Origin of an alternative genetic code in the extremely small and GC-rich genome of a bacterial symbiont

Abstract

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop-->Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an alpha-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC-biased base composition (58.4%), and a coding reassignment of UGA Stop-->Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.

Figure 1. Relationship between genome size and GC content for sequenced Bacterial and Archaeal genomes.

Obligately intracellular insect symbionts are shown as red circles, obligately intracellular α-Proteobacteria as dark blue circles, Hodgkinia as a purple circle (as it is both an obligately intracellular α-Proteobacteria and an insect symbiont), and all other α-Proteobacteria as light blue circles. Most other Bacteria and Archaea are represented by small gray circles, although some have been removed for clarity, and the plot is truncated at 10 Mb.

Figure 2. Sulcia (green) and Hodgkinia (red) both have large tubular cell morphologies and are closely associated within the same bacteriocytes.

Scale bar is 10 µm.

Figure 3. Conserved positions encoded by UGA in Hodgkinia correspond to tryptophan (W) in other Proteobacteria.

M. loti (Mloti), C. crescentus (Ccres), P. denitricans (Pdeni), R. rubrum (Rrubr), E. litoralis (Elito), P. ubique (Pubiq), and R. rickettsii (Rrick) are all α-Proteobacteria; E. coli (Ecoli), γ-Proteobacteria; N. meningitidis (Nmeni), β-Proteobacteria; and G. metallireducens (Gmeta), δ-Proteobacteria. Partial sequences from the proteins DnaE (DNA polymerase III, α subunit), RpoB (RNA polymerase, β subunit), and RpoC (RNA polymerase, β′ subunit) are shown; the positions indicated at the top of the alignments are from the Hodgkinia proteins.

Figure 4. Relationship of Hodgkinia to other α-Proteobacteria based on small subunit ribosomal DNA sequences.

By itself, this maximum likelihood tree gives moderate support (81/100 bootstrap trees) for the grouping of Hodgkinia with the Rhizobiales. The twenty highest scoring positions for the Hodgkinia clade under a non-homogenous GC content model are indicated with black circles, and provide additional support for Hodgkinia's grouping in the Rhizobiales. Abbreviations are Mcas, Magicicada cassini; Dswa, Diceroprocta swalei; and Dsem, Diceroprocta semicincta. Asterisks indicate 100% bootstrap support; values less than 70% are not shown. Scale bar denotes substitutions per site.

Figure 5. Relationship of Hodgkinia to other α-Proteobacteria based on protein sequences.

Shown is a maximum likelihood tree based on an alignment of DnaE (DNA polymerase III, α subunit). This tree strongly supports (97/100 bootstrap trees) the grouping of Hodgkinia within the Rhizobiales. Asterisks indicate 100% bootstrap support; values less than 70% are not shown. Scale bar denotes substitutions per site.

Figure 6. The count for all sense codons in the Hodgkinia genome covered by a peptide in the proteomic analysis.

All sense codons were covered at least once. Codons in yellow are known to have undergone a recoding or been completely lost in other genomes but were shown here to be present and follow the universal code in Hodgkinia. The recoded UGA codon is colored in blue.

Figure 7. Gene order analysis shows that Hodgkinia is not within the Rickettsiales.

Homologous individual genes in the trnW-fusA block (as ordered in Hodgkinia) are color-coded to highlight differences in gene order; genes in the tufA-rplN block (as ordered in Hodgkinia) are all colored pink as there are no gene order changes in this set of genes. Unrelated gene insertions are indicated with unlabeled lightly shaded boxes. Grey lines link up homologous genes. The S10 gene is indicated at the top of the figure. Genomic positions are indicated with black numbers; note that in Rickettsiales the trnW-fusA and tufA-rplN gene blocks are not contiguous on the genome. The gene order of Hodgkinia is compatible with the Rhizobiales and Rhodobacteraceae (with some gene loss in Hodgkinia), but not with Rickettsiales. Additional sequenced Rhizobiales (Brucella melitensis 16 M), Rhodobacteraceae (Jannaschia sp. CCS1) and Rickettsiales (Wolbachia endosymbiont of Drosophila melanogaster, Ehrlichia canis str. Jake, and Anaplasma marginale str. St. Maries) were examined; only one is depicted as the representative gene order for these groups.

Figure 8. Model showing the mechanism of the UGA Stop→Trp recoding in the Hodgkinia genome.

The asterisks refers to a tRNA that is identical in anticodon sequence to the canonical version but underwent a distal mutation which produced a structural change allowing A-C mismatches at the indicated position. Evidence suggesting that UGG codons are being changed to UGA codons comes from the Hodgkinia coding regions: of the 701 tryptophans in Hodgkinia proteins, almost half (48%) are coded by UGA.