David A Baltrus

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Scholarly Contributions

Chapters

• Arnold, A. E., Spraker, J., & Baltrus, D. A. (2018). Quantifying re-association of a facultative endohyphal bacterium with a filamentous fungus. In Plant Pathogenic Fungi and Oomycetes(pp 1-12). Humana.
• Baltrus, D. A. (2018). What Is a Pseudomonas syringae Population. In Population Genomics: Microorganisms. Springer, Cham. doi:10.1007/13836_2018_25
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  Although they are often best known as causative agents of agricultural disease, many phytopathogen lineages, like Pseudomonas syringae, have been sampled across a wide range of environmental contexts. These may be frequently isolated as epiphytes on disease-free plants as well as from sources associated with the water cycle like rivers, lakes, rain, snow, and clouds. The ability of these bacteria to persist across such diverse environments poses a great challenge for understanding population dynamics because adaptation likely occurs across numerous distinct niches and evolutionary parameters and will likely differ widely depending on specific contexts. Within the literature, there is an intrinsic tendency to treat all strains within these lineages the same, but such a treatment likely obscures interesting and important nuances between isolates. In this chapter, I will focus on P. syringae and explore what is known about the evolutionary dynamics of this group at the levels of genomes, phylogroups, and (broadly defined) species. I will highlight many ways in which populations could differ and will touch upon what is known and has been learned from numerous genome sequencing efforts, which hopefully shine a light toward a path forward to resolve numerous nomenclatural challenges. I will point toward the generality of what is known about P. syringae and how this may apply to other environmental systems. While there remains much to learn, the ever-increasing rate of accumulation of genomic data from diverse sources has certainly helped our ability to at least frame the evolutionarily important questions. Building from these, an impending wave of future data promises to be a powerful tool for resolving some of these discussions.
• Baltrus, D. A., Hendry, T. A., & Hockett, K. L. (2014). Ecological genomics of pseudomonas syringae. In Genomics of Plant-Associated Bacteria. Springer-Verlag Berlin Heidelberg. doi:10.1007/978-3-642-55378-3_3
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  Pseudomonas syringae is well known as a model bacterial phytopathogen in the laboratory, environment, and the field. A focus on understanding mechanisms of virulence in planta has motivated extensive research into genetic, genomic, and evolutionary factors that influence disease. However, in recent years, appreciation has grown for the life cycle of P. syringae outside of the context of plant disease. This bacterial species survives and thrives across many environments, with its broad ecology shaped through interactions with phage, bacteria, fungi, and insects in addition to traditional host plants. Here we explore what is known about the genetic and genomic basis of these diverse ecological interactions. We highlight how both new and old approaches can be used to unify our understanding of these relationships and map a path forward enabled by high-throughput genomics.
• Baltrus, D. A., Blaser, M. J., & Guillemin, K. (2009). Helicobacter pylori Genome Plasticity.. In Genome Dynamics. Genome Dyn. doi:10.1159/000235764
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  Helicobacter pylori, a Gram-negative pathogen associated with ulcers, chronic gastritis, and gastric cancers, has been a resident of the human stomach since early human history [1]. This association has only recently begun to erode with the advent of antibiotics and modern lifestyles, but even today H. pylori colonizes approximately half the world's population. To have remained a successful colonizer of humans during thousands of years of association, populations of H. pylori must have been able to survive and adapt to countless evolutionary challenges within and between hosts. As a species, H. pylori possesses one of the most fluid genomes within the prokaryotic kingdom [2], a characteristic that has likely aided its continued success. H. pylori exhibits exceptionally high rates of DNA point mutations, intragenomic recombination (facilitated by repetitive elements common in H. pylori genomes), and intergenomic recombination (mediated by natural transformation), all of which contribute to the high genomic variability between isolates. Previous reviews have focused on these processes as agents of evolutionary change within H. pylori [2-8]. The mechanisms of both mutation and natural transformation, and the evolutionary processes that retain genetic variation generated by these mechanisms, dictate the extent to which each contributes to genomic diversity in the context of different bacterial population structures [9-13]. Unlike well-studied evolutionary systems, such as Salmonella and Escherichia coli, H. pylori is notable in its lack of an environmental reservoir outside of human and other primate stomachs, suggesting that between-host survival is a relatively weak determinant of selection pressures [14, 15]. Given that H. pylori exist largely as distinct host-associated populations, it is possible to begin to model the evolutionary mechanisms that affect the long-term persistence of this species. In this chapter, we consider how the attributes of H. pylori's natural history as a long-term resident of the human stomach and the specific mechanisms of mutation and genetic exchange in this organism have shaped the H. pylori genome. We begin with a survey of genome plasticity in H. pylori. We then discuss mechanisms of mutation and natural transformation in H. pylori and examine experimental evidence for the generation of genomic changes within populations. Finally, we consider how different models of H. pylori population structure affect the relative contributions of mutation and recombination to the evolutionary success of this organism. By bridging evolutionary studies with investigations of pathogenesis from a molecular perspective, we hope to shed new light on how H. pylori has and continues to evolve with its human hosts.

Journals/Publications

• Baltrus, D. A., Feng, Q., & Kvitko, B. H. (2022). Genome Context Influences Evolutionary Flexibility of Nearly Identical Type III Effectors in Two Phytopathogenic Pseudomonads. Frontiers in microbiology, 13, 826365.
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  Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site-specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen , the impact of ICE carriage and transfer across the entire species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and highlight that this element contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in active ICEs. Taken together, our study sheds light on a set of ICE elements from pv. maculicola ES4326 and suggests how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.
• Hall, J. P., Botelho, J., Cazares, A., & Baltrus, D. A. (2022). What makes a megaplasmid?. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 377(1842), 20200472.
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  Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger. Historically, research has tended to focus on smaller plasmids that are usually easier to isolate, manipulate and sequence, but with improved genome assemblies made possible by long-read sequencing, there is increased appreciation that very large plasmids-known as megaplasmids-are widespread, diverse, complex, and often encode key traits in the biology of their host microorganisms. Why are megaplasmids so big? What other features come with large plasmid size that could affect bacterial ecology and evolution? Are megaplasmids 'just' big plasmids, or do they have distinct characteristics? In this perspective, we reflect on the distribution, diversity, biology, and gene content of megaplasmids, providing an overview to these large, yet often overlooked, mobile genetic elements. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.
• Hall, J. P., Harrison, E., & Baltrus, D. A. (2022). Introduction: the secret lives of microbial mobile genetic elements. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 377(1842), 20200460.
• Shaffer, J. P., Carter, M. E., Spraker, J. E., Clark, M., Smith, B. A., Hockett, K. L., Baltrus, D. A., & Arnold, A. E. (2022). Transcriptional Profiles of a Foliar Fungal Endophyte (, Ascomycota) and Its Bacterial Symbiont (, ) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction. mSystems, 7(2), e0009122.
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  Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by sp. and sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of sp. in coculture was greater than in axenic culture, but the opposite was true for sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and .
