencysyndrome patients; M. avium ssp. paratuberculosis (Map),causing Johne’s disease among ruminants and implicated inCrohn’s disease among humans; and M. avium ssp. silvaticum,a pathogen affecting birds that may cause chronic enteritisamong calves but has not yet been shown to be associated withhuman disease (27). Mle, a close relative of tubercle bacillus,causes leprosy, a chronic human neurological disease. Mma,the mycobacterium most closely related to the members of Mtbcomplex, causes systemic chronic tuberculosis in ectothermsand peripheral granulomatous disease in humans (9). Msm, afast-growing saprophytic mycobacterium, is used as a heterol-ogous model system to study gene expression and virulencemechanisms of Mtb.

Microbial genomics has provided a foundation for under-standing the basic biological processes, protein-protein inter-actions, and host-pathogen interactions and for designing novelantimicrobial compounds and vaccines. A recent study usingcomparative analysis of various microbial species demon-strated that the microbial genome is a dynamic entity shapedby multiple forces (19). Therefore, comparative functionalgenomics of the sequenced mycobacterial species can serveas an important tool in predicting the physiological impor-tance of certain distinct set of genes. Six mycobacterialspecies, including pathogenic and nonpathogenic, have beensequenced: Mtb H37Rv (7), Mtb CDC1551 (18), Mtb F11,and Mtb C (www.broad.mit.edu/seq/msc/); Mle (8); Msmmc2155 (www.tigr.org); Mbo AF2122/97 (20); Map K-10(35); and Mma (www.sanger.ac.uk).

Serine/threonine protein kinases (STPKs) have been shownto be important virulence factors in various pathogenic bacteria(11). Reversible protein phosphorylation by these STPKs playsa key role in regulating plethora of cellular processes includingstress response, regulation of cell cycle, and development. Thisregulatory phenomenon is unambiguously preserved during thecourse of evolution in all forms of life (31, 34). The genomesequence of Mtb H37Rv revealed the presence of 11 STPKs (7).Most of the mycobacterial STPKs have been biochemicallycharacterized (3, 6, 22, 23, 33, 37–40, 46, 52, 54, 61), andsome of them have been investigated for their physiologicalroles as well (10, 12, 14, 15, 30, 41, 55, 61). Some of thesekinases have been implicated in the pathogenesis and survivalof the tubercle bacillus within host (45, 63).

In this study, a comprehensive approach has been taken toidentify specific functional ortholog(s) of each Mtb kinase inmycobacterial species. This is particularly important in thecontext of various recent reports giving evidence of in vitrophosphorylation of a substrate by multiple kinases, implyingthat the signaling in mycobacterium could be more complex

Article published online before print. See web site for date of publication(http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: Y. Singh, Inst. ofGenomics and Integrative Biology, Mall Road, Delhi 110 007, India (e-mail:[email protected]).

Physiol Genomics 29: 66–75, 2007.First published December 5, 2006; doi:10.1152/physiolgenomics.00221.2006.

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than expected (23, 30, 37, 61). This study, in particular, wouldadd another dimension to the in vitro phosphotransfer studiesin identification of physiological substrates and thereby func-tion, while resolving the existing complexities. Another prac-tical utility of this approach would be to help determinewhether STPKs are discrete genomic units or instead a con-tinuum across various mycobacterial species that varies ac-cording to a number of properties, including but not limited tovirulence and host preference. So far, there is no comparativegenomic study elucidating the signaling network in mycobac-terial genus. Also, the number of kinases annotated in theavailable databases does not reflect the correct number ofkinases with characteristic structural motifs. The principlesapplied herein toward mining the mycobacterial kinome gamutmay be extended to other prokaryotes in assigning functions toorphan kinases.

