A comprehensive genetic characterization of bacterial motility.

We have developed a powerful experimental framework that combines competitive selection and microarray-based genetic footprinting to comprehensively reveal the genetic basis of bacterial behaviors. Application of this method to Escherichia coli motility identifies 95% of the known flagellar and chemotaxis genes, and reveals three dozen novel loci that, to varying degrees and through diverse mechanisms, affect motility. To probe the network context in which these genes function, we developed a method that uncovers genome-wide epistatic interactions through comprehensive analyses of double-mutant phenotypes. This allows us to place the novel genes within the context of signaling and regulatory networks, including the Rcs phosphorelay pathway and the cyclic di-GMP second-messenger system. This unifying framework enables sensitive and comprehensive genetic characterization of complex behaviors across the microbial biosphere.

High diversity of vegetative compatibility types in Cryphonectria parasitica in Japan and China.

We found high diversity of vegetative compatibility (vc) types in native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China; almost every isolate was in a unique vc type. In Japan we found 71 vc types in a sample of 79 isolates pooled from six populations. Within two populations in China, all isolates (n=28 and 11) had unique vc types; we found 15 vc types among 25 isolates in a third Chinese population where multiple isolates were collected from some trees. None of the isolates from China and only three isolates in the 71 vc types from Japan were compatible with any of 64 vc type testers from Europe, which have known vegetative incompatibility genotypes. To our knowledge this is the first report of vc type diversity for C. parasitica in Japan or of any comparisons of vc types between Asia and Europe. The most significant result of this survey is the identification fungal isolates for expanding knowledge of the genetics of vegetative incompatibility.

Recombination and migration of Cryphonectria hypovirus 1 as inferred from gene genealogies and the coalescent.

Genealogy-based methods were used to estimate migration of the fungal virus Cryphonectria hypovirus 1 between vegetative compatibility types of the host fungus, Cryphonectria parasitica, as a means of estimating horizontal transmission within two host populations. Vegetative incompatibility is a self/non-self recognition system that inhibits virus transmission under laboratory conditions but its effect on transmission in nature has not been clearly demonstrated. Recombination within and among different loci in the virus genome restricted the genealogical analyses to haplotypes with common mutation and recombinational histories. The existence of recombination necessitated that we also use genealogical approaches that can take advantage of both the mutation and recombinational histories of the sample. Virus migration between populations was significantly restricted. In contrast, estimates of migration between vegetative compatibility types were relatively high within populations despite previous evidence that transmission in the laboratory was restricted. The discordance between laboratory estimates and migration estimates from natural populations highlights the challenges in estimating pathogen transmission rates. Genealogical analyses inferred migration patterns throughout the entire coalescent history of one viral region in natural populations and not just recent patterns of migration or laboratory transmission. This application of genealogical analyses provides markedly stronger inferences on overall transmission rates than laboratory estimates do.

Predictive behavior within microbial genetic networks.

The homeostatic framework has dominated our understanding of cellular physiology. We question whether homeostasis alone adequately explains microbial responses to environmental stimuli, and explore the capacity of intracellular networks for predictive behavior in a fashion similar to metazoan nervous systems. We show that in silico biochemical networks, evolving randomly under precisely defined complex habitats, capture the dynamical, multidimensional structure of diverse environments by forming internal representations that allow prediction of environmental change. We provide evidence for such anticipatory behavior by revealing striking correlations of Escherichia coli transcriptional responses to temperature and oxygen perturbations-precisely mirroring the covariation of these parameters upon transitions between the outside world and the mammalian gastrointestinal tract. We further show that these internal correlations reflect a true associative learning paradigm, because they show rapid decoupling upon exposure to novel environments.

Diversity of viruses in Cryphonectria parasitica and C. nitschkei in Japan and China, and partial characterization of a new chrysovirus species.

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.

Whole-genome discovery of transcription factor binding sites by network-level conservation.

Comprehensive identification of DNA cis-regulatory elements is crucial for a predictive understanding of transcriptional network dynamics. Strong evidence suggests that these DNA sequence motifs are highly conserved between related species, reflecting strong selection on the network of regulatory interactions that underlie common cellular behavior. Here, we exploit a systems-level aspect of this conservation-the network-level topology of these interactions-to map transcription factor (TF) binding sites on a genomic scale. Using network-level conservation as a constraint, our algorithm finds 71% of known TF binding sites in the yeast Saccharomyces cerevisiae, using only 12% of the sequence of a phylogenetic neighbor. Most of the novel predicted motifs show strong features of known TF binding sites, such as functional category and/or expression profile coherence of their corresponding genes. Network-level conservation should provide a powerful constraint for the systematic mapping of TF binding sites in the larger genomes of higher eukaryotes.