• Smith, B. A., Dougherty, K., Clark, M., & Baltrus, D. A. (2022). Experimental evolution of the megaplasmid pMPPla107 in enables identification of genes contributing to sensitivity to an inhibitory agent. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 377(1842), 20200474.
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  Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.
• Stillson, P. T., Baltrus, D. A., & Ravenscraft, A. (2022). Prevalence of an Insect-Associated Genomic Region in Environmentally Acquired Symbionts. Applied and environmental microbiology, 88(9), e0250221.
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  Microbial symbionts are critical for the development and survival of many eukaryotes. Recent research suggests that the genes enabling these relationships can be localized in horizontally transferred regions of microbial genomes termed "symbiotic islands." Recently, a putative symbiotic island was found that may facilitate symbioses between true bugs and numerous species, based on analysis of five symbionts. We expanded on this work by exploring the putative island's prevalence, origin, and association with colonization across the bacterial family . We performed a broad comparative analysis of 229 genomes, including 8 new genomes of insect- or soil-associated sequenced for this study. We detected the region in 23% of the genomes; these were located solely within two clades. Our analyses suggested that the contiguous region arose at the common ancestor of plant- and insect-associated clades, but the genes themselves are ancestral. Although the region was initially discovered on plasmids and we did detect two likely instances of horizontal transfer within , we found that the region is almost always localized to a chromosome and does not possess any of the mobility elements that typify genomic islands. Finally, to attempt to deduce the region's function, we combined our data with information on several strains' abilities to colonize the insect's symbiotic organ. Although the region was associated with improved colonization of the host, this relationship was confounded with, and likely driven by, clade membership. These findings advance our understanding of the genomic underpinnings of a widespread insect-microbe symbiosis. Many plants and animals form intricate associations with bacteria. These pairings can be mediated by genomic islands, contiguous regions containing numerous genes with cohesive functionality. Pathogen-associated islands are well described, but recent evidence suggests that mutualistic islands, which benefit both host and symbiont, may also be common. Recently, a putative symbiosis island was found in symbionts of insects. We determined that this genomic region is located in only two clades of (the plant- and insect-associated species) and that although it has undergone horizontal transfer, it is most likely a symbiosis-associated region rather than a true island. This region is associated with improved host colonization, although this is may be due to specific clades' abilities to colonize rather than presence of the region. By studying the genomic basis of the insect- symbiosis, we can better understand how mutualisms evolve in animals.
• Weaver, S. L., Zhu, L., Ravishankar, S., Clark, M., & Baltrus, D. A. (2022). Interspecies killing activity of tailocins. Microbiology (Reading, England), 168(11).
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  Tailocins are ribosomally synthesized bacteriocins, encoded by bacterial genomes, but originally derived from bacteriophage tails. As with both bacteriocins and phage, tailocins are largely thought to be species-specific with killing activity often assumed to be directed against closely related strains. Previous investigations into interactions between tailocin host range and sensitivity across phylogenetically diverse isolates of the phytopathogen have demonstrated that many strains possess intraspecific tailocin activity and that this activity is highly precise and specific against subsets of strains. However, here we demonstrate that at least one strain of USA011R, defies both expectations and current overarching dogma because tailocins from this strain possess broad killing activity against other agriculturally significant phytopathogens such as and as well as against the clinical human pathogen serovar Choleraesui. Moreover, we show that the full spectrum of this interspecific killing activity is not conserved across closely related strains with data suggesting that even if tailocins can target different species, they do so with different efficiencies. Our results reported herein highlight the potential for and phenotypic divergence of interspecific killing activity of tailocins and establish a platform for further investigations into the evolution of tailocin host range and strain specificity.
• Baltrus, D. A., White, A., Smith, C., & Clark, M. (2021). Draft Genome Sequences of Multiple Isolates from Arizona. Microbiology resource announcements, 10(26), e0020821.
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  strains are bacteria that are well known for their distinctive physiology, behaviors, and ecology, as well as for being prodigious producers of diverse antibiotics. Here, we report draft genome sequences for eight strains that were isolated from multiple sky islands in Arizona and sequenced using an Oxford Nanopore Technologies Flongle adapter and MinION system.
• Smith, C., Rosenthal, Z., Moore, A., Mollico, M., Leligdon, C., Derrick, M., Clark, M., & Baltrus, D. A. (2021). Genomic Background Governs Opposing Responses to Nalidixic Acid upon Megaplasmid Acquisition in Pseudomonas.. mSphere, 6(1). doi:10.1128/msphere.00008-21
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  Horizontal gene transfer is a significant driver of evolutionary dynamics across microbial populations. Although the benefits of the acquisition of new genetic material are often quite clear, experiments across systems have demonstrated that gene transfer events can cause significant phenotypic changes and entail fitness costs in a way that is dependent on the genomic and environmental context. Here, we test for the generality of one previously identified cost, sensitization of cells to the antibiotic nalidixic acid after acquisition of an ∼1-Mb megaplasmid, across Pseudomonas strains and species. Overall, we find that the presence of this megaplasmid sensitizes many different Pseudomonas strains to nalidixic acid but that this same horizontal gene transfer event increases resistance of Pseudomonas putida KT2440 to nalidixic acid across assays as well as to ciprofloxacin under competitive conditions. These phenotypic results are not easily explained away as secondary consequences of overall fitness effects and appear to occur independently of another cost associated with this megaplasmid, sensitization to higher temperatures. Lastly, we draw parallels between these reported results and the phenomenon of sign epistasis for de novo mutations and explore how context dependence of effects of plasmid acquisition could impact overall evolutionary dynamics and the evolution of antimicrobial resistance.IMPORTANCE Numerous studies have demonstrated that gene transfer events (e.g., plasmid acquisition) can entail a variety of costs that arise as by-products of the incorporation of foreign DNA into established physiological and genetic systems. These costs can be ameliorated through evolutionary time by the occurrence of compensatory mutations, which stabilize the presence of a horizontally transferred region within the genome but which also may skew future adaptive possibilities for these lineages. Here, we demonstrate another possible outcome, that phenotypic changes arising as a consequence of the same horizontal gene transfer (HGT) event are costly to some strains but may actually be beneficial in other genomic backgrounds under the right conditions. These results provide a new viewpoint for considering conditions that promote plasmid maintenance and highlight the influence of genomic and environmental contexts when considering amelioration of fitness costs after HGT events.
• Weaver, S., Smith, C., Mollico, M., Hockett, K., Clark, M., & Baltrus, D. (2021). Prophylactic Application of Tailocins Prevents Infection by Pseudomonas syringae.. Phytopathology. doi:10.1094/phyto-06-21-0269-r
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  Tailocins are phage-derived bacteriocins that demonstrate great potential as agricultural antimicrobials given their high killing efficiency and their precise strain-specific targeting ability. Our group has recently categorized and characterized tailocins produced by and tailocin sensitivities of the phytopathogen Pseudomonas syringae, and here we extend these experiments to test whether prophylactic tailocin application can prevent infection of Nicotiana benthamiana by P. syringae pv. syringae B728a. Specifically, we demonstrate that multiple strains can produce tailocins that prevent infection by strain B728a and engineer a deletion mutant to prove that tailocin targeting is responsible for this protective effect. Lastly, we provide evidence that heritable resistance mutations do not explain the minority of cases where tailocins fail to prevent infection. Our results extend previous reports of prophylactic use of tailocins against phytopathogens, and establish a model system with which to test and optimize tailocin application for prophylactic treatment to prevent phytopathogen infection.
