ABSTRACT Klebsiella pneumoniae is a Gram-negative human pathogen capable of causing hospital-acquired infections with an increasing risk to human health. The total DNA from four clinically relevant strains was sequenced to >100× coverage, providing high-quality genome assemblies for K. pneumoniae strains ATCC 13883, KP4640, 101488, and 101712.

Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials. Received 27 March 2014 Accepted 10 April 2014 Published 27 May 2014 Citation Jacobs AC, Thompson MG, Black CC, Kessler JL, Clark LP, McQueary CN, Gancz HY, Corey BW, Moon JK, Si Y, Owen MT, Hallock JD, Kwak YI, Summers A, Li CZ, Rasko DA, Penwell WF, Honnold CL, Wise MC, Waterman PE, Lesho EP, Stewart RL, Actis LA, Palys TJ, Craft DW, Zurawski DV. 2014. AB5075, a highly virulent isolate of Acinetobacter baumannii, as a model strain for the evaluation of pathogenesis and antimicrobial treatments. mBio 5(3):e01076-14. doi:10.1128/mBio.01076-14. Editor Howard Shuman, University of Chicago Copyright © 2014 Jacobs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Daniel V. Zurawski, daniel.v.zurawski.ctr@mail.mil. Acinetobacter baumannii is an opportunistic, Gram-negative pathogen that thrives in clinical settings and is often multidrug resistant (MDR), factors which earn it a place among the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens of clinical importance (1). Some recent isolates are resistant to all typically used antibiotics except colistin and tigecycline and thus are called extensively or extremely drug-resistant (XDR) A. baumannii (2). MDR/XDR A. baumannii strains are a worldwide problem for clinicians and caregivers in the hospital setting, particularly in the intensive care unit (ICU) (3). A. baumannii is also often isolated from infections of severe wounds sustained in military combat. These infections are responsible for increased morbidity, with prolonged wound RESEARCH ARTICLE May/June 2014 Volume 5 Issue 3 e01076-14 ® mbio.asm.org 1 m b.asm .rg on July 5, 2014 P ubished by m b.asm .rg D ow nladed fom healing and amputations of extremities when limbs cannot be salvaged (4, 5). A. baumannii was a predominant isolate from wounded soldiers serving in Iraq (4, 5) and was associated with wartime polytrauma injuries in the past (6). Additionally, there may be a link between A. baumannii and crush injuries, as A. baumannii infections were also prevalent after the recent large earthquakes in Haiti (7) and China (8). Another disturbing development that has increased the clinical importance of A. baumannii infections is that many strains have become highly antibiotic resistant. For example, in previous decades, A. baumannii isolates obtained from both military and civilian settings were often carbapenem sensitive. Now, the majority of U.S. military isolates are carbapenem resistant (9). This trend has also been mirrored in civilian hospitals around the world (10). Recently, even colistin-resistant strains have emerged in the military health care system (11). The latter development is deeply troubling, as colistin is considered the last line of defense against these MDR isolates. Exacerbating this problem is the lack of new treatments in the pharmaceutical pipeline (12); therefore, research on A. baumannii virulence factors is urgently needed, as they could constitute potentially novel targets for future antimicrobials. While previous studies attempted to examine the virulence of different clinical A. baumannii strains utilizing in vivo model systems (13, 14), the majority of A. baumannii researchers still use two American Type Culture Collection (ATCC) strains, ATCC 19606T and ATCC 17978, which were isolated more than 50 years ago and are not significantly antibiotic resistant. These strains are certainly more amenable to genetic manipulation than most clinical isolates (15, 16) and share considerable genome homology ( 90%) to current A. baumannii isolates (17), but they are not representative of contemporary isolates of this rapidly evolving pathogen. Some researchers, recognizing that the ATCC isolates are dated, have performed studies with more recent clinical isolates; however, genetic manipulation of such isolates has depended on susceptibility to aminoglycosides (18–20), which is often not found in clinical strains (21). Therefore, our goal was to carry out a systematic study of our own contemporary clinical strains isolated from patients in the U.S. military health care system to identify a strain that is more representative of current clinical isolates, that is highly virulent in established model infections, and that can be genetically manipulated without a potential sacrifice with respect to virulence and antibiotic resistance. Not only does identifying such a strain account for more recent clinical outcomes, but the increased virulence in animal models allows greater statistical power in screening new therapeutics. Moreover, the ability to manipulate the genome allows the study of virulence factors, some of which may be responsible for the emergence of this pathogen in more recent years.

