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Transformation is an important mechanism of microbial evolution through which bacteria have been observed to rapidly adapt in response to clinical interventions; examples include facilitating vaccine evasion and the development of penicillin resistance in the major respiratory pathogen Streptococcus pneumoniae. To characterise the process in detail, the genomes of 124 S. pneumoniae isolates produced through in vitro transformation were sequenced and recombination events detected. Those recombinations importing the selected marker were independent of unselected events elsewhere in the genome, the positions of which were not significantly affected by local sequence similarity between donor and recipient or mismatch repair processes. However, both types of recombinations were sometimes mosaic, with multiple non-contiguous segments originating from the same molecule of donor DNA. The lengths of the unselected events were exponentially distributed with a mean of 2.3 kb, implying that recombinations are stochastically resolved with a fixed per base probability of 4.4×10−4 bp−1. This distribution of recombination sizes, coupled with an observed under representation of large insertions within transferred sequence, suggests transformation has the potential to reduce the size of bacterial genomes, and is unlikely to act as an efficient mechanism for the uptake of accessory genomic loci., Author Summary Transformation is the process by which cells take up DNA from the environment and integrate it into their genome. It was first observed in the bacterium Streptococcus pneumoniae, a common cause of pneumonia and meningitis. This ability has allowed S. pneumoniae to evolve resistance to penicillin and to change its surface antigens to evade vaccines. To characterise this process in detail, we transformed an S. pneumoniae strain in the laboratory and sequenced the genomes of the resultant mutants. This showed that multiple recombinations, arising from different molecules of imported DNA, could occur around the genome at the same time. Some individual imported molecules donated multiple segments of sequence into the recipient. The positions of the recombinations were not significantly affected by the level of sequence similarity between donor and recipient, as had previously been observed for transfers between species, or by the mismatch repair process, which had previously been found to inhibit the transfer of small numbers of mutations. The recombinations' lengths were exponentially distributed with a mean length of 2.3 kb. This implies that the smallest version of a genetic locus will become the most common in the bacterial population in the absence of selection for a longer alternative.

The development of next-generation sequencing platforms is set to reveal an unprecedented level of detail on short-term molecular evolutionary processes in bacteria. Here we re-analyse genome-wide single nucleotide polymorphism (SNP) datasets for recently emerged clones of methicillin resistant Staphylococcus aureus (MRSA) and Clostridium difficile. We note a highly significant enrichment of synonymous SNPs in those genes which have been affected by recombination, i.e. those genes on mobile elements designated “non-core” (in the case of S. aureus), or those core genes which have been affected by homologous replacements (S. aureus and C. difficile). This observation suggests that the previously documented decrease in dN/dS over time in bacteria applies not only to genomes of differing levels of divergence overall, but also to horizontally acquired genes of differing levels of divergence within a single genome. We also consider the role of increased drift acting on recently emerged, highly specialised clones, and the impact of recombination on selection at linked sites. This work has implications for a wide range of genomic analyses., Author Summary As bacteria diversify, many of the nucleotide changes that emerge will render the cell slightly less competitive, and these mutations will tend to be removed by natural selection. However, this purging process does not happen instantaneously, and this delay allows deleterious mutations to survive in the population long enough to be sampled. Genomes at the very initial stages of diversification therefore exhibit a relatively high proportion of slightly deleterious mutations and, as most of these are non-synonymous mutations, this is manifest as a high dN/dS ratio. However, the effective population size will also impact on this ratio, as will selection operating on neighbouring SNPs. Here we examine the distribution of synonymous and non-synonymous SNPs within recently emerged clones of two important nosocomial pathogens, methicillin resistant Staphylococcus aureus (MRSA) and Clostridium difficile. In both species, we note a much higher proportion of synonymous changes in those single nucleotide polymorphisms (SNPs) likely to have emerged through recombination compared to de novo mutations. We argue that this effect is explained by the very recent emergence of the mutational SNPs combined with a reduction in the efficiency of selection due to niche specialisation.

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology., Author Summary In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called α or β, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis. The Giardia enzyme is unique among eukaryotes because it has undergone reductive evolution to function efficiently as a single β-subunit monomer. We also show that the recent claim that the equivalent enzyme in the hydrogenosome, another type of reduced mitochondrion of the human parasite Trichomonas vaginalis, functions as a homodimer of two β-subunits, is not supported. The Trichomonas enzyme requires both an α- and a β-subunit to function most efficiently. Computational analysis of the Giardia and Trichomonas enzymes reveals that their structures and surface charge distributions have co-evolved to match the peculiar properties of the targeting signals that they process. The Giardia mitosome is an ideal model for studying the limits of mitochondrial reductive evolution and, because it makes cofactors that are essential for Giardia survival, is a potential therapeutic target for this important human parasite.