• Baltrus, D. A. (2020). Bacterial dispersal and biogeography as underappreciated influences on phytobiomes.. Current opinion in plant biology, 56, 37-46. doi:10.1016/j.pbi.2020.02.010
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  Bacterial strains are not distributed evenly throughout the environment. Here I explore how differential distribution and dispersal patterns of bacteria could affect interactions and coevolutionary dynamics with plants, and highlight ways that variation could be taken advantage of to develop robust and effective microbial consortia to inoculate crops. Questions about biogeographical patterns in viruses, fungi, and other eukaryotes are equally as prevalent and important for agriculture, and are in some cases more thoroughly explored. For simplicity as well as to bring attention to bacterial biogeography and dispersal in the context of plant interactions, I focus solely on bacterial patterns and questions for this article. The next few years will no doubt bring great advances in our understanding of dispersal capabilities and population dynamics for many plant-associated bacteria, and one of the next looming challenges will be learning to harvest this diversity in ways that can benefit agriculture.
• Baltrus, D. A., & Clark, M. (2020). Complete Genome Sequence of Pseudomonas coronafaciens pv. oryzae 1_6. Microbiology resource announcements, 9(3).
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  pv. oryzae 1_6 was originally isolated as a phytopathogen of rice. Here, we report a complete genome sequence for this strain, containing a circular chromosome and one circular plasmid, assembled using a hybrid approach combining Illumina paired-end reads and longer reads sequenced on an Oxford Nanopore Flongle flow cell.
• Baltrus, D. A., Clark, M., Humphrey, P. T., & Whiteman, N. K. (2020). Complete Genome Sequences for sp. Strains 29A and 43A. Microbiology resource announcements, 9(39).
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  sp. strains 29A and 43A were originally isolated from the phyllosphere of individual plants of (Brassicaceae). Here, we report complete genome sequences for these two closely related strains, assembled using a hybrid approach combining Illumina paired-end reads and longer reads sequenced on an Oxford Nanopore MinION flow cell.
• Dunning Hotopp, J. C., Baltrus, D. A., Bruno, V. M., Dennehy, J. J., Gill, S. R., Maresca, J. A., Matthijnssens, J., Newton, I. L., Putonti, C., Rasko, D. A., Rokas, A., Roux, S., Stajich, J. E., Stedman, K. M., Stewart, F. J., & Thrash, J. C. (2020). Best Practices for Successfully Writing and Publishing a Genome Announcement in. Microbiology resource announcements, 9(36).
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  (MRA) provides peer-reviewed announcements of scientific resources for the microbial research community. We describe the best practices for writing an announcement that ensures that these publications are truly useful resources. Adhering to these best practices can lead to successful publication without the need for extensive revisions.
• Johnson, M. D., David, J. M., Gardy, J., Gardy, J., David, J. M., David, J. M., Baltrus, D. A., & Baltrus, D. A. (2020). Crowdsourcing virtual summer research opportunities to support minorities in microbiology.. Nature microbiology, 5(11), 1311-1313. doi:10.1038/s41564-020-00807-6
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  Summer research programmes are renowned for encouraging underrepresented minorities (URMs) to pursue STEM careers, but COVID-19 left many students in the United States unable to participate. We created the National Summer Undergraduate Research Project to matchmake students with mentors, enabling 250 URM students to do summer research.
• Kandel, P. P., Hockett, K. L., & Baltrus, D. A. (2020). Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms.. Journal of bacteriology, 202(13). doi:10.1128/jb.00142-20
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  Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.
• Thao, K. K., Stice, S. P., Kvitko, B. H., Khang, C. H., Dutta, B., & Baltrus, D. A. (2020). Thiosulfinate Tolerance Is a Virulence Strategy of an Atypical Bacterial Pathogen of Onion.. Current biology : CB, 30(16), 3130-3140.e6. doi:10.1016/j.cub.2020.05.092
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  Unlike most characterized bacterial plant pathogens, the broad-host-range plant pathogen Pantoea ananatis lacks both the virulence-associated type III and type II secretion systems. In the absence of these typical pathogenicity factors, P. ananatis induces necrotic symptoms and extensive cell death in onion tissue dependent on the HiVir proposed secondary metabolite synthesis gene cluster. Onion (Allium. cepa L), garlic (A. sativum L.), and other members of the Allium genus produce volatile antimicrobial thiosulfinates upon cellular damage. However, the roles of endogenous thiosulfinate production in host-bacterial pathogen interactions have not been described. We found a strong correlation between the genetic requirements for P. ananatis to colonize necrotized onion tissue and its capacity for tolerance to the thiosulfinate "allicin" based on the presence of an eleven-gene, plasmid-borne, virulence cluster of sulfur redox genes. We have designated them "alt" genes for allicin tolerance. We show that allicin and onion thiosulfinates restrict bacterial growth with similar kinetics. The alt gene cluster is sufficient to confer allicin tolerance and protects the glutathione pool during allicin treatment. Independent alt genes make partial phenotypic contributions indicating that they function as a collective cohort to manage thiol stress. Our work implicates endogenous onion thiosulfinates produced during cellular damage as major mediators of interactions with bacteria. The P. ananatis-onion pathosystem can be modeled as a chemical arms race of pathogen attack, host chemical counterattack, and pathogen defense.
• Thrash, J. C., Stewart, F. J., Stedman, K. M., Stajich, J. E., Roux, S., Rokas, A., Rasko, D. A., Putonti, C., Newton, I. L., Matthijnssens, J., Maresca, J. A., Hotopp, J. C., Gill, S. R., Dennehy, J. J., Bruno, V. M., & Baltrus, D. A. (2020). Best Practices for Successfully Writing and Publishing a Genome Announcement in Microbiology Resource Announcements.. Microbiology resource announcements, 9(36). doi:10.1128/mra.00763-20
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  Microbiology Resource Announcements (MRA) provides peer-reviewed announcements of scientific resources for the microbial research community. We describe the best practices for writing an announcement that ensures that these publications are truly useful resources. Adhering to these best practices can lead to successful publication without the need for extensive revisions.
• Baltrus, D. A., & Clark, M. (2019). A complete genome sequence for Pseudomonas syringae pv. pisi PP1 highlights the importance of multiple modes of horizontal gene transfer during phytopathogen evolution. Molecular plant pathology, 20(7), 1013-1018.
