Journal of Bacteriology, September 2004, p . 6316-6319, Vol . 186, No . 18

Bacterial Thymidylate Synthase with Intein, Group II Intron, and Distinctive ThyX Motifs

Xiang-Qin Liu^* and Jing Yang

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Received 8 April 2004/ Accepted 25 May 2004

 
The ThyX class of thymidylate synthases was previously characterized by a common ThyX motif, RHRX₇S . We report bacterial ThyX sequences having distinctive ThyX motifs, suggesting a more general ThyX motif, R/THRX_7-8S . One ThyX sequence has an intein in its ThyX motif that was shown to do protein splicing and a group II intron in its gene, suggesting a hot spot for these self-splicing mobile elements.

 
The recently discovered ThyX class of thymidylate synthasesis not similar to ThyA in its structure [16] or in its reductive mechanism [10, 19], revealing complexity in the evolution ofthymidine production in DNA synthesis . ThyX exists in organismslacking ThyA and shows a sporadic phylogenetic distributionindicating lateral gene transfers [19] . Its presence in manypathogenic bacteria and absence in humans make ThyX an attractivetarget for potential antibacterial drugs . To realize this potential,knowledge is needed regarding important structural elementsincluding the common ThyX motif found in previously known ThyXsequences.

Inteins and introns are rare and have not been found previously with ThyX . An intein is a protein intervening sequence thatcan self-excise and concomitantly splice together its flankingextein sequences [21] . Many inteins also harbor an endonuclease domain that initiates intein homing, which confers genetic mobility on the intein [3, 5] and explains its sporadic phylogeneticdistribution [20] . Group II introns are another type of self-splicingmobile element, and most bacterial group II introns encode areverse transcriptase-like [RTL] protein [23] that assists intronsplicing and mobility, including retrotransposition [2, 11, 12] . Group II introns are believed to be evolutionary ancestorsof nuclear spliceosomal introns [4], but they are strongly excludedfrom conserved protein-coding genes in bacteria [6-8], althoughsome bacteriophage genes encode both inteins and group I introns[9, 13, 14] . Surprisingly, a bacterial ribonucleotide reductase[RIR]-encoding gene was found recently to encode multiple inteinsand group II introns [18] . To explore and understand this newphenomenon, we searched for similar inteins and introns in relatedgenes.

An intein- and intron-encoding thyX gene was found during a BLAST search [1] of the GenBank database . This gene is from the oceanic N₂-fixing cyanobacterium Trichodesmium erythraeum.As illustrated in Fig . 1, the three exon and extein coding sequencesare 196, 80, and 444 bp long, respectively, and they togetherpredicted a 240-amino-acid ThyX sequence that is very similarto known ThyX sequences . The intron was identified by its strongsequence similarity to known group II introns in this organism,which included the T.er.I4 intron in an RIR-encoding gene [18]and the Tr.e.I1 intron in an intergenic sequence [6] . It ismore than 80% identical to the other introns in the 680-nucleotide folded region [data not shown], although it lacks the RTL coding sequence present in domain IV of the other introns.

 
The intein was recognized by its intein sequence motifs [Fig. 2], and its boundaries were readily identified through comparisonswith inteinless ThyX sequences . The intein clearly has sequencemotifs [A, B, F, and G] for a splicing domain, but it either lacks or has incomplete sequence motifs [C, D, E, and H] fora homing endonuclease domain . It showed less than 15% sequenceidentity and no insertion site similarity to other known inteins.For example, it showed 14% sequence identity and 28% sequencesimilarity to the Synechocystis sp . strain PCC6803 DnaB intein.Nevertheless, it showed efficient protein splicing in a recombinantprotein in Escherichia coli [Fig . 3].

 
The T . erythraeum thyX gene is only the second bacterial gene [except for phage genes] known to encode inteins and introns, following an RIR-encoding gene [18] . It is not certain why thesetwo genes appear to be hot spots for intein and intron insertions,which most likely involve intein homing and group II intronretrotransposition . The ThyX intein likely had a homing endonucleasedomain and later lost it, on the basis of observation of putativeremnants of this domain in the intein . The ThyX intron and theT.er.I4 intron of the RIR-encoding gene, showing strong sequenceidentity, are likely related through recent retrotransposition,and it is tempting to speculate that the RTL-less ThyX intronis assisted in retrotransposition by the RTL protein encodedin the T.er.I4 intron . It is interesting that the thyX geneand the RIR-encoding gene both encode proteins involved in nucleicacid metabolism, because such genes have been recognized asfavored homes of inteins and introns for various reasons [8,17] . We further noticed that the ThyX intein is inserted inthe conserved ThyX motif, which prompted further analysis ofThyX motifs.