EXPERIMENTAL PROCEDURES

Database Search

We searched the completely sequenced genomes of the Mycobac-terium species (Mtb H37Rv, Mtb C, Mtb F11, Mtb CDC1551, Mle,Mbo, Map, Msm, and Mma) for the presence of STPKs using each of11 full-length STPK sequences of Mtb as well as a consensus kinasedomain sequence (calculated from kinase domains of 11 Mtb STPKsusing a script available at http://www.bork.embl-heidelberg.de/Align-ment/consensus.html) as query. In addition, individual genomes werealso searched using the words “Pkn” and “serine-threonine kinase” toidentify the annotated kinase sequences. The Pkn2 type sequences(34) thus retrieved were further critically evaluated for the presence of11 Hank’s type kinase subdomains. Mtb H37Rv, Mtb C, Mtb F11, MtbCDC1551, Mle, Map, and Mbo genome sequences, available atNational Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) were searched for thepresence of STPKs by employing PSI-BLAST (1) and BLASTP intothe NR database using the default settings. We limited the Entrez toa particular mycobacterial species while performing PSI-BLAST andBLASTP at NCBI. Msm and Mma genome sequences available atTIGR (http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi) andSanger Centre (http://www.sanger.ac.uk/Projects/Microbes/), respec-tively, were searched with BLASTP. Mma kinases were searchedagainst the MM.pep database (http://www.sanger.ac.uk/DataSearch/blast.shtml), while Msm kinases were searched against M. smegmatisMC2.pep (http://tigrblast.tigr.org/cmr-blast/) using the default set-tings. The critical residues in kinase subdomains were carefullyspotted in each retrieved sequence, such as Rossmann-fold motif(GXGXXGXV), lysine in subdomain II, and aspartic acid residues insubdomain VIb and VII known to be involved in the ATP binding/catalysis (5, 24). Domain boundaries and structural organization of theretrieved STPK sequences were analyzed using SMART (51), Inter-ProScan (http://www.ebi.ac.uk/InterProScan/), and Conserved Do-main Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).Transmembrane segments were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and HMMTOP (http://www.enzim.hu/hmmtop/). Multiple sequence alignments were constructedusing CLUSTALX (1.83) (59) or T-COFFEE (v. 4.59) (44). Full-length protein kinase sequences, which give a better representation ofthe catalytic domain as well as regulatory regions compared withdomains alone, were used for the phylogenetic tree analysis. Thephylogenetic analysis was carried out using CLUSTALX andPHYLIP (ver. 3.6) programs PROTDIST, NEIGHBOR, SEQBOOT,and CONSENSE (17). The Jones-Taylor-Thomton amino acid substi-tution matrix was used in PROTDIST. The input order of sequencesfor phylogenetic analysis was randomized, wherever this option wasgiven. Phylogenetic tree was visualized with TREEVIEW. A region

of at least 10-Kb on either side of each kinase gene in all mycobac-terial genomes was gleaned to identify the neighboring genes, partic-ularly any cluster of genes proposed to be involved in any particularprocess/pathway. The genes/clusters thus obtained were then com-pared across all mycobacterial genomes for the conservation ofgenomic location with respect to a given kinase.