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  Hybrid assembly strategies that combine long-read sequencing reads from Oxford Nanopore's MinION device combined with high-depth Illumina paired-end reads have enabled completion and circularization of both plasmids and chromosomes from multiple bacterial strains. Here we demonstrate the utility of supplementing Illumina paired-end reads from a previously published draft genome of P. syringae pv. pisi PP1 with long reads to generate a complete genome sequence for this strain. The phylogenetic placement and genomic repertoire of virulence factors within this strain provides a unique perspective on virulence evolution within P. syringae phylogroup 2, and highlights that strains can rapidly acquire virulence factors through horizontal gene transfer by acquisition of plasmids as well as through chromosomal recombination.
• Baltrus, D. A., Clark, M., Inderbitzin, P., Pignatta, D., Knight-Connoni, V., & Arnold, A. E. (2019). Complete Genome Sequence of MAH-14. Microbiology resource announcements, 8(29).
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  Diverse strains of () have been isolated from a variety of environments, most frequently in association with both plants and fungi. Motivated by the lack of genomic information for strains throughout the genus we report here a complete genome sequence for strain MAH-14.
• Baltrus, D. A., Clark, M., Smith, C., & Hockett, K. L. (2019). Localized recombination drives diversification of killing spectra for phage-derived syringacins. The ISME journal, 13(2), 237-249.
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  To better understand the potential for antagonistic interactions between members of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage-derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage-derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage-derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.
• Baltrus, D. A., Cuomo, C. A., Dennehy, J. J., Dunning Hotopp, J. C., Maresca, J. A., Newton, I. L., Rasko, D. A., Rokas, A., Roux, S., & Stajich, J. E. (2019). Future-Proofing Your Genome Assembly for Reproducibility and Clarity. Microbiology resource announcements, 8(36).
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  Descriptions of resources, like the genome assemblies reported in , are often frozen at their time of publication, yet they will need to be interpreted in the midst of continually evolving technologies. It is therefore important to ensure that researchers accessing published resources have access to all of the information required to repeat, interpret, and extend these original analyses. Here, we provide a set of suggestions to help make certain that published resources remain useful and repeatable for the foreseeable future.
• Lu-Irving, P., Harenčár, J. G., Sounart, H., Welles, S. R., Swope, S. M., Baltrus, D. A., & Dlugosch, K. M. (2019). Native and Invading Yellow Starthistle (Centaurea solstitialis) Microbiomes Differ in Composition and Diversity of Bacteria. mSphere, 4(2).
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  Invasive species could benefit from being introduced to locations with more favorable species interactions, including the loss of enemies, the gain of mutualists, or the simplification of complex interaction networks. Microbiomes are an important source of species interactions with strong fitness effects on multicellular organisms, and these interactions are known to vary across regions. The highly invasive plant yellow starthistle () has been shown to experience more favorable microbial interactions in its invasions of the Americas, but the microbiome that must contribute to this variation in interactions is unknown. We sequenced amplicons of 16S rRNA genes to characterize bacterial community compositions in the phyllosphere, ectorhizosphere, and endorhizosphere of yellow starthistle plants from seven invading populations in California, USA, and eight native populations in Europe. We tested for the differentiation of microbiomes by geography, plant compartment, and plant genotype. Bacterial communities differed significantly between native and invading plants within plant compartments, with consistently lower diversity in the microbiome of invading plants. The diversity of bacteria in roots was positively correlated with plant genotype diversity within both ranges, but this relationship did not explain microbiome differences between ranges. Our results reveal that these invading plants are experiencing either a simplified microbial environment or simplified microbial interactions as a result of the dominance of a few taxa within their microbiome. Our findings highlight several alternative hypotheses for the sources of variation that we observe in invader microbiomes and the potential for altered bacterial interactions to facilitate invasion success. Previous studies have found that introduced plants commonly experience more favorable microbial interactions in their non-native range, suggesting that changes to the microbiome could be an important contributor to invasion success. Little is known about microbiome variation across native and invading populations, however, and the potential sources of more favorable interactions are undescribed. Here, we report one of the first microbiome comparisons of plants from multiple native and invading populations, in the noxious weed yellow starthistle. We identify clear differences in composition and diversity of microbiome bacteria. Our findings raise new questions about the sources of these differences, and we outline the next generation of research that will be required to connect microbiome variation to its potential role in plant invasions.
• Morimoto, J., & Baltrus, D. A. (2019). The Extended Genotype: To What Extent? A Comment on Carthey et al. Trends in ecology & evolution.
• Sharma, R., Pielstick, B. A., Bell, K. A., Nieman, T. B., Stubbs, O. A., Yeates, E. L., Baltrus, D. A., & Grose, J. H. (2019). A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against. Frontiers in microbiology, 10, 1533.
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  is a plant pathogen from the family and a causative agent of the devastating agricultural disease fire blight. Here we characterize eight lytic bacteriophages of that we isolated from the Wasatch front (Utah, United States) that are highly similar to vB_EamM_Ea35-70 which was isolated in Ontario, Canada. With the genome size ranging from 271 to 275 kb, this is a novel jumbo family of bacteriophages. These jumbo bacteriophages were further characterized through genomic and proteomic comparison, mass spectrometry, host range and burst size. Their proteomes are highly unstudied, with over 200 putative proteins with no known homologs. The production of 27 of these putative proteins was confirmed by mass spectrometry analysis. These bacteriophages appear to be most similar to bacteriophages that infect and rather than bacteria by protein similarity, however, we were only able to detect infection of and the closely related strains of .
• Smith, B. A., Leligdon, C., & Baltrus, D. A. (2019). Just the Two of Us? A Family of Pseudomonas Megaplasmids Offers a Rare Glimpse into the Evolution of Large Mobile Elements. Genome biology and evolution, 11(4), 1192-1206.
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  Pseudomonads are ubiquitous group of environmental proteobacteria, well known for their roles in biogeochemical cycling, in the breakdown of xenobiotic materials, as plant growth promoters, and as pathogens of a variety of host organisms. We have previously identified a large megaplasmid present within one isolate of the plant pathogen Pseudomonas syringae, and here we report that a second member of this megaplasmid family is found within an environmental Pseudomonad isolate most closely related to Pseudomonas putida. Many of the shared genes are involved in critical cellular processes like replication, transcription, translation, and DNA repair. We argue that presence of these shared pathways sheds new light on discussions about the types of genes that undergo horizontal gene transfer (i.e., the complexity hypothesis) as well as the evolution of pangenomes. Furthermore, although both megaplasmids display a high level of synteny, genes that are shared differ by over 50% on average at the amino acid level. This combination of conservation in gene order despite divergence in gene sequence suggests that this Pseudomonad megaplasmid family is relatively old, that gene order is under strong selection within this family, and that there are likely many more members of this megaplasmid family waiting to be found in nature.
• Baltrus, D. A., & Orth, K. N. (2018). Understanding genomic diversity in Pseudomonas syringae throughout the forest and on the trees. The New phytologist, 219(2), 482-484.
