although 1224 bacterial genomes have been sequences, there are <10 that have been well-characterized experimentally - Ruchira S. Datta

one is at the mercy of the assumption that similarity to model species sequences preserves function - Ruchira S. Datta

project goal: "annotate" a non-model genome using phenotyping - Ruchira S. Datta

Francisella novicida is an infectious gamma proteobacterium, work instead with surrogae Francisella tularensis which infects mice. Small genome. - Ruchira S. Datta

used transposon library to identify every non-essential gene, use highly parallel phenotyping of mutants to infer genotype-phenotype associations - Ruchira S. Datta

the transposon mutant analysis gave mutants in 1457 of 1742 genes, 3.1 mutants/gene, 97.5% confirmed by re-sequencing - Ruchira S. Datta

redundancy w/ 3 mutants was important for confidence in the genotype/phenotype associations - Ruchira S. Datta

phenotypes checked: carbon utilization, small molecule biosynthesis, and antibiotic and stress sensitivites - Ruchira S. Datta

e.g., growth on carbon sources: glucose, fructose, glycerol, glutamine - Ruchira S. Datta

e.g., specific for glycerol: had mutated glycerol kinase - Ruchira S. Datta

check phenotype profile of each mutant in each condition, measure relative growth - Ruchira S. Datta

of genes with mutant phenotypes, 55% had fully predicted functions, 25% had no predicted function, 20% had partially predicted function - Ruchira S. Datta

e.g., mutant phenotype was fructose utilization, gene annotation was already fructose kinase, then not that helpful - Ruchira S. Datta

but fructose utilization of gene annotated as hypothetical is more useful - Ruchira S. Datta

fructose utilization of gene annotated as sugar-proton symporter is more useful - Ruchira S. Datta

in existing proline biosynthesis annotation, the pathway appeared to be incomplete as proC was missing. but mutant phenotype showed it was indeed competent to produce proline. used alternative pathway: arginine via amidinotransferase to ornithine, and orthinithine via ornithine cyclodeaminase to proline - Ruchira S. Datta

quinolone antibiotic resistance (e.g., ciprofloxacin, nalidixic acid): found genes involved in recombination, replication and repair, efflux, and several novel genes not previously known to be involved - Ruchira S. Datta

partial overlap with Jeffrey Miller's list of quinolone resistance - could be due to technical issues, but also due to only partial conservation of the antibiotic resistance mechanisms - Ruchira S. Datta

detergent sensitivity/resistance: ABC transporter responsible for keeping phospholipids out of outer leaflet in E. coli. this is the mla system. But here in this small genome, there are two mla-gene related systems for keeping phospholipids out of outer membrane. Also found novel genes associated with this phenotype. - Ruchira S. Datta

can cluster phenotypes by their mutant profiles. e.g., ciprofloxacin clusters with nalidixic acid. but aminoglycoside antibiotics cluster with certain stresses, 46C or ph8.3 - Ruchira S. Datta

limitations of this approach: polar effects (don't know about downstream genes on same operon). however, these genes should be working together anyway, so may not be as big an issue. essential genes and unpredicted genes are missing from the analysis. - Ruchira S. Datta

large-scale phenotyping is a rich source of functional information and is feasible for non-model prokaryotes - Ruchira S. Datta

requires a mutant library, of which there are 10 or 12 published ones? not limited by that, TnSeq should open up many possibilities - Ruchira S. Datta

Bruno Sobral: vocabulary to describe phenotypes? is this controlled, can it be queried? A: just using traditional genetic language. assigning a number to each phenotype of each gene. Bruno Sobral: assay has a name? A: yes, presence of stressor or absence of nutrient. Bruno Sobral: how often was the starting annotation actually useful? A: hard to estimate. on average, verifying predicted phenotype or attaching new one to hypothetical. - Ruchira S. Datta

Q: phenotype of proC mutant? A: not yet. Q: if have something particular interested in? A: send an email - Ruchira S. Datta

Jonathan Eisen: why didn't do the unpredicted genes? A: did include some in gaps between genes, had phenotypes, not very systematically - Ruchira S. Datta