RESULTS AND DISCUSSION

The wealth of sequence information compiled from micro-bial genome projects has opened new avenues for studying theimportance of a protein in survival and virulence of tuberclebacillus and develop strategies to target these molecules/path-ways for therapeutic interventions (53, 62). In the presentstudy, we aim to assign a parallel nomenclature to all themycobacterial STPK-like sequences as ortholog pairs, therebyaiding the prediction of their functions in mycobacterium. Weused the symmetrical/bidirectional best hits (SymBet) method(32) to define an orthologous relationship between STPKsacross mycobacterial species. To resolve issues, where morethan one SymBet was obtained, auxiliary criteria such aspercentage identity cut-off values, conservation of order ofgenes (synteny), and domain composition and organizationwithin the protein were used to define orthologs and/or paral-ogs. Synteny is particularly important in defining correct or-thologs, when there are a large number of highly relatedparalogs present at different genomic locations. As synteny isknown to be considerably disrupted, when the protein sequenceidentity is �50% (26), a cutoff of �50% identity in full-lengthsequence was used to define candidate orthologs. Simulta-neously, a stringent cut off value of �70% was used for kinasedomain sequence identity, because intracellular kinase domainparticularly is under much tighter selective constraint than thejuxtamembrane and extracellular domains (29). Aforemen-tioned strategy helped us annotate certain kinases, which arecurrently either missannotated with different symbols or, insome cases, not even annotated as a kinase. Analysis ofneighboring genes in the vicinity of STPKs revealed conser-vation of certain gene clusters involved in physiological pro-cesses/pathways. Dandekar et al. (13) proposed that the pro-teins encoded by conserved gene pairs appear to interactphysically. Based on the conservation of gene clusters withrespect to a particular kinase across mycobacterial species, wepropose role of some of the STPKs in regulation of mycobac-terial physiology.

Distribution of STPK-like Sequences inMycobacterial Genomes

Earlier reports suggested the presence of 11 STPKs in Mtb(H37Rv) and 4 STPKs in Mle (7, 8). Using a combination ofsearch tools (PSI-BLAST and BLASTP), we identified 13STPKs in Msm, 11 STPKs in Mbo, 10 STPKs in Map, 24STPKs in Mma, and 11 STPKs in all Mtb strains (CDC1551,F11, and C) (Fig. 1). A careful analysis of kinase domains andtheir subdomain characteristic residues in all the sequencesretrieved in our search helped us annotate certain hypotheticalproteins as kinases and also discard some of the sequencesannotated as kinases in the genome database in our study.

Classification and Analysis of Mycobacterial STPKs

The full-length kinase sequence of all the retrieved STPKsof Mtb (H37Rv), Mbo, Msm, Map, Mle, and Mma was used to

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Fig. 1. Original genome annotations (protein identifiers) have been listed, and the names of organisms have been abbreviated as follows: Mtb, M. tuberculosis; Mbo,M. bovis; Mle, M. leprae; Msm, M. smegmatis; Map, M. avium subsp. paratuberculosis; Mma, M. marinum. The absence of orthologs of various kinases is shown by(–). The table is split into clades separated by dashed lines as described in Fig. 2. Representative schematic domain compositions are shown where vertical blue coloredbars represent transmembrane helices and other abbreviations are as follows: S_TKc, serine threonine protein kinase catalytic domain; PASTA, penicillin-binding proteinand serine/threonine kinase-associated domain; DsbG, protein-disulfide isomerase domain; NHL, NHL repeat; PBPb, periplasmic binding proteins; cyclophilin,cyclophilin-type peptidylprolyl cis, trans isomerase domain; WD40, protein-protein interaction module; DUF, domain of unknown function; MalT, ATP-dependenttranscriptional regulator domain; COG3903, P-loop containing ATPase domain; Ad_Cycl_assoc, adenylate cyclase associated; COG3118, thioredoxin domain-containing protein. *Truncated protein kinases. � Sequencing error that resulted in a codon and misannotation of pknA ORF as 2 ORFs.

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generate the phylogenetic tree. The phylogenetic tree sug-gested the clustering of all mycobacterial STPKs into fivegroups or clades (Fig. 2) as described below:

Clade I (PknA, PknB, PknL, ABL group). Clade I representsthe group of transmembrane sensor kinases from differentmycobacterial species that cluster along with PknA, PknB, andPknL of Mtb. Signal transduction mediated by PknA and PknBof Mtb has recently been shown to regulate cell shape inmycobacteria, a process that may be conserved among gram-positive bacteria (2, 14, 30).

