Identification of erythritol loci

The data set of erythritol loci utilized in this work was constructed in atwo-step process. First BLASTN was used to identify sequenced genomes containinghomologs to the core erythritol catabolic genes R. leguminosarum andS. meliloti[18]. The use of BLASTN rather than BLASTP at this stage allowed us torefine the search to bacteria with sequenced genomes. Furthermore, limiting thesearch to genes with highly similar sequences by using BLASTN allowed us tolimit our search to only genes that are likely involved in erythritolcatabolism, since all of these genes encode proteins in highly ubiquitousfamilies found throughout bacterial genomes. Initially BLASTN searches wereperformed using all the core erythritol genes shared between R.leguminosarum and S. meliloti (eryA, eryB, eryC anderyD). However, the search using eryA provided the mostdiverse data set that also showed a sharp drop in E-value and query coverage.Using either eryA from R. leguminosarum, or eryA fromS. meliloti for the BLASTN search resulted in an identical dataset. Genomes containing homologs to eryA were selected on the basis ofE-values less than 1.00E-5. In cases where multiple strains of the samebacterial species were found to have highly homologous putative erythritol genes(>99% identity) only a single representative of the species was used to avoidredundancy. Additionally B. melitensis 16M and B. suis 1330were chosen as representatives of the Brucella lineage despite a largenumber of Brucella species that were identified in our search due tothe high degrees of similarity between their erythritol catabolic genes.

Second, the genetic region containing eryA in these organisms wasidentified and analyzed using the IMG Ortholog Neighborhood Viewer(http://img.jgi.doe.gov) [19] in order to construct the gene maps (loci). The amino acid sequenceof EryA from S. meliloti was used as a query for the IMG OrthologNeighborhood Viewer search.

To analyze the genetic content of organisms in our data set, the amino acidsequence encoded by each gene involved in erythritol catabolism in R.leguminosarum, or in erythritol, adonitol or L-arabitol catabolism inS. meliloti, was individually used in a BLASTP search of the 19genomes in the data set. The sugar binding proteins of the S. melilotiand R. leguminosarum transporter were used as representatives of theentire ABC transporter. Identity cut-off values that were used to delineatepotential homologs to erythritol proteins were unique to each query amino acidsequence. Cut-off values were as follows: MptA: 56%, EryD: 44%, EryA: 46%, RbtA:50%, EryB: 65%, LalA: 49%, RbtB: 51%, RbtC: 40%, EryC: 68%, TpiB: 69%, EryR:61%, EryG: 73%. These values were manually determined and generally correlatedto a large drop in percentage identity within the BLASTP hits.

Homologs identified that were not within the primary eryA containingloci were used as a query within IMG-Ortholog neighborhood viewer to analyze theregion surrounding them. Secondary loci containing homologs to some of thesegenes were identified in Mesorhizobium sp. and Sinorhizobiumfredii. These loci are putative erythritol loci based on homology toknown loci involved in erythritol catabolism in Sinorhizobium meliloti[15, 16], Rhizobium leguminosarum[20]and Brucella abortus[21]. Despite not having been experimentally verified we will refer to allloci in our data set as erythritol loci for the purpose of this manuscript.

Phylogenetic analysis

Amino acid sequences of homologs to proteins previously shown to play a role inerythritol, adonitol or L-arabitol catabolism from each of the organisms in thedata set were collected and used for phylogenetic analysis. The 16SrDNA and RpoD sequences were also extracted from the NCBI databasefor species examined in this study in order to obtain a potential species treethat could be compared with the various phylogenetic gene trees obtained fromthe individual genes located within the polyol (i.e. erythritol, arabitol, andadonitol) utilization loci. Amino acid sequences were aligned using Clustal-X [22] and PRALINE [23] the resulting alignments were refined manually with the GeneDocprogram v2.5.010 [24].

Phylogenies were generated with maximum likelihood analysis (ML) as implementedin the Molecular Evolutionary Genetic Analysis package (MEGA5) [25] and with MrBayes [26]. MEGA5 was used to identify the most suitable substitution models forthe aligned data sets. In order to evaluate support for the nodes observed inthe ML phylogenetic trees bootstrap analysis [27] was conducted by analysing 1000 pseudo replicates.

The MrBayes program (v3.1) was used for Bayesian analysis [26, 28] and the parameters set for amino acid alignments were mixed modelsand for the 16S rDNA gamma distribution with 4 rate categories. The models used(setting mixed model) for generating the final 50% majority rule trees wereestimated by the program itself. The Bayesian inference of phylogenies wasinitiated from a random starting tree and four chains were run simultaneouslyfor 1 000 000 generations; trees were sampled every 100 generations. The first25% of trees generated were discarded (“burn-in”) and the remainingtrees were used to compute the posterior probability values.

Phylogenetic trees were constructed for RpoD, 16S rDNA and all the key genesassociated with the EryA genes. Phylogenetic trees were plotted with theTreeView program [29] using MEGA5 and/or MrBayes tree outfiles. Final trees were annotatedusing Adobe Illustrator.