• Baltrus, D. A., Dougherty, K., Diaz, B., & Murillo, R. (2018). Evolutionary Plasticity of AmrZ Regulation in. mSphere, 3(2).
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  encodes a master regulator protein conserved across pseudomonads, which can be either a positive or negative regulator of swimming motility depending on the species examined. To better understand plasticity in the regulatory function of AmrZ, we characterized the mode of regulation for this protein for two different motility-related phenotypes in As in , AmrZ functions as a positive regulator of swimming motility within , which suggests that the functions of this protein with regard to swimming motility have switched at least twice across pseudomonads. Shifts in mode of regulation cannot be explained by changes in AmrZ sequence alone. We further show that AmrZ acts as a positive regulator of colony spreading within this strain and that this regulation is at least partially independent of swimming motility. Closer investigation of mechanistic shifts in dual-function regulators like AmrZ could provide unique insights into how transcriptional pathways are rewired between closely related species. Microbes often display finely tuned patterns of gene regulation across different environments, with major regulatory changes controlled by a small group of "master" regulators within each cell. AmrZ is a master regulator of gene expression across pseudomonads and can be either a positive or negative regulator for a variety of pathways depending on the strain and genomic context. Here, we demonstrate that the phenotypic outcomes of regulation of swimming motility by AmrZ have switched at least twice independently in pseudomonads, so that AmrZ promotes increased swimming motility in and but represses this phenotype in and Since examples of switches in regulatory mode are relatively rare, further investigation into the mechanisms underlying shifts in regulator function for AmrZ could provide unique insights into the evolution of bacterial regulatory proteins.
• Baltrus, D. A., Spraker, J., & Arnold, A. E. (2018). Quantifying Re-association of a Facultative Endohyphal Bacterium with a Filamentous Fungus. Methods in molecular biology (Clifton, N.J.), 1848, 1-11.
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  We present here a method to quantify reassociation between facultative endohyphal bacteria and filamentous fungal hosts. Our method takes advantage of the capabilities of fungal cell walls to selectively protect internal bacteria from gentamicin treatment, an assay adapted from studies of internalized bacterial pathogens in cell culture. We report the efficacy of gentamicin to kill planktonic bacteria treated during fungal coculture, and also describe and characterize a sampling scheme to recover and quantify culturable bacteria from the growing edge of fungal mycelium in vitro. This assay enables qualitative and quantitative tests of reassociation capabilities for facultative endohyphal bacteria with host fungi and provides a means to investigate the genetic basis for these associations in a repeatable way.
• Fitzpatrick, C. R., Lu-Irving, P., Copeland, J., Guttman, D. S., Wang, P. W., Baltrus, D. A., Dlugosch, K. M., & Johnson, M. T. (2018). Chloroplast sequence variation and the efficacy of peptide nucleic acids for blocking host amplification in plant microbiome studies. Microbiome, 6(1), 144.
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  The ability to efficiently characterize microbial communities from host individuals can be limited by co-amplification of host organellar sequences (mitochondrial and/or plastid), which share a common ancestor and thus sequence similarity with extant bacterial lineages. One promising approach is the use of sequence-specific peptide nucleic acid (PNA) clamps, which bind to, and block amplification of, host-derived DNA. Universal PNA clamps have been proposed to block host plant-derived mitochondrial (mPNA) and plastid (pPNA) sequences at the V4 16S rRNA locus, but their efficacy across a wide range of host plant species has not been experimentally tested.
• Justin, S. P., Zalamea, P., Sarmiento, C., Gallery, R. E., Dalling, J. W., Davis, A. S., Baltrus, D. A., & Arnold, A. E. (2018). Context-dependent and variable effects of endohyphal bacteria on interactions between fungi and seeds. Fungal Ecology, 36, 117-127. doi:https://doi.org/10.1016/j.funeco.2018.08.008
• Sharma, R., Berg, J. A., Beatty, N. J., Choi, M. C., Cowger, A. E., Cozzens, B. J., Duncan, S. G., Fajardo, C. P., Ferguson, H. P., Galbraith, T., Herring, J. A., Hoj, T. R., Durrant, J. L., Hyde, J. R., Jensen, G. L., Ke, S. Y., Killpack, S., Kruger, J. L., Lawrence, E. E., , Nwosu, I. O., et al. (2018). Genome Sequences of Nine Erwinia amylovora Bacteriophages. Microbiology resource announcements, 7(14).
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  Erwinia amylovora is a plant pathogen belonging to the Enterobacteriaceae family, a family containing many plant and animal pathogens. Herein, we announce nine genome sequences of E. amylovora bacteriophages isolated from infected apple trees along the Wasatch Front in Utah.
• Araldi-Brondolo, S. J., Spraker, J., Shaffer, J. P., Woytenko, E. H., Baltrus, D. A., Gallery, R. E., & Arnold, A. E. (2017). Bacterial Endosymbionts: Master Modulators of Fungal Phenotypes. Microbiology spectrum, 5(5).
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  The ecological modes of fungi are shaped not only by their intrinsic features and the environment in which they occur, but also by their interactions with diverse microbes. Here we explore the ecological and genomic features of diverse bacterial endosymbionts-endohyphal bacteria-that together are emerging as major determinants of fungal phenotypes and plant-fungi interactions. We first provide a historical perspective on the study of endohyphal bacteria. We then propose a functional classification of three main groups, providing an overview of their genomic, phylogenetic, and ecological traits. Last, we explore frontiers in the study of endohyphal bacteria, with special attention to those facultative and horizontally transmitted bacteria that associate with some of the most diverse lineages of fungi. Overall, our aim is to synthesize the rich literature from nearly 50 years of studies on endohyphal bacteria as a means to highlight potential applications and new research directions.
• Baltrus, D. A. (2017). Adaptation, specialization, and coevolution within phytobiomes.. Current opinion in plant biology, 38, 109-116. doi:10.1016/j.pbi.2017.04.023
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  Growth patterns of individual plants and evolutionary trajectories of plant communities are intimately linked with and are critically affected by host-associated microbiomes. Research across systems has begun to shed light on how these phytobiomes are established under laboratory and natural conditions, and have cultivated hope that a better understanding of the governing principles for host-microbe interactions can guide attempts to engineer microbiomes to boost agricultural yields. One important, yet relatively understudied, parameter in regards to phytobiome membership is the degree to which specialization and coevolution between plant species and microbes provides structure to these communities. In this article, I provide an overview of mechanisms enabling adaptation and specialization of phytobiome communities to host plants as well as the potential for plants themselves to recruit and cultivate beneficial interactions. I further explore the possibility of host-beneficial microbe coevolution and suggest particular situations that could promote the evolution of such close-knit partnerships. It is my hope that this overview will encourage future experiments that can begin to fill in this black box of ecological and evolutionary interactions across phytobiomes.