Transposon mutagenesis experiments by Sassetti et al. (50)propose that pknA and pknB genes are individually required forgrowth of Mtb in culture, suggesting them to be essentialgenes. Interestingly, search for orthologs of pknA in Mapindicated the presence of two successive open reading frames(ORFs), MAP0018 and MAP0017, showing homology withthe amino and carboxy termini of pknA, respectively (35). Acloser examination revealed that these two ORFs in Map areseparated by a stop codon generated from the deletion of anucleotide that resulted in the formation of a new ORFMAP0017. Since the Mtb pknA gene is essential, resequencingof the region containing deletion was done, and indeed asequencing error was found, confirming the existence of anintact pknA gene in Map (Supplemental Fig. S1). (The onlineversion of this article contains supplemental material.) A pknAand pknB locus, which includes pstP, rodA, pbpA, and two

FHA-domain-containing genes, was found to be conservednear the origin of replication throughout the mycobacteriumgenus (Figs. 1, 3).

Bioinformatic analysis revealed that pknL of Mtb is con-served in all sequenced mycobacterial species (Fig. 1). Myco-bacterial PknL orthologs lack a substantial extracellular do-main (Fig. 1); however, PknL orthologs in other actinobacterialike Corynebacterium glutamicum ATCC13032 (CG2388, aPknL ortholog) show the presence of a penicillin-bindingprotein and serine/threonine kinase-associated (PASTA) do-main at the carboxy terminus, an extracellular sensor domaincharacteristic of Mtb PknB. The significant bootstrap value(434) of the tree branch (Fig. 2) and the presence of commondomains suggest a common origin and lineage-specific expan-sion of PknL and PknB. Interestingly, we identified a �30-kblocus encompassing pknL and several genes encoding a dcwcluster (division cell wall gene cluster involved in cell enve-lope biosynthesis and cell division) that was found to beconserved throughout the genus (Fig. 4). The conservation ofregions proposed to be involved in cell division and cell wallsynthesis in the neighborhood of both pknB and pknL substan-tiates the notion that the two kinases may regulate commonprocesses. A recent study indicates that an STPK mutantlacking an extracellular sensor domain comprising threePASTA domains displayed distinct pleiotropic effects on cellgrowth/division/septation and virulence factor expression in

Fig. 2. Phylogenetic analysis of serine/threoninekinases from 6 completely sequenced mycobacte-rial genomes. Analysis was performed by theneighbor-joining method of PHYLIP (ver. 3.63),and the tree was viewed by TREEVIEW. Thenumber at the nodes indicates bootstrap supportvalues after 500 bootstrap cycles. Unrooted treewas drawn using most distinct M. smegmatisprotein kinase MSMEG5417 as out-group. (Thekinase domain of this gene has no flanking se-quences at either NH2 or COOH-terminal endsbut has an insertion sequence of 22 amino acidsbetween subdomains VII and VIII.) The serine/threonine protein kinases (STPKs) were dividedinto 5 clades according to a number of criteria asdescribed in text. The STPKs in M. tuberculosisstrains CDC1551, F11, and C are identical orhighly similar to those in M. tuberculosis strainH37Rv and have not been enlisted.



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Streptococcus pyogenes (28). In view of this report, it seemsplausible that PknL in mycobacteria may have adapted adifferent mechanism to activate itself in vivo in the absence ofa sensor domain. A recent report by Kang et al. (30) suggestedthat PknB and PknA can cross-phosphorylate each other invitro. In the light of the evidences of cross-phosphorylation inmycobacterial signaling network and the necessity of phos-phorylation in activation of kinase activity (4, 16), a cross-talkof PknL with PknA and PknB cannot be ruled out. Importantly,the activation loop of PknL has a consensus sequence of apreferred phosphorylation site (I178LGTAAYLSP187) for PknAand PknB (30).