Phylogenetic distribution of putative erythritol loci

Genetic content of loci

The genetic content of each of the organisms ery loci were analyzed byconducting a BLASTP search to the 19 genomes in our data set of the amino acidsequence of each gene associated with erythritol catabolism in R.leguminosarum, or erythritol, adonitol or L-arabitol catabolism inS. meliloti. The results of the BLAST search are presented inTable  2, depicting the presence or absence ofhomologs to erythritol, adonitol or L-arabitol catabolic genes in each of thegenomes that was investigated. Gene maps of erythritol loci were constructedbased on the output of our IMG Ortholog Neighborhood Viewer searches and aredepicted in Figure  1.

Genes encoding homologs to the core erythritol proteins EryA, EryB and EryD wereubiquitous throughout our data set (Table  2). Withrespect to the remaining genes, the genetic content of the species can begrouped into three broad categories. (1) Species that contain genes encodinghomologs associated with erythritol, adonitol and L-arabitol catabolism. Thisincludes S. meliloti, S. medicae, S. fredii, M. loti, M. opportunism, M.ciceri, R. denitrificans and R. litoralis. These genomescontained homologs to genes that encode enzymes specifically involved erythritolcatabolism such as EryC, and TpiB as well as specifically involved in adonitoland L-arabitol catabolism including LalA, and RbtBC. They also containgenes encoding an ABC transporter homologous to the S. melilotierythritol, adonitol and L-arabitol transporter (MptABCDE) and do not encodehomologs to the R. leguminosarum erythritol transporter (EryEFG). Onenotable exception is M. ciceri which encodes EryEFG homologs ratherthan MptABCDE (Table  2). (2) Species that containall the genes associated with erythritol catabolism, but lack the genesassociated with adonitol or L-arabitol catabolism. These species include R.leguminosarum bvs. viciae and trifolii, A.radiobacter, O. anthropi, B. suis, B. melitensis, and E.fergusonii. These loci encode EryABCDR-TpiB as well as homologs to theR. leguminosarum ABC transporter EryEFG, but lack genes encodinghomologs to enzymes associated specifically with adonitol and L-arabitolcatabolism or the S. meliloti transport protein MptABCDE. E.fergusonii contains the most minimal set of homologs to erythritolgenes of all the genomes investigated, and did not encode EryR and TpiB. (3)Species that do not encode the specifically erythritol associated EryC, EryR,and TpiB, but encode the adonitol/L-arabitol catabolic complement LalA-RbtABCand homologs to the S. meliloti polyol transporter MptABCDE. Theseinclude Bradyrhizobium spp. BTAi1 and ORS278, A. multivorum, A.cryptum and V. eiseniae.

The genetic structure of erythritol loci

The genetic context of eryA in each of the genomes in our data setsupported that each of these organisms contained an erythritol locus. A physicalmap of the loci in each of these organisms is depicted in Figure  1. Of note, a number of putative erythritol loci wereidentified in organisms with incomplete genome sequences at the time ofanalysis, and thus are not discussed here, including: Octadecabacterantarcticus, Pelagibaca bermudensis Enterobacter hormaechei,Fulvimarina pelagi, Aurantimonas sp. SI85-9A1, Roseibium sp.TrichSKD4, Burkholderia thailandensis and Stappia aggregata.

The putative erythritol loci of bacteria in our data set ranged in geneticcomplexity with the loci from S. meliloti and S. medicaecontaining 17 different genes, to the simplest being the locus of E.fergusonii, which contained only two divergently transcribed operonsthat are homologous to the eryEFG and eryABCD loci of R.leguminosarum. A number of species contained loci that were identicalin content and arrangement to the R. leguminosarum erythritol locusincluding members of the Brucella, Ochrobacterum, andAgrobacterium. The only species that contains a locus identical incontent and arrangement to S. meliloti is the closely relatedSinorhizobium medicae. The locus of Sinorhizobium frediiNGR234, contains all but one of the genes (fucA1) found in the otherSinorhizobium loci (Figure  2).

The loci of Mesorhizobium species were varied, however all threeMesorhizobium sp. contained an independent locus with homologs tolalA and rbtBC elsewhere in the genome (Figure  1). Interestingly, while Mesorhizobium loti andMesorhizobium opportunism both contain transporters homologous tomptABCDE, Mesorhizobium ciceri bv. biserrulaecontains a transporter homologous to eryEFG. This operon also containsthe same hypothetical gene that is found at the beginning of the R.leguminosarum eryEFG transcript. The transporters however, arearranged in a manner similar to that seen in S. meliloti and the geneencoding the regulator eryD, is found ahead of the transporter genes,whereas in R. leguminosarum and Brucella, eryD isfound following eryC (Figure  1). We alsonote that whereas M. loti and M. opportunism both contain aputative fructose 1,6 bis phosphate aldolase gene between theeryR-tpiB-rpiB operon and eryC, a homolog to this is alsogene is found adjacent to the rpiB in Brucella.

