Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy

Abstract

Background: The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis.

Results: In 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions. A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource. The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway. Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis. Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently).

Conclusions: (i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing. There are currently seven tryptophan congruency groups in the Bacteria. Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer). (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced. The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis. Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Figure 1

Positioning of 47 complete-genome organisms on a 16S rRNA tree (phylogram view). Among these, seven 16S rRNA subtree regions are color-coded to facilitate comparison with the TCG regions on the Trp-protein concatenate tree of Fig. 2. Dashed lines in orange indicate organisms representing lineages where genomes of close relatives have not yet been sequenced. A grey box indicates a region of minimal current genome representation that is expected to become a region of subtree congruency. The subdivisions of the Proteobacteria are labelled at the lower right. Note that it is an idiosyncracy of tree presentation that Tfe and Xfa appear to group more closely with beta-proteobacteria than with other gamma-proteobacteria. However, it is a distance tree and close inspection of the distances reveal the identity of the gamma-proteobacteria as a single, cohesive group. The horizontal bar corresponds to 0.1 substitutions per site on the distance tree.

Figure 2

Phylogenetic tree (radial view) constructed using the seven-domain (TrpAa/Ab/B/C/D/Ea/Eb) concatenated sequences of Trp proteins that specifically participate in primary biosynthesis. The Trp-pathway concatenates are from the same 47 organisms shown in Fig. 1. Dashed, orange lines indicate the lineage positions of concatenated sequences from organisms that presently lack any close relatives whose genomes have been sequenced. TCG clusters are color-coded as in Fig. 1, where full bacterial names matching the corresponding abbreviations can be found. The Cje lineage, marked in aqua, represents a probable TCG grouping. Nodes marked with solid black circles are supported by bootstrap values of 100%. Concatenates of LGT origin within TCG-1 are outlined with a grey pattern.

Figure 3

Phylogenetic tree (phylogram view) of TrpAa sequences. Nodes occupied by a solid, black circle are supported by bootstrap values of 100%. Other bootstrap values are given within unfilled circles. Xenologs are shown with grey candy-striped bars, and paralogs are shown with lavender/white patterning. A specialized-pathway paralog from Streptomyces coelicolor (Sco_CDA) is shown, as well as a probable specialized-pathway xenolog (Xfa_2) from Xylella [7]. Genes in C. diptheriae, C. glutamicum, and H. pylori that belong to whole-pathway operons originating via LGT are shown within TCG-1. TCG-1 also includes PhnA, encoded by a gene of a partial-pathway operon from P. aeruginosa. TCG-7 is fragmented, the Ban and Sau TrpAa sequences being too divergent to fall within TCG-7.

Figure 4

Phylogenetic tree (radial view) showing that free-standing TrpAa domains, TrpAa components of TrpAa•TrpAb fusions, and TrpAa components of TrpAa•TrpAb_phz fusions are all distinct from one another. The position of some TCG groups are marked at the top. TrpA• fusion domains are color-coded to indicate their expected TCG placements if convergent evolution were not a factor. The TCG-1 grouping is distinctly divergent from all of the other free-standing TrpAa sequences.

Figure 5

Phylogenetic tree of TrpAb sequences. Xfa_2 is a probable specialized-pathway xenolog from Xylella. Sco_CDA is a specialized-pathway paralog within TCG-5. TCG-1 contains a xenolog (PhnB) from Pseudomonas aeruginosa that (together with PhnA) is encoded by genes of a partial-pathway operon. Trp Ab genes from Helicobacter pylori, Corynebacterium diptheriae, and C. glutamicum are xenolog members of whole-pathway operons. TCG-2 and TCG-7 are fragmented. (Consult Fig. 2 for intact TCGs.) Asterisks (Tma, Cje, and Eco) indicate domains that exist as part of TrpAb•TrpB fusions.

Figure 6

Phylogenetic tree of TrpB sequences. Det_2 is diagrammed with both patterns, indicating it could be either an ancient paralog or a xenolog. Sco_CDA is a specialized-pathway paralog (antibiotic). Asp, Asp_2, Npu, and Npu_2 are paralogs from a gene duplication that preceded speciation of Npu and Asp. The Asp and Npu sequences were arbitrarily used for input into the concatenate tree of Fig. 2. TCG-7 is fragmented. TCG-1 contains xenolog members of whole-pathway operons from H. pylori, C. diptheriae, and C. glutamicum. Asterisks (Tma, Cje, and Eco) indicate domains existing as part of TrpAb•TrpB fusions.

Figure 7

Phylogenetic tree of TrpC sequences. TCG-1 contains xenolog members of whole-pathway operons from H. pylori, C. glutamicum and C. diptheriae. TCG-7 is fragmented. All members of TCG-1 shown (except Cje, whose position is probably coincidental) possess •TrpC as a fusion domain with TrpD•.

Figure 8

Phylogenetic tree of HisA sequences. Congruency groupings that match TCG clusters are labelled 'HCG' for histidine congruency group. Helicobacter pylori and Streptococcus pneumoniae are not represented on the tree because they have lost the histidine pathway. C. jejuni HisA appears to be a xenologous member of TCG-1. Similar to the relatively loose TCG-2 and TCG-7 groupings, HCG-2 and HCG-8 are fragmented. Actinomycete bacteria possess a tightly clustered section of dual-pathway HisA (PriA) sequences within HCG-5.

Figure 9

Phylogenetic tree of TrpD sequences. N. punctiforme and Anabaena sp. each possess a set of three paralogs. The Asp_2 and Npu_2 paralog sequences were used for input into the concatenates of Fig. 2. In addition to the xenolog TrpD• domain of Cgl and Cdi that is present in TCG-1, Cgl and Cdi also possess 'remnant' TrpD proteins denoted Cgl_r and Cdi_r that cluster in TCG-5.

Figure 10

Phylogenetic tree of TrpEa sequences. TrpEa proteins from Pae and Psy fall into TCG-3 instead of into TCG-2. Paralogs Asp_1, Npu_1, Asp_2 and Npu_2 were generated by a gene duplication that preceded speciation of Asp and Npu. Asp_2 and Npu_2 were arbitrarily used for concatenate input (Fig. 2). TCG-7 is not very cohesive.

Figure 11

Phylogenetic tree of TrpEb sequences. Cdi_2 is encoded by a paralog copy of trpEb_1 that has been inserted ahead of the Cdi trp operon. TrpEb_2 is encoded by a highly divergent paralog subclass of trpEb that probably has a specialized function [28]. No TrpEb_2 sequences were used to construct concatenates (Fig. 2). Rsp_2 is shown with two patterns, indicating uncertainty about whether it is an ancient paralog or a xenolog.

Figure 12

Schematic portrayal of two whole-pathway trp operon transfers and two partial-pathway trp operon transfers. The partial tree shown is taken from Fig. 1, which identifies the organisms. In C. diptheriae, the vertical evolutionary events of gene duplication and insertion that occurred following LGT can be visualized by comparing the gene organization shown to the right (including intergenic spacing) with the gene organization originally received from the enteric donor (bottom right). Gene insertions that followed LGT are shown in white.

Figure 13

Comparison of trpL leader regions in species of coryneform bacteria and in a representative enteric bacterium. In the coryneform bacteria, the start codon for trpL is uncertain, and second start sites at an internal position for C. glutamicum and C. efficiens are indicated by black, vertical arrows.