The conservation of dcw cluster in the vicinity of pknLacross genus implicates a possible regulation of this cluster byPknL in mycobacterium. The conservation of an �30-kb locusencompassing a pknL and dcw gene cluster (Fig. 4), phyloge-netic clustering of PknL with PknA/PknB (Fig. 2), and possiblecross talk between these kinases suggest that clade I is involvedin the signal relay regulating cell division. Moreover, thepresence of a putative DNA binding transcriptional regulator(Rv2175) upstream of pknL across the mycobacterium genus asa conserved gene pair suggests a probable mechanism ofregulation of the dcw gene cluster by Rv2175 in a phosphory-lation-dependent manner. The possibility of Rv2175 serving asa substrate of PknL is further augmented by various reports of

in vitro as well as in vivo phosphorylation of proteins coded bygenes conserved in the vicinity of a kinase in mycobacterium(12, 30, 39, 40).

A distinctive member of clade I, MSMEG6166, showsgenomic organization quite similar to that of PknA and PknB.Genes located in close proximity to MSMEG6166 encodeputative transcriptional regulators, MSMEG6158 andMSMEG6159 (whiB1), and the predicted cell division andelongation protein, MSMEG6160 (pbpA or ponA). The or-thologs of all these genes except for STPK (MSMEG6166)were found to be present in all mycobacterial species. Thegenetic organization of Msm-specific STPK, MSMEG6166,suggests its plausible role in regulation of cell division.

Clade II (PknH, PknE, PknD, HED group). Clade II repre-sents the group of integral membrane receptor as well ascytoplasmic kinases that cluster along with PknH, PknE, andPknD (Fig. 2). PknH regulates intracellular bacterial growth atlater stages of the infection process, particularly during thechronic phase (45). Recently, it has also been shown that PknHregulates embCAB operon, thereby affecting ethambutol resis-tance and LAM/LM ratio, an important determinant of viru-lence (55). Phylogenetic tree analysis showed that MM1982,MM4171, MM4169, MM4156, MAP2504, and Mb1297 clus-ter with PknH of Mtb (Fig. 2). Except for MM1982, theprimary amino acid sequences of these full-length proteins

Fig. 3. Comparison of pknA and pknB locusacross mycobacterial genomes. Arrows indi-cate the orientation of open reading frames(ORFs). Vertical bars across arrows representpseudogenes in Mle. This gene cluster is con-served in all mycobacterial genomes. Originalgenome annotations have been listed, and thenames of organisms have been abbreviated asfollows: Mbo, Mycobacterium bovis; Mtb,Mycobacterium tuberculosis; Mle, Mycobac-terium leprae; Msm, Mycobacterium smeg-matis; Map, Mycobacterium avium ssp. para-tuberculosis K-10; Mma, Mycobacterium ma-rinum.



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show �50% similarity with PknH (Supplemental Table S1).Upstream of MM4156, an embR ortholog, MM4155, is present,suggesting the conservation of pknH synteny in Mma. Thus,the true ortholog of PknH in Mma is MM4156, althoughMM4171 shows higher (79%) identity compared withMM4156 (62%) with intact PknH (Supplemental Table S1).This is in agreement with an earlier report, where the investi-gators found that in 29–38% of investigated co-ortholog rela-tionships, the less-similar gene pair retained the ancestral genein the neighborhood (43). The remaining two kinases of Mma(MM4171, MM4169) can be classified as inparalogs (32), asthese proteins are more similar to each other than any otherkinase sequence from the other mycobacterial genomes (Sup-plemental Tables S1 and S2). A gene encoding FHA domaincontaining ABC transporter, MM4170, lies between two of thepknH coorthologs, MM4169 and MM4171, encoded in thesame orientation, indicating the possible regulation of thetransporter by these kinases. An earlier report has shown invitro phosphorylation of an Mtb FHA domain containing ABCtransporter Rv1747 by multiple protein kinases (23). In Map,the pknH ortholog present downstream from embR (MAP2503)is truncated, leading to the formation of two ORFs, MAP2504and MAP2505, corresponding to the kinase and sensor do-mains, respectively. The PknH ortholog of Mbo Mb1297 hasdeletions in the juxtamembranal region encompassing a pro-line-rich region (Supplemental Fig. S2). Earlier, this proline-rich region was predicted to be involved in protein-proteininteraction (52).

