Sequence conservation and functional constraint on intergenic spacers in reduced genomes of the obligate symbiont...

Analyses of genome reduction in obligate bacterial symbionts typically focus on the removal and retention of protein-coding regions, which are subject to ongoing inactivation and deletion. However, these same forces operate on intergenic spacers (IGSs) and affect their contents, maintenance, and rates of evolution. IGSs comprise both non-coding, non-functional regions, including decaying pseudogenes at varying stages of recognizability, as well as functional elements, such as genes for sRNAs and regulatory control elements. The genomes of Buchnera and other small genome symbionts display biased nucleotide compositions and high rates of sequence evolution and contain few recognizable regulatory elements. However, IGS lengths are highly correlated across divergent Buchnera genomes, suggesting the presence of functional elements. To identify functional regions within the IGSs, we sequenced two Buchnera genomes (from aphid species Uroleucon ambrosiae and Acyrthosiphon kondoi) and applied a phylogenetic footprinting approach to alignments of orthologous IGSs from a total of eight Buchnera genomes corresponding to six aphid species. Inclusion of these new genomes allowed comparative analyses at intermediate levels of divergence, enabling the detection of both conserved elements and previously unrecognized pseudogenes. Analyses of these genomes revealed that 232 of 336 IGS alignments over 50 nucleotides in length displayed substantial sequence conservation. Conserved alignment blocks within these IGSs encompassed 88 Shine-Dalgarno sequences, 55 transcriptional terminators, 5 Sigma-32 binding sites, and 12 novel small RNAs. Although pseudogene formation, and thus IGS formation, are ongoing processes in these genomes, a large proportion of intergenic spacers contain functional sequences.