and other carbapenem-resistant are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. Based on an independent Tn-seq assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity using custom phenotypic microarray plates. The screen identified several mutations increasing sensitivity to ß-lactams (in ), and found that two-component regulator mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone and several ß-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism. and other carbapenem-resistant are recognized as a top public health threat by the Centers for Disease Control. The analysis of these major nosocomial pathogens has been limited by the experimental resources available for studying them. The work presented here describes a sequence-defined mutant library for a strain (KPNIH1) which represents an attractive model for studies of this pathogen because it is a recent isolate of the major sequence type that causes infection, the epidemiology of the outbreak it caused is well-characterized, and an annotated genome sequence is available. The ready availability of defined mutants for nearly all nonessential genes of the model strain should facilitate the genetic dissection of complex traits like pathogenesis and antibiotic resistance.

To help define the biological functions of nonessential genes of Francisella novicida , we measured the growth of arrayed members of a comprehensive transposon mutant library under a variety of nutrition and stress conditions. Mutant phenotypes were identified for 37% of the genes, corresponding to ten carbon source utilization pathways, nine amino acid- and nucleotide-biosynthetic pathways, ten intrinsic antibiotic resistance traits, and six other stress resistance traits. The greatest surprise of the analysis was the large number of genotype-phenotype relationships that were not predictable from studies of Escherichia coli and other model species. The study identified candidate genes for a missing glycolysis function (phosphofructokinase), an unusual proline-biosynthetic pathway, parallel outer membrane lipid asymmetry maintenance systems, and novel antibiotic resistance functions. The analysis provides an evaluation of annotation predictions, identifies cases in which fundamental processes differ from those in model species, and helps create an empirical foundation for understanding virulence and other complex processes.