PknE has been shown to phosphorylate various FHA domaincontaining proteins like GarA and ABC transporter in vitro(23, 61). Mtb PknE orthologs have been found only in Mma(MM2581) and Mbo (Mb1772) (Fig. 1). PknD has been shownto phosphorylate a transporter of RND family, MmpL7, inaddition to various FHA domain-containing proteins (23, 49,61). The locus organization of pknD in Mtb has a downstreampstS gene shown to be involved in phosphate uptake (47).Importantly, PknD has NHL-repeat �-propeller domain at thecarboxy terminus (Fig. 1), which likely functions as anchoringsensor domain (21). The pknD ortholog in Mbo (Mb0955) hasbeen shown to be truncated due to the insertion of an A atposition 830, resulting in the loss of a transmembrane andsensor domain (48). The pknD ortholog with the downstreampstS gene is conserved in Map (MAP3387) and Mma(MM4577). One of the clade II members, MM3398, has adomain organization similar to that of PknD (Fig. 1) but cannotbe classified as a PknD ortholog because it does not fulfillmany important criteria described earlier for assigning orthologstatus. It is not a SymBet of PknD, shows �70% identity inkinase domain (Supplemental Table S2), and does not haveconserved synteny. This gene might have evolved as an inde-pendent fusion event of kinase domain with �-propellerdomain.

A cluster of kinases branches out in parallel to the HEDgroup. These group members show very high percent identitywith the HED group members in their kinase domain, which isevident by the bootstrap values of the nodes in phenogram

Fig. 4. Comparison of pknL locus across mycobacterial genomes. Genetic organization of a �30-kb region in mycobacterial genome encoding pknL and putativedivision cell wall (dcw) gene cluster. Arrows indicate the orientation of ORFs. This gene cluster is conserved in all mycobacterial genomes sequenced to date.Original genome annotations have been listed and the names of organisms have been abbreviated as described in Fig. 3 legend.



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(Fig. 2, Supplemental Table S2). This cluster is represented bysoluble and transmembrane receptor kinases from Map, Msm,and Mma (Fig. 1). Though these kinases show high bootstrapvalues (Fig. 2), none of them meets the criteria laid down toclassify them as an ortholog of any kinase in any species (Fig.1). MAP2031 has an upstream gene (MAP2032) encodingtranscriptional regulator of Tet-R family. A unique solublekinase in Mma, MM3405 branches out from MAP1049 andMSMEG1194 in phenogram and possesses WD repeats (aprotein-protein interaction module) at the carboxy terminus(Fig. 1). Downstream of MM3405 is a possible operon (over-lapping gene sequences in same orientation) encoding genes ofthe UMP biosynthesis pathway (MM3407, MM3408, andMM3409).

Interestingly, MAP1049 was found to have a distinct pepti-dylprolyl cis-trans isomerase (PPIase)/cyclophilin domain at itscarboxy terminus (Fig. 1). Also, just 31 bp upstream ofMAP1049 is a PPIase B gene (MAP1050) in the same orien-tation (68% similarity between two cyclophilin domains).PPIases play a role in folding, transport, and assembly ofproteins and are also implicated in cell cycle control throughtranscriptional regulation. Their activity is directed via target-ing to prior phosphorylated proteins in yeast and mammals(56). Also a PPIase, Pin 1 activity has been shown to beregulated by phosphorylation at serine residue in mammaliancells (36). Importantly, in mycobacterium, another PPIasegene, namely PPIase A (Rv0009), is conserved throughoutmycobacterial species downstream from pknA and pknBoperon, a locus that has already been shown to be involved incell division regulation in mycobacteria (Fig. 3). Recent workin computational genomics has shown that a functional asso-ciation between two genes can be derived from the existence ofa fusion of the two as one continuous sequence in anothergenome (64). Presence of a cyclophilin domain fused with akinase as one continuous sequence as well as an independentconserved gene pair of kinases hint at the existence of a similarphosphorylation mediated signaling mechanism regulatingPPIase activity and subsequently cell division in mycobacteria.

