Lateral gene transfer and ancient paralogy of operons containing redundant copies of tryptophan-pathway genes in Xylella species and in heterocystous cyanobacteria

Abstract

Background: Tryptophan-pathway genes that exist within an apparent operon-like organization were evaluated as examples of multi-genic genomic regions that contain phylogenetically incongruous genes and coexist with genes outside the operon that are congruous. A seven-gene cluster in Xylella fastidiosa includes genes encoding the two subunits of anthranilate synthase, an aryl-CoA synthetase, and trpR. A second gene block, present in the Anabaena/Nostoc lineage, but not in other cyanobacteria, contains a near-complete tryptophan operon nested within an apparent supraoperon containing other aromatic-pathway genes.

Results: The gene block in X. fastidiosa exhibits a sharply delineated low-GC content. This, as well as bias of codon usage and 3:1 dinucleotide analysis, strongly implicates lateral gene transfer (LGT). In contrast, parametric studies and protein tree phylogenies did not support the origination of the Anabaena/Nostoc gene block by LGT.

Conclusions: Judging from the apparent minimal amelioration, the low-GC gene block in X. fastidiosa probably originated by LGT at a relatively recent time. The surprising inability to pinpoint a donor lineage still leaves room for alternative, albeit less likely, explanations other than LGT. On the other hand, the large Anabaena/Nostoc gene block does not seem to have arisen by LGT. We suggest that the contemporary Anabaena/Nostoc array of divergent paralogs represents an ancient ancestral state of paralog divergence, with extensive streamlining by gene loss occurring in the lineage of descent representing other (unicellular) cyanobacteria.

Figure 1

Protein tree for TrpR. Bootstrap values are shown at internal branch positions as percentages (1,000 replicates).

Figure 2

Genomic organization of aromatic-pathway genes in cyanobacteria. Genes relevant to the common pathway segment, the tryptophan branch, the tyrosine branch, and the phenylalanine branch are color-coded, as indicated. A system for uniform genomic naming of Trp-pathway genes or domains has been used as previously implemented [23,57]. Fused catalytic domains are joined by solid black linkers. Gene positions along the entire chromosomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 are shown. The qualitative presence or absence of genes in Nostoc punctiforme, an unfinished genome, is also indicated. Detailed zoom-in schematics are shown for the gene organizations within the supraoperons of Anabaena and Nostoc, regions spanning 13,000-14,000 bp. In the latter regions, intergenic spacing is shown, with negative values indicating the extent of genic overlap.

Figure 3

Fitch diagram [29] illustrating the origin and distribution of ortholog and paralogs of trpD in cyanobacteria. Paralogs, originating by gene duplication events (Dp1 and Dp2), track back to a horizontal line, whereas orthologs, originating by speciation (Sp1, Sp2, Sp3 and Sp4), track back to an inverted Y. The six trpD genes of Nostoc (Npu) and Anabaena (Asp) comprise a paralog set, and each of those comprises a four-member ortholog set with respect to the trpD genes from P. marinus (Pmu), Synechococcus sp. (Syn), and Synechocystis sp. (Ssp).

Figure 4

Block of genes acquired by lateral gene transfer (LGT) in Xylella fastidiosa. The gene map at the top shows the LGT block of genes with a green bar. The gene block begins with trpAa on the left and ends with trpR on the right. Intergenic spacing is given. The vertical pale green bar in the lower panel shows the corresponding genes from bottom to top. The GC% for each gene is shown, and the gene products are named. The hypothetical protein belongs to pfam00583, the acetyltransferase (GNAT) family. The low-GC gene block of the X. fastidiosa genome corresponds to gene numbers XF1914 (trpAa)-XF1920 (trpR).

Figure 5

Three-to-one dinucleotide analysis of the putative LGT-block of X. fastidiosa genes shown in Figure 4. For easier viewing, four of the 16 dinucleotide combinations have been selected. The frequency variation of each gene is shown as positive variation (upward-pointing bars) or negative variation (downward-pointing bars) with respect to the average genomic frequencies (set to a value of zero at the midline), the absolute values of which can be seen in Table 1. treg, transcriptional regulator; hypo, hypothetical gene.

