Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCEAcinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.A cinetobacter baumannii is a Gram-negative opportunistic pathogen that causes infections with serious morbidity and mortality and is one of a group of six pathogens responsible for most multidrug-resistant (MDR) nosocomial infections (the ESKAPE pathogens, i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (1, 2). The pathogen is infamous for its ability to persist in hospital settings, a feature that reflects its capacity for long-term survival on abiotic surfaces through resistance to desiccation and disinfectants (3).Genomic and molecular epidemiological studies of A. baumannii isolates have helped define the pathogen's global population structure, its antibiotic resistance gene repertoire, the size and content of its pangenome, and phylogenetic relationships among outbreak strains (3-6). Three primary clonal lineages (GC1 to GC3) appear responsible for the majority of hospital outbreaks globally (7). Although these lineages display restricted genetic diversity among core genes (7), the species' genome is actually quite dynamic. Strains display striking variability in accessory gene content (5, 8), including antibiotic resistance genes (9), even among related isolates of a single outbreak (10). This genomic variability presumably reflects the actions of transmissible plasmids, insertion elements, phage, integrons, natural transformation, and reco...

SummaryThe TonB system of Escherichia coli (TonB/ExbB/ ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library. Comprehensive sequence-defined mutant libraries have facilitated the genetic dissection of complex processes in several bacterial species (7). In principle, such libraries can be screened directly for mutants exhibiting a given phenotype to provide relatively complete identification of nonessential functions responsible for the trait. In practice, however, such screens can fail to achieve completeness and may even suggest incorrect genotypephenotype associations. There are several potential contributing factors that arise from how mutant libraries are created and quality control tested.Most defined mutant libraries have been generated by largescale transposon mutagenesis and sequencing. Relatively complete genome coverage requires that an average of 5 to 10 unique insertions per gene be identified (7). Such large primary libraries serve as a source of mutants for smaller secondary libraries that retain genome coverage and facilitate phenotype screening (1-3, 6). Secondary libraries are usually made up of one or more colonypurified mutants per gene, with insertions situated toward the centers of coding regions to help ensure inactivation. A limitation of most secondary libraries is that mutant identities have not been verified by resequencing. In rare cases in which they have been checked, many assignments (typically on the order of 10%) have been found to be incorrect (3). An additional limitation of secondary libraries consisting of only one mutant per gene is that screens using them are at risk of missing genotype-phenotype associations due to mutant cross-contamination and insertion alleles that fail to inactivate. Libraries with more than one mutant per gene provide more than one chance to identify an association.This report describes a Pseudomonas aeruginosa PAO1 secondary transposon mutant library with two mutants for most genes in which mutant identities have been confirmed by multiple resequencings. The redundancy and verification of mutant identities should make the library particularly useful...

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.