Clade III (PknF PknI, PknJ, FIJ group). Clade III representsthe group of kinases that cluster along with PknF, PknI, andPknJ (Fig. 2). PknF has been shown to play a role in theregulation of glucose transport, cell growth, and septum for-mation in Mtb (15). Four STPKs of Msm (MSMEG0879,MSMEG5491, MSMEG1183, and MSMEG3682), one STPKof Map (MAP1332), seven STPKs of Mma (MM2606,MM4923, MM2941, MM2408, MM4174, MM1794, andMM1423) and three STPKs of Mtb (PknI, PknJ, and PknF)show very high similarity in their kinase domains (Supplemen-tal Table S2). On the basis of bidirectional best hits (SymBet),locus organization (presence of ABC transporter, Rv1747 or-tholog), and percent identity, it can be concluded thatMM2606, Mb1775, and MAP1332 are orthologs of PknF (Fig.1). Only syntenic evidence can determine the correct orthologwhen there are a number of highly related paralogs at differentgenomic locations. Since none of the clade III members fromMsm show conservation of synteny of PknF, it is difficult tomake an ortholog assertion. MAP1332, the only representativekinase of Map in clade III, is a SymBet of PknF; however, itscarboxy terminus (374–561 amino acid residues) shows 57%similarity with that of PknI. Thus, MAP1332 may represent anexample of a fusion event between two kinases in Map.

Moreover, upstream from MAP1332 there is an ABC trans-porter (Rv1747) ortholog, MAP1331, suggesting that PknI andPknF may share common substrates as well.

pknI is one of the genes that have been shown to bedownregulated during infection of Mtb in THP-1 macrophages(58). Although the pknI ortholog has been reported in Msmbased on Southern blot analysis (22), the closest gene to pknIat the DNA sequence level, MSMEG1183, shows markeddifferences at the protein sequence level (Supplemental TablesS1 and S2). In fact, MSMEG1183 is not a SymBet of PknI andshows only 54% identity in kinase domain at the amino acidsequence level (Supplemental Table S2). The greater similarityof MSMEG1183 to other kinases of clade III at the proteinsequence level, lack of asparagine in VIb subdomain, a char-acteristic feature of PknI (VIb, DXKPXN-DXNPXN) (seeSupplemental Table S3), and dissimilar flanking genes argueagainst the presence of a PknI ortholog in Msm. The analysisof PknI ortholog in Mma, MM1794, shows deleterious substi-tutions at critical residues in subdomain I (G13R, G23D), II(K3E), VIb (R3G, N3D), and VII (D3G) (SupplementalTable S3) that have been shown to be essential for catalysis(25, 60). Since these residues are involved in catalysis, theprospects of catalytic competence of MM1794 must be viewed

Fig. 5. Comparison of pknG locus across mycobacterial genomes. Arrowsindicate the orientation of ORFs. Vertical bars across arrows represent pseu-dogenes. Original genome annotations have been listed, and the names oforganisms have been abbreviated as described in Fig. 3 legend.



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with a higher degree of caution. An ortholog of PknJ is absentin all sequenced mycobacterial species except for Mbo (Fig. 1).The presence of PknJ in the sequenced members of Mtbcomplex alone suggests its unique repertoire of signalingspecific to the Mtb complex.