• Baltrus, D. A., Dougherty, K., Arendt, K. R., Huntemann, M., Clum, A., Pillay, M., Palaniappan, K., Varghese, N., Mikhailova, N., Stamatis, D., Reddy, T. B., Ngan, C. Y., Daum, C., Shapiro, N., Markowitz, V., Ivanova, N., Kyrpides, N., Woyke, T., & Arnold, A. E. (2017). Absence of genome reduction in diverse, facultative endohyphal bacteria. Microbial genomics, 3(2), e000101.
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  Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.
• Baltrus, D. A., Dougherty, K., Arendt, K., Huntemann, M., Clum, A., Pillay, M., Palaniappan, K., Vargese, N., Mikhailova, N., Stamatis, D., Reddy, T., Ngan, C. Y., Daum, C., Shapiro, N., Markowitz, V., Ivanova, N., Kyrpides, N., Woyke, T., Arnold, A. E., , Baltrus, D. A., et al. (2017). Absence of genome reduction in diverse, facultative endohyphal bacteria. Microbial Genomics, 3, e000101.
• Hockett, K. L., & Baltrus, D. A. (2017). Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds. Journal of visualized experiments : JoVE.
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  The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.
• Mccann, H. C., Guttman, D. S., & Baltrus, D. A. (2017). Evolution, genomics and epidemiology of Pseudomonas syringae: Challenges in Bacterial Molecular Plant Pathology.. Molecular plant pathology, 18(1), 152-168. doi:10.1111/mpp.12506
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  A remarkable shift in our understanding of plant-pathogenic bacteria is underway. Until recently, nearly all research on phytopathogenic bacteria was focused on a small number of model strains, which provided a deep, but narrow, perspective on plant-microbe interactions. Advances in genome sequencing technologies have changed this by enabling the incorporation of much greater diversity into comparative and functional research. We are now moving beyond a typological understanding of a select collection of strains to a more generalized appreciation of the breadth and scope of plant-microbe interactions. The study of natural populations and evolution has particularly benefited from the expansion of genomic data. We are beginning to have a much deeper understanding of the natural genetic diversity, niche breadth, ecological constraints and defining characteristics of phytopathogenic species. Given this expanding genomic and ecological knowledge, we believe the time is ripe to evaluate what we know about the evolutionary dynamics of plant pathogens.
• Shaffer, J. P., U'Ren, J. M., Baltrus, D. A., Gallery, R. E., Arnold, A. E., Shaffer, J. P., U'Ren, J. M., Baltrus, D. A., Gallery, R. E., Arnold, A. E., Shaffer, J. P., U'Ren, J. M., Baltrus, D. A., Gallery, R. E., & Arnold, A. E. (2017). An endohyphal bacterium (Chitinophaga, Bacteroidetes) influences carbon source use by Fusarium keratoplasticum (F. solani species complex, Nectriaceae). Frontiers in Microbiology, 8, e350.
• Shaffer, J. P., U'Ren, J. M., Gallery, R. E., Baltrus, D. A., & Arnold, A. E. (2017). An Endohyphal Bacterium (Chitinophaga, Bacteroidetes) Alters Carbon Source Use by Fusarium keratoplasticum (F. solani Species Complex, Nectriaceae). Frontiers in microbiology, 8, 350.
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  Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog® phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant- and seed-fungus interactions, such as D-trehalose, myo-inositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusarium species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus-bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.
• Smee, M. R., Baltrus, D. A., & Hendry, T. A. (2017). Entomopathogenicity to Two Hemipteran Insects Is Common but Variable across Epiphytic Pseudomonas syringae Strains. Frontiers in plant science, 8, 2149.
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  Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci, and the pea aphid, Acyrthosiphon pisum, both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be non-pathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.
• Williams, L. E., Baltrus, D. A., O'Donnell, S. D., Skelly, T. J., & Martin, M. O. (2017). Complete Genome Sequence of the Predatory Bacterium Ensifer adhaerens Casida A. Genome announcements, 5(47).
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  We report here the complete genome sequence of the facultative predatory bacterium Ensifer adhaerens strain Casida A. The genome was assembled into three circular contigs, with a main chromosome as well as two large secondary replicons, that totaled 7,267,502 bp with 6,641 predicted open reading frames.
• Arendt, K. R., Hockett, K. L., Araldi-Brondolo, S. J., Baltrus, D. A., & Arnold, A. E. (2016). Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations. Applied and environmental microbiology, 82(10), 2943-9.
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  Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish in vitro conditions favoring reintroduction of two strains of EHB (Luteibacter sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes: Microdiplodia sp. (Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl2 prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.
• Arnold, A. E., Shaffer, J. P., Sarmiento, C., Zalamea, P., Gallery, R. E., Davis, A. S., & Baltrus, D. A. (2016). Diversity, Specificity, and Phylogenetic Relationships of Endohyphal Bacteria in Fungi that Inhabit Tropical Seeds and Leaves. Frontiers Ecology and Evolution. doi:https://doi.org/10.3389/fevo.2016.00116
• Baltrus, D. A. (2016). Divorcing Strain Classification from Species Names. Trends in microbiology (Opinion, Not a Review), 24(6), 431-9.
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  Confusion about strain classification and nomenclature permeates modern microbiology. Although taxonomists have traditionally acted as gatekeepers of order, the numbers of, and speed at which, new strains are identified has outpaced the opportunity for professional classification for many lineages. Furthermore, the growth of bioinformatics and database-fueled investigations have placed metadata curation in the hands of researchers with little taxonomic experience. Here I describe practical challenges facing modern microbial taxonomy, provide an overview of complexities of classification for environmentally ubiquitous taxa like Pseudomonas syringae, and emphasize that classification can be independent of nomenclature. A move toward implementation of relational classification schemes based on inherent properties of whole genomes could provide sorely needed continuity in how strains are referenced across manuscripts and data sets.
• Hendry, T. A., Clark, K. J., & Baltrus, D. A. (2016). A highly infective plant-associated bacterium influences reproductive rates in pea aphids. Royal Society open science, 3(2), 150478.
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  Pea aphids, Acyrthosiphon pisum, have the potential to increase reproduction as a defence against pathogens, though how frequently this occurs or how infection with live pathogens influences this response is not well understood. Here we determine the minimum infective dose of an environmentally common bacterium and possible aphid pathogen, Pseudomonas syringae, to determine the likelihood of pathogenic effects to pea aphids. Additionally, we used P. syringae infection to investigate how live pathogens may alter reproductive rates. We found that oral bacterial exposure decreased subsequent survival of aphids in a dose-dependent manner and we estimate that ingestion of less than 10 bacterial cells is sufficient to increase aphid mortality. Pathogen dose was positively related to aphid reproduction. Aphids exposed to low bacterial doses showed decreased, although statistically indistinguishable, fecundity compared to controls. Aphids exposed to high doses reproduced significantly more than low dose treatments and also more, but not significantly so, than controls. These results are consistent with previous studies suggesting that pea aphids may use fecundity compensation as a response to pathogens. Consequently, even low levels of exposure to a common plant-associated bacterium may therefore have significant effects on pea aphid survival and reproduction.
