Genomic characterization of ribitol teichoic acid synthesis in Staphylococcus aureus: genes, genomic organization and gene duplication
© Qian et al; licensee BioMed Central Ltd. 2006
Received: 08 October 2005
Accepted: 05 April 2006
Published: 05 April 2006
Staphylococcus aureus or MRSA (Methicillin Resistant S. aureus), is an acquired pathogen and the primary cause of nosocomial infections worldwide. In S. aureus, teichoic acid is an essential component of the cell wall, and its biosynthesis is not yet well characterized. Studies in Bacillus subtilis have discovered two different pathways of teichoic acid biosynthesis, in two strains W23 and 168 respectively, namely teichoic acid ribitol (tar) and teichoic acid glycerol (tag). The genes involved in these two pathways are also characterized, tarA, tarB, tarD, tarI, tarJ, tarK, tarL for the tar pathway, and tagA, tagB, tagD, tagE, tagF for the tag pathway. With the genome sequences of several MRSA strains: Mu50, MW2, N315, MRSA252, COL as well as methicillin susceptible strain MSSA476 available, a comparative genomic analysis was performed to characterize teichoic acid biosynthesis in these S. aureus strains.
We identified all S. aureus tar and tag gene orthologs in the selected S. aureus strains which would contribute to teichoic acids sythesis.Based on our identification of genes orthologous to tarI, tarJ, tarL, which are specific to tar pathway in B. subtilis W23, we also concluded that tar is the major teichoic acid biogenesis pathway in S. aureus. Further analyses indicated that the S. aureus tar genes, different from the divergon organization in B. subtilis, are organized into several clusters in cis. Most interesting, compared with genes in B. subtilis tar pathway, the S. aureus tar specific genes (tarI,J,L) are duplicated in all six S. aureus genomes.
In the S. aureus strains we analyzed, tar (teichoic acid ribitol) is the main teichoic acid biogenesis pathway. The tar genes are organized into several genomic groups in cis and the genes specific to tar (relative to tag): tarI, tarJ, tarL are duplicated. The genomic organization of the S. aureus tar pathway suggests their regulations are different when compared to B. subtilis tar or tag pathway, which are grouped in two operons in a divergon structure.
Staphylococcus. Aureus (S. aureus) is a Gram-positive bacterium, which causes a variety of suppurative infections and toxinoses in humans. The death rate associated with S. aureus infection is still high even with antimicrobial drug treatments due to the development of antibiotic resistance in Methicillin Resistant Staphylococcus Aureus (MRSA) strains. Current developments in antimicrobial therapeutics show little efficacy in treating S. aureus and this bacterium remains a major human health threat. S. aureus, and in particular its cell wall, remain a major target of glycopeptide antibiotics and focus of bacteriology research.
Teichoic acids, polymers of alternating phosphate and alditol groups, in addition to peptidoglycan are an essential component of bacterial cell walls. Teichoic acid biosynthesis in S. aureus has not been well characterized. B. subtilis and S. aureus are both phylogenetically classified into Bacillus/Staphylococcus group. Unlike that in S. aureus, the teichoic acid biogenesis in B. subtilis is well understood[1, 2]. There are two major types of cell wall teichoic acid in B. subtilis, poly(glycerol phosphate) [poly(GroP)] in strain 168  and poly(ribitol phosphate) [poly(RboP)] in strain W23 . In strain 168, the biosynthesis of teichoic acid poly(GroP) involves genes tagA, tagB, tagD, tagE, and tagF. These tag genes are organized in a divergon of two divergently transcribed operons, tagAB and tagDEF[4, 5]. In W23, the biosynthesis of teichoic acids poly(RboP) involves genes tarA, tarB, tarD, tarF, tarI, tarJ, tarK and tarL. The tar genes, similar to the tag genes, are also organized in a divergon, tagAB-tagDEF . The biogenesis of essential wall teichoic acid in B. subtilis differs in these two strains, yet still shares similar enzymatic steps. Functions of tagB, tagD, tagF were identified biochemically [1, 6]. Based on high sequence similarity, tarA, tarB, tarD, and tarF are believed to perform similar enzymatic reactions as tagA, tagB, tagD, and tagF do respectively , while tarI, tarJ, tarK and tarL carry functions specific to poly(RboP) teichoic acid biosynthesis .
