Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Background

The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results

Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions.

Conclusions

Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.     

Evolution of petaloid sepals independent of shifts in B-class MADS box gene expression

Attractive petals are an integral component of animal-pollinated flowers and in many flowering plant species are restricted to the second floral whorl. Interestingly, multiple times during angiosperm evolution, petaloid characteristics have expanded to adjacent floral whorls or to extra-floral organs. Here, we investigate developmental characteristics of petaloid sepals in Rhodochiton atrosanguineum, a close relative of the model species Antirrhinum majus (snapdragon). We undertook this in two ways, first using scanning electron microscopy we investigate the micromorphology of petals and sepals, followed by expression studies of genes usually responsible for the formation of petaloid structures. From our data, we conclude that R. atrosanguineum petaloid sepals lack micromorphological characteristics of petals and that petaloid sepals did not evolve through regulatory evolution of B-class MADS box genes, which have been shown to specify second whorl petal identity in a number of model flowering plant species including snapdragon. These data, in conjunction with other studies, suggests multiple convergent pathways for the evolution of showy sepals.     

Patterns of shoot architecture in locally adapted populations are linked to intraspecific differences in gene regulation

? Shoot architecture, including the number and location of branches, is a crucial aspect of plant function, morphological diversification, life history evolution and crop domestication. ? Genes controlling shoot architecture are well characterized in, and largely conserved across, model flowering plant species. The role of these genes in the evolution of morphological diversity in natural populations, however, has not been explored. ? We identify axillary meristem outgrowth as a primary driver of divergent branch number and life histories in two locally adapted populations of the monkeyflower, Mimulus guttatus. ? Furthermore, we show that MORE AXILLARY GROWTH (MAX) gene expression strongly correlates with natural variation in branch outgrowth in this species, linking modification of the MAX-dependent pathway to the evolutionary diversification of shoot architecture.     

Evolution of GCYC, a Gesneriaceae homolog of CYCLOIDEA, within Gesnerioideae (Gesneriaceae)

Through recent advances in molecular developmental biology it has become clear that similar morphological traits may sometimes arise from different genetic bases. The molecular developmental biology of floral symmetry has been examined recently in detail and several genes important in controlling floral symmetry in diverse Asteridae have been identified. One of the most important among these is the floral symmetry gene CYCLOIDEA (CYC). We compared GCYC (the Gesneriaceae homolog of CYC) sequences in Gesneriaceae genera with the typical bilaterally symmetric flowers and genera with radial or near radial symmetry. Parsimony, Bayesian and maximum likelihood analyses of GCYC sequences among members of Gesnerioideae are mostly congruent with previous phylogenetic hypotheses, but suggest two unexpected generic positions: Diastema as sister to Gesneria, and Bellonia within Gloxinieae. In order to evaluate whether these results might be artifactual we obtained new gene sequences from chloroplast and nuclear ribosomal regions. These data disagree with GCYC regarding the placement of Diastema, but agree with GCYC regarding Bellonia. We did not find any mutations in GCYC that could explain the shift in symmetry and there were no consistent differences in molecular evolution between taxa with bilaterally or radially symmetric flowers. Likewise taxa with radial floral symmetry are not sister to each other showing that the loss of bilateral symmetry has occurred multiple times in parallel. Further investigations of GCYC expression will be necessary to determine if any of these independent events involved changes in the regulation of GCYC.     

Functional analyses of genetic pathways controlling petal specification in poppy

MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved.     

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.