Motivation: Gene blocks are genes co-located on the chromosome. In many cases, gene blocks are conserved between bacterial species, sometimes as operons, when genes are co-transcribed. The conservation is rarely absolute: gene loss, gain, duplication, block splitting and block fusion are frequently observed. An open question in bacterial molecular evolution is that of the formation and breakup of gene blocks, for which several models have been proposed. These models, however, are not generally applicable to all types of gene blocks, and consequently cannot be used to broadly compare and study gene block evolution. To address this problem, we introduce an event-based method for tracking gene block evolution in bacteria. Results: We show here that the evolution of gene blocks in proteobacteria can be described by a small set of events. Those include the insertion of genes into, or the splitting of genes out of a gene block, gene loss, and gene duplication. We show how the event-based method of gene block evolution allows us to determine the evolutionary rateand may be used to trace the ancestral states of their formation. We conclude that the event-based method can be used to help us understand the formation of these important bacterial genomic structures. Availability and implementation: The software is available under GPLv3 license on http://github.com/reamdc1/gene_block_evolution.git. Supplementary online material: http://iddo-friedberg.net/operon-evolution Contact: i.friedberg@miamioh.edu Supplementary information: Supplementary data are available at Bioinformatics online.

ABSTRACT Acinetobacter baumannii is a bacterial pathogen with serious implications on human health, due to increasing reports of multidrug-resistant strains isolated from patients. Total DNA from the multidrug-resistant A. baumannii strain A155 clinical isolate was sequenced to greater than 65× coverage, providing high-quality contig assemblies.

Genome replication by the baculovirus DNA polymerase often generates errors in mononucleotide repeat (MNR) sequences due to replication slippage. This results in the inactivation of genes that affects different stages of the cell infection cycle. Here we mapped these MNRs in the 59 baculovirus genomes. We found that the MNR frequencies of baculovirus genomes are different and not correlated with the genome sizes. Although the average A/T content of baculoviruses is 58.67%, the A/T MNR frequency is significantly higher than that of the G/C MNRs. Furthermore, the A7/T7 MNRs are the most frequent of those we studied. Finally, MNR frequencies in different classes of baculovirus genes, such as immediate early genes, show differences between baculovirus genomes, suggesting that the distribution and frequency of different MNRs are unique to each baculovirus species or strain. Therefore, the results of this study can help select appropriate baculoviruses for the development of biological insecticides.

The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the "few articles - many proteins" phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments.

ABSTRACT The autotrophic communities in the lakes of the McMurdo Dry Valleys, Antarctica, have generated interest since the early 1960s owing to low light transmission through the permanent ice covers, a strongly bimodal seasonal light cycle, constant cold water temperatures, and geographical isolation. Previous work has shown that autotrophic carbon fixation in these lakes provides an important source of organic matter to this polar desert. Lake Bonney has two lobes separated by a shallow sill and is one of several chemically stratified lakes in the dry valleys that support year-round biological activity. As part of an International Polar Year initiative, we monitored the diversity and abundance of major isoforms of RubisCO in Lake Bonney by using a combined sequencing and quantitative PCR approach during the transition from summer to polar winter. Form ID RubisCO genes related to a stramenopile, a haptophyte, and a cryptophyte were identified, while primers specific for form IA/B RubisCO detected a diverse autotrophic community of chlorophytes, cyanobacteria, and chemoautotrophic proteobacteria. Form ID RubisCO dominated phytoplankton communities in both lobes of the lake and closely matched depth profiles for photosynthesis and chlorophyll. Our results indicate a coupling between light availability, photosynthesis, and rbcL mRNA levels in deep phytoplankton populations. Regulatory control of rbcL in phytoplankton living in nutrient-deprived shallow depths does not appear to be solely light dependent. The distinct water chemistries of the east and west lobes have resulted in depth- and lobe-dependent variability in RubisCO diversity, which plays a role in transcriptional activity of the key gene responsible for carbon fixation.