It has been reported that S. aureus H contains ribitol in its cell wall as does B. subtilis W23 [7, 8]. It has also been shown that a TarD like enzyme exists in S. aureus with catalytic characteristics different from B. subtilis TagD . These studies suggest poly(RboP) could be one of the cell wall teichoic acids in some S. aureus strains, yet this has not been unequivocally demonstrated.
Recently the genomes of MRSA strains Mu50, MW2, N315, MRSA252, and COL as well as methicillin susceptible strain MSSA476 have been sequenced [9–12]. The available sequence information enables us to take a comparative genomics approach to study the genomic requirements of wall teichoic acid in S. aureus by comparing them to the genes involved in B. subtilis teichoic acid synthesis.
We took all B. subtilis tar and tag genes, computationally to identify all orthologous genes supposedly involved in wall teichoic acid biogenesis in the S. aureus strains Mu50, MW2, N315, MRSA252, MSSA476 and COL. Our results suggest that poly(RboP), rather than poly(GroP), is the major teichoic acid in these strains. We also report the genomic organization of the teichoic acid biogenesis genes, which is different from the divergon organization in B. subtilis W23 and 168.
Identify genes involved in wall teichoic acid synthesis in S. aureus through comparative genomics analysis
The BLAST homology between tar/tag genes and their orthologs in S. aureus strains. Newly identified TarB (named new_tarB) is also included. Orthologs of tagA/tarA are merged together. Same for tagD/tarD, tarF/tagF, tarB/tagB. Detail Blast Results are shown in Additional File 2 and annotations of these genes are shown in Additional file 3.
S. aureus contains tarF instead of tagF
In B. subtilis, despite sharing 60% identity, the size difference between TarF and TagF implies a functional difference. TagF and TarF both use CDP-glycerol as a substrate but do not carry out identical functions. In strain W23, TarF is likely to be responsible for the addition of the second glycerol-phosphate which completes the linkage unit process. While in 168, TagF polymerizes the complete glycerol-phosphate chain onto the first residue , which requires a bigger protein. Thus the size difference correlates with their functional difference between TagF and TarF. The size difference between TagF and TarF may also be used to differentiate tar or tag pathway in teichoic acid synthesis .
In the analyzed S. aureus strains, the size of tagF/tarF orthologs suggests the existence of TarF like function rather than TagF like function, and additional enzymes are required to complete teichoic acid synthesis.
S. aureus utilize tar pathway instead of tag pathway for teichoic acid biosynthesis
In B. subtilis strain W23, tarI, tarJ. tarK and tarL are specific to tar pathway, which are responsible for the synthesis of RboP and the addition of poly(RboP) to the linkage unit  and are absent in strain 168's tag pathway. The identification of tarI, tarJ and tarL orthologs (Figure 1 and Table 1) and the tarF kind of function (instead of that of tagF) strongly suggest that the tar rather than the tag pathway is employed for cell wall teichoic acid synthesis in analyzed S. aureus strains. In the rest of the paper, these S. aureus wall teichoic acid synthesis genes are all referred as tar genes (Tar prefix will be used to refer to their protein products).
There are two copies for each tar J, tarI and tarL gene in those S. aureus strains (Figure 1, Table 1 and Figure 2). Interestingly, the tarK ortholog is missing from the analyzed S. aureus strains. In W23, tarK and tarL are identified to catalyze a similar function, but tarL could take a bigger substrate enzymatically (poly(Rbop)) . Thus the absence of tarK ortholog in S. aureus could either mean it is functionally replaced by one of the two tarL or be compensated for by the extra copy of tarL.