New ThyX sequences having distinctive ThyX motifs were found through BLAST searches of the GenBank database . Although nothaving inteins or introns, they showed ThyX motifs that aresimilar to but distinct from the previously defined ThyX motifRHRX₇S [Table 1 and Fig . 4] . Most of the new ThyX sequences[T . erythraeum, Nostoc punctiforme, Nostoc sp . strain PCC7120,Synechococcus sp . strain WH8102, mycobacteriophage Bxz1, Streptomycescoelicolor, and Streptomyces avermitilis MA-4680] are readilyidentified because they are more than 20% identical and 30%similar to the functionally identified Helicobacter pylori J99ThyX protein and to the structurally determined Thermotoga maritimaThyX protein . ThyX sequences from the two thermophilic sources[Thermosynechococcus elongatus and thermophilic bacteriophageRM378], however, were less easy to identify because of theirstriking differences from other ThyX sequences at the N andC termini [Fig . 4B] . Nevertheless, they are approximately 18%identical and 34% similar to T . erythraeum ThyX over four-fifthsof the T . erythraeum ThyX sequence, which is quite significantin light of the generally low levels of sequence conservationamong ThyX proteins . For example, the ThyX sequences of twocyanobacterial species [T . erythraeum and Synechocystis sp.strain PCC6803] are only 17% identical and 33% similar . Theshortened N-terminal sequence of the T . elongatus and thermophilicbacteriophage RM378 ThyX proteins, relative to that of otherThyX proteins, may be compensated for by the extended C-terminalsequence, although the extended C-terminal sequence does notshow similarity to that of other ThyX sequences . T . elongatusThyX is also the only recognizable thymidylate synthase [whichis functionally essential] that could be predicted from thecomplete genome sequence of this organism.

 
These ThyX sequences, when grouped by their ThyX motifs as inTable 1, showed higher sequence identities within a group than between groups . In particular, the ThyX sequences of four cyanobacteria [T . erythraeum, N . punctiforme, Nostoc sp . strain PCC7120, andSynechococcus sp . strain WH8102], having the same ThyX motif,THRX₈S, have more than 60% sequence identity with each other.But they show less than 20% sequence identity with the ThyXsequences of other cyanobacteria [e.g., Synechocystis sp . strainPCC6803] that have a different ThyX motif . The previously identifiedThyX motif RHRX₇S has the widest distribution, encompassingbacteria, archaea, and eucarya; therefore, it likely is theoriginal ThyX motif present in the common ancestor of ThyX proteins.The new ThyX motifs THRX₈S and THRX₇S, found in some cyanobacterialspecies, likely represent later divergence from the presumedoriginal ThyX motif, and they could also have been acquiredthrough lateral gene transfer . Interestingly, three of the fourdistinctive ThyX motifs have been found with bacteriophage,which could have facilitated lateral transfer of thyX genes.

We suggest a more general ThyX motif, R/THRX_7-8S, in order to accommodate all four of the distinctive ThyX motifs [Table 1]. While this report was under review, others reported the functional characterization of two residues of the ThyX motif RHRX_7-8S located at the catalytic site [15] . The absolutely conservedS residue [S84] was shown to function as a nucleophile, and the first R residue [R74] was shown to participate in flavin adenine dinucleotide and dUMP binding . But our findings showthat the first R residue is not absolutely conserved . However,when a ThyX motif begins with T instead of an R, it either hasan R immediately before a T [in T . erythraeum, N . punctiforme,Nostoc sp . strain PCC7120, and Synechococcus sp . strain WH8102]or has a second R internally [in T . erythraeum and thermophilic bacteriophage RM378], and this could suggest functional replacement of alternative R residues.

 
This work was supported by research grants from the NationalScience and Engineering Research Council of Canada.

 
* Corresponding author . Mailing address: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada . Phone: [902] 494-1208 . Fax: [902] 494-1355 . E-mail: pxqliu@dal.ca .

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