Brad Goodner

Genome Sequences of Three Agrobacterium Biovars Help Elucidate the Evolution of Multichromosome Genomes in Bacteria

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

Optical mapping as a routine tool for bacterial genome sequence finishing.

BackgroundIn sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.ResultsWhole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.ConclusionOur experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.

Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum

Community Page Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum Jayna L. Ditty 1 , Christopher A. Kvaal 2 , Brad Goodner 3 , Sharyn K. Freyermuth 4 , Cheryl Bailey 5 , Robert A. Britton 6 , Stuart G. Gordon 7 , Sabine Heinhorst 8 , Kelynne Reed 9 , Zhaohui Xu 10 , Erin R. Sanders-Lorenz 11 , Seth Axen 12 , Edwin Kim 12 , Mitrick Johns 13 , Kathleen Scott 14 , Cheryl A. Kerfeld 12,15 * 1 Department of Biology, University of St. Thomas, St. Paul, Minnesota, United States of America, 2 Department of Biological Sciences, St. Cloud State University, St. Cloud, Minnesota, United States of America, 3 Department of Biology, Hiram College, Hiram, Ohio, United States of America, 4 Biochemistry Department, University of Missouri- Columbia, Columbia, Missouri, United States of America, 5 Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 6 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America, 7 Department of Biology, Presbyterian College, Clinton, South Carolina, United States of America, 8 Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi, United States of America, 9 Biology Department, Austin College, Sherman, Texas, United States of America, 10 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America, 11 Department of Microbiology, Immunology and Molecular Genetics, University of California – Los Angeles, Los Angeles, California, United States of America, 12 Department of Energy-Joint Genome Institute, Walnut Creek, California, United States of America, 13 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America, 14 Department of Integrative Biology, University of South Florida, Tampa, Florida, United States of America, 15 Department of Plant and Microbial Biology, University of California Berkley, Berkeley, California, United States of America Introduction Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these basic scientific methodologies in under- graduate education leads to increased undergraduate and post-graduate reten- tion in the sciences [13–16]. Toward this end, many undergraduate teaching orga- nizations offer training and suggestions for faculty to update and improve their teaching approaches to help students learn as scientists, through design and discovery (e.g., Council of Undergraduate Research [www.cur.org] and Project Kaleidoscope [ www.pkal.org]). With the advent of genome sequencing and bioinformatics, many scientists now formulate biological questions and inter- pret research results in the context of genomic information. Just as the use of bioinformatic tools and databases changed the way scientists investigate problems, it must change how scientists teach to create new opportunities for students to gain experiences reflecting the influence of genomics, proteomics, and bioinformatics on modern life sciences research [17–41]. Educators have responded by incorpo- rating bioinformatics into diverse life science curricula [42–44]. While these published exercises in, and guidelines for, bioinformatics curricula are helpful and inspirational, faculty new to the area of bioinformatics inevitably need training in the theoretical underpinnings of the algo- rithms [45]. Moreover, effectively inte- grating bioinformatics into courses or independent research projects requires infrastructure for organizing and assessing student work. Here, we present a new platform for faculty to keep current with the rapidly changing field of bioinfor- matics, the Integrated Microbial Genomes Annotation Collaboration Toolkit (IMG- ACT) (Figure 1). It was developed by instructors from both research-intensive and predominately undergraduate institu- tions in collaboration with the Department of Energy-Joint Genome Institute (DOE- JGI) as a means to innovate and update undergraduate education and faculty de- velopment. The IMG-ACT program pro- vides a cadre of tools, including access to a clearinghouse of genome sequences, bioin- formatics databases, data storage, instruc- tor course management, and student notebooks for organizing the results of their bioinformatic investigations. In the process, IMG-ACT makes it feasible to provide undergraduate research opportu- nities to a greater number and diversity of students, in contrast to the traditional mentor-to-student apprenticeship model for undergraduate research, which can be too expensive and time-consuming to provide for every undergraduate. The IMG-ACT serves as the hub for the network of faculty and students that use the system for microbial genome analysis. Open access of the IMG-ACT infrastructure to participating schools en- sures that all types of higher education institutions can utilize it. With the infra- structure in place, faculty can focus their efforts on the pedagogy of bioinformatics, involvement of students in research, and use of this tool for their own research agenda. What the original faculty mem- bers of the IMG-ACT development team present here is an overview of how the IMG-ACT program has affected our Citation: Ditty JL, Kvaal CA, Goodner B, Freyermuth SK, Bailey C, et al. (2010) Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum. PLoS Biol 8(8): e1000448. doi:10.1371/journal.pbio.1000448 Published August 10, 2010 Copyright: s 2010 Ditty et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No specific funding was received for this work. The Community Page is a forum for organizations and societies to highlight their efforts to enhance the dissemination and value of scientific knowledge. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: IMG-ACT; Integrated Microbial Genomes Annotation Collaboration Toolkit * E-mail: ckerfeld@lbl.gov PLoS Biology | www.plosbiology.org August 2010 | Volume 8 | Issue 8 | e1000448

Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium

ABSTRACT Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.

Functional Genes That Distinguish Maize Phyllosphere Metagenomes in Drought and Well-Watered Conditions

Abstract The phyllosphere epiphytic microbiome is composed of microorganisms that colonize the external aerial portions of plants. Relationships of plant responses to specific microorganisms–both pathogenic and beneficial–have been examined, but the phyllosphere microbiome functional and metabolic profile responses are not well described. Changing crop growth conditions, such as increased drought, can have profound impacts on crop productivity. Also, epiphytic microbial communities provide a new target for crop yield optimization. We compared Zea mays leaf microbiomes collected under drought and well-watered conditions by examining functional gene annotation patterns across three physically disparate locations each with and without drought treatment, through the application of short read metagenomic sequencing. Drought samples exhibited different functional sequence compositions at each of the three field sites. Maize phyllosphere functional profiles revealed a wide variety of metabolic and regulatory processes that differed in drought and normal water conditions and provide key baseline information for future selective breeding.The phyllosphere microbiome consists of the genetic content of the microorganisms that colonize the external aerial portions of plants. Relationships of plant responses to specific microorganisms‐‐both pathogenic and beneficial‐‐have been examined, but responses of the phyllosphere microbiome overall are not well described. Changing crop growth conditions such as increased drought can have profound impacts on crop productivity and epiphytic microbial communities provide a new target for crop yield optimization. We provide the first comparison of Zea mays leaf microbiomes between drought and well-watered conditions. We examined the maize leaf phyllosphere microbiome from three physically disparate locations with and without drought treatment, through the application of deep sequencing with short sequence read technology. Functional profiles revealed a wide variety of metabolic and regulatory processes including the ability to adapt to changing environmental conditions associated with, and external to the plant and the presence of potential plant growth promoting traits suggesting possible beneficial plant-microbiome interactions. Both specific field site and drought affected taxonomic and functional composition of these leaf epiphyte communities.

FULL GENOME SEQUENCE OF AZOTOBACTER VINELANDII: PRELIMINARY ANALYSIS

Department of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ, USA; Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA; Hiram College, Hiram, OH, USA; Monsanto Company, St. Louis, MO, USA; Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA; Department of Applied Biological Sciences, Arizona State University, Tempe, AZ, USA; Department of Biology, Seattle Pacific University, Seattle, WA, USA