Figure 6

Organization of trp-pathway genes in X. fastidiosa and its nearest phylogenetic neighbors. The position of the organisms indicated on a 16S rRNA subtree is shown at the far left. To enhance the presentation, the trp-gene acronyms are shortened. Thus, trpAa is shown as Aa, etc. Intergenic spacing is indicated. dmt refers to a putative DNA methyltransferase. TrpAa in Nitrosomonas europeae and trpC in Bordetella parapertussis are located in other chromosomal positions, unlinked to other trp-pathway genes. X. fastidiosa and N. europeae, but not the other organisms shown in the figure, possess truA (encoding tRNA pseudouridine synthase A) upstream of trpC. truA and trpC are translationally coupled with 31-bp and 105-bp overlaps in X. fastidiosa and N. europeae, respectively. The gene organizations shown for a given organism is identical to the other organisms shown in parentheses as follows: Ralstonia metallidurans (R. solanacearum), Burkholderia fungorum (B. pseudomallei, B. mallei), and B. parapertussis (B. pertussis, B. bronchiseptica). R. solanacearum, in addition to the genes shown, has adjacent paralogs of trpB and trpD located on a large plasmid. The trpAaAbBD and trpCEbEa operons of the X. fastidiosa 9a5c genome correspond to gene numbers XF0210-XF0213 and XF1374-XF1376, respectively.

Figure 7

Three-to-one dinucleotide analysis. (a) The aroA_Iβ-tyrAc gene block in Anabaena. Deviations from genomic frequencies are expressed as positive (upward-pointing bars) or negative (downward-pointing bars) percentages. (b) For comparison, the results obtained for the low-GC gene block of X. fastidiosa (of which Figure 4 is a subset). The gene blocks of interest are highlighted in yellow, and the flanking genes are indicated by numbers.

Figure 8

Codon usage for the pairs of TrpAa domains in the genomes of Anabaena sp. (Asp) and Xylella fastidiosa (Xfa). (a) Leucine; (b) serine; (c) arginine; (d) glycine; (e) valine and (f) proline. From left-to-right, Xfa TrpAa_1 is encoded from the low-GC gene block (In) and Xfa TrpAa_2 is encoded from outside (Out) the gene block; Asp TrpAa_1 is encoded from within the aroA_IβtyrA_(p) gene block (In) and Asp TrpAa_2 is encoded from outside (Out) the latter gene block. Synonymous codons are shown at the right of each amino acid set and color-coded to match the percent usage indicated by the bars.

Figure 9

16S rRNA tree showing the phylogenetic distribution (highlighted in yellow) of trpAa•trpAb fusions. The gene fusions unlinked to any other trp genes are shown to the right of the highlighted name. The remaining trp-operon gene organizations are shown at the right. The white arrows indicate gene insertions that encode the following: Thermomonospora, integral membrane protein; Streptomyces, three membrane proteins: Corynebacterium, membrane protein, pantoate 3-alanine ligase (panC), and 3-methyl-2-oxobutanoate hydroxymethyl transferase (panB); Mycobacterium, conserved hypothetical protein; Cytophaga, conserved hypothetical protein; Sphingomonas, conserved hypothetical protein and outer-membrane protein; Rhodobacter, and acetyltransferase yibQ; Ralstonia, DNA methyltransferase (dmt); Burkholdaria, DNA methyltransferase (dmt). In addition aroR in R. sphaeroides is a putative regulatory gene [58]. The lineage relationships of three organisms that have maintained the putative ancestral trp operon are shown with heavy, gray lines.

Figure 10

Comparison of TrpAa•TrpAb linker regions. The seven independent fusions that are suggested were aligned with free-standing TrpAa and TrpAb proteins in order to visualize the inter-domain linker regions. Amino-acid residue numbering is indicated at the left and right margins.