• Hockett, K. L., Renner, T., & Baltrus, D. A. (2015). Independent Co-Option of a Tailed Bacteriophage into a Killing Complex in Pseudomonas. mBio, 6(4), e00452.
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  Competition between microbes is widespread in nature, especially among those that are closely related. To combat competitors, bacteria have evolved numerous protein-based systems (bacteriocins) that kill strains closely related to the producer. In characterizing the bacteriocin complement and killing spectra for the model strain Pseudomonas syringae B728a, we discovered that its activity was not linked to any predicted bacteriocin but is derived from a prophage. Instead of encoding an active prophage, this region encodes a bacteriophage-derived bacteriocin, termed an R-type syringacin. This R-type syringacin is striking in its convergence with the well-studied R-type pyocin of P. aeruginosa in both genomic location and molecular function. Genomic alignment, amino acid percent sequence identity, and phylogenetic inference all support a scenario where the R-type syringacin has been co-opted independently of the R-type pyocin. Moreover, the presence of this region is conserved among several other Pseudomonas species and thus is likely important for intermicrobial interactions throughout this important genus.
• Baltrus, D. A., Dougherty, K., Beckstrom-Sternberg, S. M., Beckstrom-Sternberg, J. S., & Foster, J. T. (2014). Incongruence between multi-locus sequence analysis (MLSA) and whole-genome-based phylogenies: Pseudomonas syringae pathovar pisi as a cautionary tale. Molecular Plant Pathology.
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  Abstract: Previous phylogenies, built using a subset of genomic loci, split Pseudomonas syringae pv. pisi into two well-supported clades and implied convergence in host range for these lineages. The analysis of phenotypic and genotypic data within the context of this phylogenetic relationship implied further convergence at the level of virulence gene loss and acquisition. We generate draft genome assemblies for two additional P.syringae strains, isolated from diseased pea plants, and demonstrate incongruence between phylogenies created from a subset of the data compared with the whole genomes. Our whole-genome analysis demonstrates that strains classified as pv. pisi actually form a coherent monophyletic clade, so that apparent convergence is actually the product of shared ancestry. We use this example to urge caution when making evolutionary inferences across closely related strains of P.syringae. © 2013 BSPP AND JOHN WILEY & SONS LTD.
• Baltrus, D. A., Yourstone, S., Lind, A., Guilbaud, C., Sands, D. C., Jones, C. D., Morris, C. E., & Dangl, J. L. (2014). Draft Genome Sequences of a Phylogenetically Diverse Suite of Pseudomonas syringae Strains from Multiple Source Populations. Genome announcements, 2(1).
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  Here, we report the draft genome sequences for 7 phylogenetically diverse isolates of Pseudomonas syringae, obtained from numerous environmental sources and geographically proximate crop species. Overall, these sequences provide a wealth of information about the differences (or lack thereof) between isolates from disease outbreaks and those from other sources.
• Bartoli, C., Berge, O., Monteil, C. L., Guilbaud, C., Balestra, G. M., Varvaro, L., Jones, C., Dangl, J. L., Baltrus, D. A., Sands, D. C., & Morris, C. E. (2014). The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits. Environmental microbiology, 16(7), 2301-15.
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  As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.
• Dougherty, K., Smith, B. A., Moore, A. F., Maitland, S., Fanger, C., Murillo, R., & Baltrus, D. A. (2014). Multiple phenotypic changes associated with large-scale horizontal gene transfer. PloS one, 9(7), e102170.
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  Horizontal gene transfer often leads to phenotypic changes within recipient organisms independent of any immediate evolutionary benefits. While secondary phenotypic effects of horizontal transfer (i.e., changes in growth rates) have been demonstrated and studied across a variety of systems using relatively small plasmids and phage, little is known about the magnitude or number of such costs after the transfer of larger regions. Here we describe numerous phenotypic changes that occur after a large-scale horizontal transfer event (∼1 Mb megaplasmid) within Pseudomonas stutzeri including sensitization to various stresses as well as changes in bacterial behavior. These results highlight the power of horizontal transfer to shift pleiotropic relationships and cellular networks within bacterial genomes. They also provide an important context for how secondary effects of transfer can bias evolutionary trajectories and interactions between species. Lastly, these results and system provide a foundation to investigate evolutionary consequences in real time as newly acquired regions are ameliorated and integrated into new genomic contexts.
• Hendry, T. A., Hunter, M. S., & Baltrus, D. A. (2014). The Facultative Symbiont Rickettsia Protects an Invasive Whitefly Against Entomopathogenic Pseudomonas syringae Strains. Applied and environmental microbiology.
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  Facultative endosymbionts can benefit insect hosts in a variety of ways, including context dependent roles such as providing defense against pathogens. The role of some symbionts in defense may be overlooked, however, when pathogen infection is transient, sporadic, or asymptomatic. The facultative endosymbiont Rickettsia increases the fitness of the sweet potato whitefly (Bemisia tabaci) in some populations through mechanisms that are not yet understood. In this study we investigated the role of Rickettsia in mediating the interaction between the sweet potato whitefly and Pseudomonas syringae, a common environmental bacterium, some strains of which are pathogenic to aphids. Our results show that P. syringae multiplies within whiteflies leading to host death and that whiteflies infected with Rickettsia show a decreased rate of death due to P. syringae. Experiments using plants coated with P. syringae confirmed that whiteflies can acquire the bacteria at a low rate while feeding, leading to increased mortality, particularly when the whiteflies are not infected with Rickettsia. These results suggest that P. syringae may affect whitefly populations in nature and that Rickettsia can ameliorate this effect. This study highlights the possible importance of interactions among opportunistic environmental pathogens and endosymbionts of insects.
• Hockett, K. L., Nishimura, M. T., Karlsrud, E., Dougherty, K., & Baltrus, D. A. (2014). Pseudomonas syringae CC1557: a highly virulent strain with an unusually small type III effector repertoire that includes a novel effector. Molecular plant-microbe interactions : MPMI, 27(9), 923-32.
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  Both type III effector proteins and nonribosomal peptide toxins play important roles for Pseudomonas syringae pathogenicity in host plants, but whether and how these pathways interact to promote infection remains unclear. Genomic evidence from one clade of P. syringae suggests a tradeoff between the total number of type III effector proteins and presence of syringomycin, syringopeptin, and syringolin A toxins. Here, we report the complete genome sequence from P. syringae CC1557, which contains the lowest number of known type III effectors to date and has also acquired genes similar to sequences encoding syringomycin pathways from other strains. We demonstrate that this strain is pathogenic on Nicotiana benthamiana and that both the type III secretion system and a new type III effector, hopBJ1, contribute to pathogenicity. We further demonstrate that activity of HopBJ1 is dependent on residues structurally similar to the catalytic site of Escherichia coli CNF1 toxin. Taken together, our results provide additional support for a negative correlation between type III effector repertoires and the potential to produce syringomycin-like toxins while also highlighting how genomic synteny and bioinformatics can be used to identify and characterize novel virulence proteins.