Clade IV (PknK). Clade IV represents soluble kinases thatcluster along with PknK (Fig. 2). Our analysis suggests thatorthologs of PknK are present in both slow- and fast-growingmycobacteria, with the exceptions of Map and Mle (Fig. 1).The PknK orthologs, like their Mtb counterpart, have regula-tory regions of the LuxR family and AAA domain at thecarboxy terminus. pknK is located adjacent to oxidoreductasesand various transcriptional regulators including virS(Rv3082c), which has been shown to regulate mymA operon(57). Four STPKs from Mma (MM2576, MM3547, MM0366,and MM0365), one from Msm (MSMEG0519), and one fromMbo (Mb3107) cluster with PknK. The locus organization ofpknK is not conserved in other species; however, in Msm,various oxidoreductase/dehydrogenase family genes (MS-MEG0515, MSMEG0516, MSMEG0517, MSMEG0518,and MSMEG0520) and transcriptional regulators (MS-MEG0514, MSMEG0525, and MSMEG0529) are present inthe vicinity of the kinase. MM2576 is the only kinase fromMma that fulfills the aforementioned criteria for orthologassertion (Fig. 1). Importantly, InterProscan result shows anAraC type HTH region (amino acid residues from 966–1009)and TPR motif (from 735 to 983 amino acid residues, 6.2 e�7)in the Mma PknK ortholog, MM2576 (Supplemental Fig. S3).These regions are found to be conserved in Mtb and MsmPknK orthologs as well. Other clade members from Mma showthe presence of autoregulatory regions like ATPase domains.Also, two of the members, MM0366 and MM3547, have anadditional terminally located adenylyl cyclase-associated re-gion (Fig. 1).

Clade V (PknG). Clade V represents soluble kinases thatcluster along with PknG. The pknG locus in Mtb includes glnH(Rv0411c) as an operon member (data not shown) and aconserved hypothetical membrane protein (Rv0412c). PknGwas reported to inhibit phagosome-lysosome fusion, thus al-lowing intracellular survival of mycobacteria (63). Reduced invitro growth and disturbed glutamine metabolism in pknGmutants of Mtb as well as Corynebacterium glutamicum implythat pknG function is not restricted to the pathogenic lifestyle(10, 42). In agreement with this notion, our analysis suggestedthe conservation of pknG and its locus throughout mycobacte-rial genus (Figs. 1, 5). Moreover, we also noticed the conser-vation of few genes of thiamine biosynthesis pathway upstreamof pknG and glnH (Fig. 5). MSMEG0781 of Msm is 79, 77,and 76% similar to PknG orthologs of Mtb (H37Rv), Map, andMle, respectively (Supplemental Table S1). Multiple sequencealignment of PknG orthologs suggests that they have similarkinase domains and carboxy termini, while PknG of MtbCDC1551, Mle, Map, and Msm has an extended amino termi-nus (Supplemental Fig. S4). The dissimilarity observed atamino terminus of PknG (Supplemental Fig. S4) may accountfor the functional differences that may be an underlying reasonfor enhanced survival observed in the case of Mtb PknGoverexpressing Msm (63). The ubiquitous presence of pknGand operon member, glnH, across both pathogenic and non-pathogenic species strongly supports the notion that pknG has

a role to play in the maintenance of general mycobacterialphysiology.

In conclusion, we have endeavored to provide an in-depthanalysis and functional ortholog designation for mycobacterialSTPKs based on a combination of criteria. This functionalclassification into clades helped us to predict certain novelphysiological functions of kinases. With knowledge recentlygained about the physiological role of mycobacterial STPKs,kinases have emerged as good drug targets for mycobacteria.Hence a holistic approach is inevitable in targeting thesemolecules, where functionally linked/similar kinases, as clas-sified in the study, can constitute a regulatory network.

ACKNOWLEDGMENTS

The M. marinum sequence data were generated by the Sanger InstitutePathogen Sequencing Unit and can be obtained from: http://www.sanger.ac.uk/Projects/M_marinum.

Thanks to Professor Vivek Kapur, University of Minnesota, for resequenc-ing Map pknA gene.

GRANTS

The work was supported by Council of Scientific and Industrial ResearchGrant No. SMM0003.

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Serine threonine protein kinases of mycobacterial genus: phylogeny to function

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