Identifying the correct full length tarB in S. aureus Mu50 and N315
According to multiple sequence alignment analysis and BLAST analysis of B. subtilis TarB/TagB (protein) with the corresponding S. aureus orthologs, the TarB ORFs in Mu50 and N315 were found to be notably shorter than B. subtilis TarB/TagB. They were also shown to lack half of the amino acid residues from the N-terminal of B.subtilis TarB/TagB (Figure 3a). One would expect that the translation start site would be further upstream in both strains. The ORF prediction was thus rerun on this genomic region (see methods), and the correct tarB/tagB in Mu50 and N315 were identified (see Additional file 1). The correct tarB in N315 is from 687949 to 689052, producing an ORF of 367 amino acids; the correct tarB In Mu50 is located between 712200 and 713303, also encoding an ORF of 367 amino acids. Both two new TarB ORFs were subsequently confirmed by a TBLASTN analysis against these two genomes with B. subtilis TarB as query. The new TarB ORFs in Mu50 and N315 strains and their alignment with the original incorrectly predicted ones are shown in Figure 3a.
Genomic organization of tar genes and duplication of tarI, tarJ and tarL in S. aureus strains
The dot blot analysis also demonstrated that a small section of the C-terminal of tarJ is not very conserved in the two copies of tarJ. The enzymatic implication of this is not yet clear. Similarly, the N-terminal of tarL (almost half the size of tarL) is neither homologous between the two tarL genes. It implies that one of the tarL is very likely to be the missing tarK.
To understand the biosynthesis of cell wall teichoic acid in S. aureus strains Mu50, MW2, N315, MRSA252, MSSA476 and COL, we took a bioinformatics approach to perform a comparative genomics analysis., We used the B. subtilis teichoic acid synthesis pathway as the base for comparison and identified all the genes essential to teichoic acid synthesis in these six S. aureus strains. Besides tarA/tagA, tarB/tagB, and tarD/tagD like genes, we identified tarF rather than tagF like gene and tar specific genes tarI, tarJ and tarL. The latter three ones are duplicated in these S. aureus strains.
In B. subtilis, tarA, tarB, tarD and tarF in W23 are the most similar to their counterparts in strain 168: tagA, tagB, tagD and tagF , whose functions in wall techoic acid synthesis are well understood [1, 2]. Since tarF does not carry out a polymerization function as tagF does, W23 tar pathways requires tarI, tarJ, tarK and tarL to add poly(RboP) to the linkage unit to complete teichoic acid synthesis . The identification of tarF like function and tarI, tarJ, tarL like genes strongly support the fact that the analyzed S. aureus strains use a tar like pathway and poly(RboP) is their major cell wall teichoic acids. This conclusion is consistent with the observations that a TarD rather than TagD like catalytic mechanism presentin S. aureus  and the identification of ribitol teichioc acid in the cell wall of S. aureus H [7, 8].
Putative enzymatic functions of S. aureus Tar proteins
N-acetylglucosaminyldiphosphoundecaprenol N-acetyl-beta-D- mannosaminyltransferase.
Putative CDP-glycerol:glycerophosphate glycerophosphotransferase tarB
Putative Glycerol-3-phosphate cytidylyltransferase.
Putative CDP-glycerol:glycerophosphate glycerophosphotransferase tarF
Putative D-ribitol-5-phosphate cytidylyltransferase.
Putative ribitol-5-phosphate dehydrogenase
Putative CDP-ribitol ribitolphosphotransferase
The genomic organizations of tar genes in S. aureus are quite different from B. subtilis. They are organized into several clusters in cis rather than the divergon in B. subtilis, and may be subjected to different regulatory mechanisms.
As we analyzed, tar (teichoic acid ribitol) is the main teichoic acid biogenesis pathway in the S. aureus strains. And, the tar genes are organized into several genomic groups in cis and the genes specific to tar (relative to tag): tarI, tarJ, tarL are duplicated. The genomic organization of the S. aureus tar pathway suggests their regulations are different when compared to B. subtilis tar or tag pathway, which are grouped in two operons in a divergon structure.
