Heather Hendrickson

Lateral gene transfer: when will adolescence end?

The scope and impact of horizontal gene transfer (HGT) in Bacteria and Archaea has grown from a topic largely ignored by the microbiological community to a hot‐button issue gaining staunch supporters (on particular points of view) at a seemingly ever‐increasing rate. Opinions range from HGT being a phenomenon with minor impact on overall microbial evolution and diversification to HGT being so rampant as to obfuscate any opportunities for elucidating microbial evolution – especially organismal phylogeny – from sequence comparisons. This contentious issue has been fuelled by the influx of complete genome sequences, which has allowed for a more detailed examination of this question than previously afforded. We propose that the lack of common ground upon which to formulate consensus viewpoints probably stems from the absence of answers to four critical questions. If addressed, they could clarify concepts, reject tenuous speculation and solidify a robust foundation for the integration of HGT into a framework for long‐term microbial evolution, regardless of the intellectual camp in which you reside. Here, we examine these issues, why their answers shape the outcome of this debate and the progress being made to address them.

Amplification-mutagenesis: Evidence that ''directed'' adaptive mutation and general hypermutability result from growth with a selected gene amplification

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac+ revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac+ revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F′ plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events—amplification, mutation, segregation—may help to explain both the origins of some cancers and the evolution of new genes under selection.

Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores.

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites

In bacteria, Ter sites bound to Tus/Rtp proteins halt replication forks moving only in one direction, providing a convenient mechanism to terminate them once the chromosome had been replicated. Considering the importance of replication termination and its position as a checkpoint in cell division, the accumulated knowledge on these systems has not dispelled fundamental questions regarding its role in cell biology: why are there so many copies of Ter, why are they distributed over such a large portion of the chromosome, why is the tus gene not conserved among bacteria, and why do tus mutants lack measurable phenotypes? Here we examine bacterial genomes using bioinformatics techniques to identify the region(s) where DNA polymerase III‐mediated replication has historically been terminated. We find that in both Escherichia coli and Bacillus subtilis, changes in mutational bias patterns indicate that replication termination most likely occurs at or near the dif site. More importantly, there is no evidence from mutational bias signatures that replication forks originating at oriC have terminated at Ter sites. We propose that Ter sites participate in halting replication forks originating from DNA repair events, and not those originating at the chromosomal origin of replication.

Order and disorder during Escherichia coli divergence.

Escherichia coli is a single species with numerous recognized roles, from lab workhorse to beneficial intestinal commensal or deadly pathogen. The extant strains have disparate lifestyles as a result of differential niche expansion since their divergence 25–40 million years ago, ten times longer than the estimated divergence between chimpanzees and humans [1,2]. Not only do these roles vary by strain (variant) of the species, but the recognition of a strain’s role in one context does not exclude radically different behaviour in another, due to differential gene expression [3]. These are organisms adapting on evolutionary and lifetime scales to myriad environments and pressures. How do these strains differ from one another and what sustains their identification as a single species? To address these questions, Touchon et al. have completely sequenced and annotated six strains of E. coli while reannotating previously sequenced strains, as discussed in this issue of PLoS Genetics [4]. Comparative genomics analyses of 20 E. coli strains and one out-group provided insights into the contributions of horizontal gene transfer (HGT) and mutation on evolution in this species. In addition, the strains were tested in a mouse model to compare their virulence.

Complete Genome Sequences of Three Novel Pseudomonas fluorescens SBW25 Bacteriophages, Noxifer, Phabio, and Skulduggery

ABSTRACT Three novel bacteriophages, two of which are jumbophages, were isolated from compost in Auckland, New Zealand. Noxifer, Phabio, and Skulduggery are double-stranded DNA (dsDNA) phages with genome sizes of 278,136 bp (Noxifer), 309,157 bp (Phabio), and 62,978 bp (Skulduggery).

Experimental Evolution of Cell Shape in Bacteria

Cell shape is a fundamental property in bacterial kingdom. MreB is a protein that determines rod-like shape, and its deletion is generally lethal. Here, we deleted the mreB homolog from rod-shaped bacterium Pseudomonas fluorescens SBW25 and found that ΔmreB cells are viable, spherical cells with a 20% reduction in competitive fitness and high variability in cell size. We show that cell death, correlated with increased levels of elongation asymmetry between sister cells, accounts for the large fitness reduction. After a thousand generations in rich media, the fitness of evolved ΔmreB lines was restored to ancestral levels and cells regained symmetry and ancestral size, while maintaining spherical shape. Using population sequencing, we identified pbp1A, coding for a protein involved in cell wall synthesis, as the primary target for compensatory mutations of the ΔmreB genotype. Our findings suggest that reducing elongasome associated PBPs aids in the production of symmetric cells when MreB is absent.

Chromosome architecture constrains horizontal gene transfer in bacteria

Despite significant frequencies of lateral gene transfer between species, higher taxonomic groups of bacteria show ecological and phenotypic cohesion. This suggests that barriers prevent panmictic dissemination of genes via lateral gene transfer. We have proposed that most bacterial genomes have a functional architecture imposed by Architecture IMparting Sequences (AIMS). AIMS are defined as 8 base pair sequences preferentially abundant on leading strands, whose abundance and strand-bias are positively correlated with proximity to the replication terminus. We determined that inversions whose endpoints lie within a single chromosome arm, which would reverse the polarity of AIMS in the inverted region, are both shorter and less frequent near the replication terminus. This distribution is consistent with the increased selection on AIMS function in this region, thus constraining DNA rearrangement. To test the hypothesis that AIMS also constrain DNA transfer between genomes, AIMS were identified in genomes while ignoring atypical, potentially laterally-transferred genes. The strand-bias of AIMS within recently acquired genes was negatively correlated with the distance of those genes from their genome’s replication terminus. This suggests that selection for AIMS function prevents the acquisition of genes whose AIMS are not found predominantly in the permissive orientation. This constraint has led to the loss of at least 18% of genes acquired by transfer in the terminus-proximal region. We used completely sequenced genomes to produce a predictive road map of paths of expected horizontal gene transfer between species based on AIMS compatibility between donor and recipient genomes. These results support a model whereby organisms retain introgressed genes only if the benefits conferred by their encoded functions outweigh the detriments incurred by the presence of foreign DNA lacking genome-wide architectural information.

Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih.

ABSTRACT Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc2155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.