Bradyrhizobium sp. BTAi1, and ORS278, A. cryptum and V.eiseniae all have similar genetic arrangement to that of S.meliloti, except that they do not contain a homolog to eryC,or an associated eryR-tpiB-rpiB operon. These loci also differprimarily in their arrangement of lalA-rbtBC (Figure  1).

The phylogenies of erythritol proteins do not correlate with speciesphylogeny

The DNA sequences of 16S rDNA (data not shown) as well as the amino acidsequences of RpoD were extracted from GenBank to analyze the phylogeneticrelationships of the organisms examined in this study, using the mostphylogenetically distant organism Verminephrobacter eiseniae as anout-group. The results of the 16S rDNA and RpoD sequence analyses werein concordance with each other and are consistent with phylogenies that havebeen previously generated [42]. Initial comparison of the operon structures with the generatedphylogenies suggested that the operon structure(s) did not correlate with thespecies phylogeny. Since the structure of some operons did not correspond wellwith the species phylogenies we wished to determine if operon structure didcorrelate with any of the erythritol genes found at the S. melilotiloci. Since homologs to EryA, EryB and EryD were ubiquitous through the dataset, it was decided to construct phylogenies based on Maximum Likelihood andBayesian analysis using the EryA, EryB and EryD data sets. The topology of thephylogenetic tree using EryA is presented in Figure  2. A tree including branch lengths is included as Additional file1: Figure S1. V. eiseniae was also themost distant member with respect to the EryA phylogeny and again used as anoutgroup. The phylogenetic trees of EryB and EryD are not shown but weregenerally consistent with the EryA phylogeny. The species tree, based on RpoD,was included as a mirror tree with the EryA tree to demonstrate possiblehorizontal gene transfer events (Figure  2).

The data show that there is a high degree of correlation between the lociconfiguration and the EryA phylogenetic tree (Figure  1, 2). We note the similarity of the loci ofA. radiobacter and R. leguminosarum to Brucellaspecies and O. anthropi but not to the more closely relatedSinorhizobium species. This suggests that a horizontal genetransfer may have occurred between these organisms. This is in agreement withwhat has been previously reported [20]. It also seems likely that a horizontal gene transfer event may haveoccurred between the Brucella and E. fergusonii. This mayexplain the unique occurrence of the loci’s presence in a member of thegamma-proteobacteria. Finally, our mirror tree suggests that a horizontal genetransfer of the more complex erythritol locus may have occurred between M.loti and an ancestral species the Sinorhizobium species(Figure  2).

Modes of evolution for the polyol utilization loci

Comparison of the phylogenetic trees of EryA, EryB and EryD to the arrangementand content of the loci led us to more thoroughly investigate the phylogenies ofa number of proteins that stood out as unique within the data set. Thesephylogenies have led us to postulate modes of evolution that may have occurredin these loci.

BLASTP analysis showed a clear distinction between the type of transporterencoded by each of the loci and the remaining genetic content. In general, locithat contained adonitol/L-arabitol type genes contained a transporter homologousto the S. meliloti MptABCDE (Table  2,Figure  1). Loci that contained only erythritol genescontained a transporter homologous to the EryEFG of R. leguminosarum.One exception to this correlation was M. ciceri bv. biserrulaewhich contained a homologous transporter to EryEFG rather than MptABCDE. This isinteresting because M. ciceri groups with the otherMesorhizobia in the EryABD trees. In order to analyze the evolutionof these transporters more clearly, phylogenetic trees were constructed ofhomologs to EryG and homologs to MptA (Figure  3). Ingeneral the phylogenies are in agreement with the EryABD phylogenies, with theexception of M. ciceri which falls on a basal branch of the EryGphylogeny. The disparities between the EryG and EryABD phylogenies of Mciceri strongly suggest that parts of its erythritol locus have adifferent origin. This may have been the result of horizontal gene transfer of asecond R. leguminosarum type erythritol locus, followed byrecombination between the two.

In two organisms, apparent duplications of genes were present. In M.loti one homolog of lalA was present in the erythritol locus,while a second copy was present elsewhere in the genome adjacent to homologuesof rbtB and rbtC, consistent with its location in the othertwo Mesorhizobium genomes. In S. fredii homologs to theapparent small operon that contains eryR-tpiB-rpiB were found both, asexpected, in the erythritol locus, but also elsewhere on the chromosome in thesame arrangement. To analyze the evolutionary history of these duplicationsphylogenetic trees were constructed for the LalA and TpiB homologs (Figure 4 and 5). The two copies of thelalA gene in M. loti are most likely an example ofparalogs, as they still group within the same clade among other lalAhomologs (Figure  4). The tpiB genes(Figure  5) in S. fredii are possibleexamples of xenologs [43] as the phylogenetic tree shows that the two versions of thetpiB gene in S. fredii are only distantly related, withone homolog grouping within the expected clade that includes S. medicaeand S. meliloti and the second homolog (not part of the main locus)showing monophyly with those found in a clade containing R.leguminosarum sp., B. suis, etc. (Figure  5).