Six bacteria strains are included in this analysis: Staphylococcus aureus Mu50, MW2, N315, MRSA252, MSSA476, COL and Bacillus subtilis 168. Their respective Refseq proteomes are downloaded from NCBI microbial genomes website . The complete geonome sequence of Bacillus subtilis W23 is not available yet. The Tar proteins in W23 (A, B, D, F, I, J, K, L) are also downloaded from NCBI microbial genomes website .
The eight Tar proteins and five Tag proteins (A, B, D, E, F) were used as queries to BLASTP against the proteomes (ORFs) of S. aureus Mu50, MW2, N315, MRSA252, MSSA476 and COL. For each querying protein, using E value cut off of 1e-10, we collected all the significant hits.
To define the identified possible Tar/Tag homoglous proteins in six S. aureus strains, we also took the above identified S. aureus proteins (ORFs) to BLAST back against proteome of B. subtilis 168. Mutual best BLAST hit is another evidence of the orthologous relationship.
Multiple sequence alignment and phylogenetic tree building
The above selected BLAST hits from the combined dataset plus the relevant Tar protein are taken to do ClustalW alignment. The multiple sequence alignment result is further taken as input to do phylogenetic analysis.
Mega3 package is used for phylogenetic analysis and tree building. First the ClustalW produced .aln files were transformed into Mega readable .meg files, followed by performing neighbour-joining phylogenetic analysis taking .meg files as inputs. The default Mega parameters were used when making NJ trees and performing bootstrap test (bootstrap replication time at 500).
Since Tar/TagB, Tar/TagF, TarL, and TarK share certain significant BLAST hits, we put them and their BLAST hits together to do ClustalW alignment and Mega analysis.
MUMmer alignment and dot plot analysis
MUMmer package V3.0 was downloaded from TIGR ftp sites , which was used to align between the B. subtilis 168 genome and each genome of the six S. aureus strains. Promer program was selected for its relatively high sensitivity. Promer generates amino acid alignments between two DNA input files which contain multiple sequences in FASTA format. Mummerplot was used to produce the dot-blot of the MUMmer alignments (Figure 2).
Identification of full length tarB in S. aureus Mu50 and N315
The TarB orthologs identified from S. aureus Mu50 and N315 Refseq proteomes are remarkably shorter than TarB in B. subtilis W23, MRSA252, MSSA476 and COL, missing the N-terminal part and only about half the size of W23 TarB. We applied NCBI's ORF finder program  to analyze the TarB regions of N315 and Mu50. Two extended ORFs were identified which were similar to W23 TarB in length. Alignment analysis confirmed they are TarB orthologs in these two S. aureus strains (Figure 3).
The ORF in N315 for TarB locates in the genome from 687949 to 689052, and ORF in Mu50 for TarB from 712200 to 713303.
The genomic organization of tar genes
The genomic localization information of Tar and Tag genes in B. subtilis 168, W23 and S. aureus strains Mu50, MW2, N315, MRSA252, MSSA476, COL were retrieved from NCBI microbial genomes website . Genomic organization maps of the five bacteria were then made (Figure 4, Additional file 4).
Analysis of tarIJL duplication in S. aureus strains
BLASTP and phylogenetic analysis identified two copies of TarI, J and L in S. aureus Mu50, N315, MW2, MRSA252, MSSA476 and COL. From genomic analysis, TarI, TarJ and TarL in the analyzed S. aureus strains, are clustered into two tarIJL regions. To confirm that it is caused by gene duplication, we further performed alignment and dot-blot analysis of those two tarIJL regions in each of the S. aureus strains. 
We first cut out the relevant genomic sequences of tarIJL regions, including the intergenic regions among I, J, K genes and upstream 275 bp. Then we used dotmatcher program from EMBOSS package to perform dot-blot analysis of the two tarIJL regions (Figure 5). We also ran NCBI bl2seq program to align the two regions (not shown).
We would like to thank Dave Figuora at Merck Research laboratories for English editing and discussion. We also wish to thank Dr. Qing Zhang at Schering Plough Research Institute for suggestions. This study was supported by Major Program of Shanghai Committee of Science and Technology (No. 04dz14004) and National 973 Key Research Program of China (No. 2001CB510209, 2002CB713807, 2003CB715900).
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