• Mucyn, T. S., Yourstone, S., Lind, A. L., Biswas, S., Nishimura, M. T., Baltrus, D. A., Cumbie, J. S., Chang, J. H., Jones, C. D., Dangl, J. L., & Grant, S. R. (2014). Variable suites of non-effector genes are co-regulated in the type III secretion virulence regulon across the Pseudomonas syringae phylogeny. PLoS pathogens, 10(1), e1003807.
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  Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better insight into the evolution of virulence across an evolutionarily diverse pathogen species.
• Romanchuk, A., Jones, C. D., Karkare, K., Moore, A., Smith, B. A., Jones, C., Dougherty, K., & Baltrus, D. A. (2014). Bigger is not always better: transmission and fitness burden of ∼1MB Pseudomonas syringae megaplasmid pMPPla107. Plasmid, 73, 16-25.
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  Horizontal gene transfer (HGT) is a widespread process that enables the acquisition of genes and metabolic pathways in single evolutionary steps. Previous reports have described fitness costs of HGT, but have largely focused on the acquisition of relatively small plasmids. We have previously shown that a Pseudomonas syringae pv. lachrymans strain recently acquired a cryptic megaplasmid, pMPPla107. This extrachromosomal element contributes hundreds of new genes to P. syringae and increases total genomic content by approximately 18%. However, this early work did not directly explore transmissibility, stability, or fitness costs associated with acquisition of pMPPla107.
• Smith, B. A., Dougherty, K. M., & Baltrus, D. A. (2014). Complete Genome Sequence of the Highly Transformable Pseudomonas stutzeri Strain 28a24. Genome announcements, 2(3).
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  Here, we report the complete genome sequence for an isolate of Pseudomonas stutzeri that is highly competent for natural transformation. This sequence enables insights into the genetic basis of natural transformation rate variations and provides an additional data point for genomic comparisons across a ubiquitous and highly diverse bacterial species.
• Baltrus, D. A. (2013). Exploring the costs of horizontal gene transfer. Trends in Ecology and Evolution, 28(8), 489-495.
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  PMID: 23706556;Abstract: Horizontal gene transfer (HGT) is one of the most important evolutionary forces within microbial populations. Although evidence for beneficial fitness effects of HGT is overwhelming, recently acquired regions often function inefficiently within new genomic backgrounds so that each transfer event has the potential to disrupt existing regulatory and physiological networks. Identifying and exploring costs is essential for guiding general discussions about the interplay between selection and HGT, as well as generating hypotheses to explain how HGT affects evolutionary potential through, for example, changing adaptive trajectories. Focusing on costs of HGT as foundations for future studies will enhance exploration at the interface between acquired regions and recipient genomes, including the process of amelioration, and enable experimental evaluation of the role of HGT in structuring genetic diversity across populations. © 2013 Elsevier Ltd.
• Baltrus, D., & Baltrus, D. A. (2013). Exploring the costs of horizontal gene transfer. Trends in ecology & evolution, 28(8).
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  Horizontal gene transfer (HGT) is one of the most important evolutionary forces within microbial populations. Although evidence for beneficial fitness effects of HGT is overwhelming, recently acquired regions often function inefficiently within new genomic backgrounds so that each transfer event has the potential to disrupt existing regulatory and physiological networks. Identifying and exploring costs is essential for guiding general discussions about the interplay between selection and HGT, as well as generating hypotheses to explain how HGT affects evolutionary potential through, for example, changing adaptive trajectories. Focusing on costs of HGT as foundations for future studies will enhance exploration at the interface between acquired regions and recipient genomes, including the process of amelioration, and enable experimental evaluation of the role of HGT in structuring genetic diversity across populations.
• Sarris, P. F., Trantas, E. A., Baltrus, D. A., Bull, C. T., Wechter, W. P., Yan, S., Ververidis, F., Almeida, N. F., Jones, C. D., Dangl, J. L., Panopoulos, N. J., Vinatzer, B. A., & Goumas, D. E. (2013). Comparative Genomics of Multiple Strains of Pseudomonas cannabina pv. alisalensis, a Potential Model Pathogen of Both Monocots and Dicots. PLoS ONE, 8(3).
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  PMID: 23555661;PMCID: PMC3610874;Abstract: Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.
• Sarris, P. F., Trantas, E. A., Baltrus, D. A., Bull, C. T., Wechter, W. P., Yan, S., Ververidis, F., Almeida, N. F., Jones, C. D., Dangl, J. L., Panopoulos, N. J., Vinatzer, B. A., & Goumas, D. E. (2013). Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots.. PLoS ONE.
• Baltrus, D. A., Nishimura, M. T., Dougherty, K. M., Biswas, S., Mukhtar, M. S., Vicente, J., Holub, E. B., & Dangl, J. L. (2012). The molecular basis of host specialization in bean pathovars of Pseudomonas syringae. Molecular Plant-Microbe Interactions, 25(7), 877-888.
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  PMID: 22414441;Abstract: Biotrophic phytopathogens are typically limited to their adapted host range. In recent decades, investigations have teased apart the general molecular basis of intraspecific variation for innate immunity of plants, typically involving receptor proteins that enable perception of pathogen-associated molecular patterns or avirulence elicitors from the pathogen as triggers for defense induction. However, general consensus concerning evolutionary and molecular factors that alter host range across closely related phytopathogen isolates has been more elusive. Here, through genome comparisons and genetic manipulations, we investigate the underlying mechanisms that structure host range across closely related strains of Pseudomonas syringae isolated from different legume hosts. Although type III secretionindependent virulence factors are conserved across these three strains, we find that the presence of two genes encoding type III effectors (hopC1 and hopM1) and the absence of another (avrB2) potentially contribute to host range differences between pathovars glycinea and phaseolicola. These findings reinforce the idea that a complex genetic basis underlies host range evolution in plant pathogens. This complexity is present even in host-microbe interactions featuring relatively little divergence among both hosts and their adapted pathogens. © 2012 The American Phytopathological Society.
• Baltrus, D., Nishimura, M., Dougherty, K., Biswas, S., Mukhtar, M., Vicente, J., Holub, E., & Dangl, J. (2012). The molecular basis of host specialization in bean pathovars of Pseudomonas syringae. Mol Plant Microbe Interact, 25(7), 877-888.
• Hao, X., Lin, Y., Johnstone, L., Baltrus, D. A., Miller, S. J., Wei, G., & Rensing, C. (2012). Draft genome sequence of plant growth-promoting rhizobium Mesorhizobium amorphae, isolated from zinc-lead mine tailings. Journal of Bacteriology, 194(3), 736-737.
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  PMID: 22247533;PMCID: PMC3264094;Abstract: Here, we describe the draft genome sequence of Mesorhizobium amorphae strain CCNWGS0123, isolated from nodules of Robinia pseudoacacia growing on zinc-lead mine tailings. A large number of metal(loid) resistance genes, as well as genes reported to promote plant growth, were identified, presenting a great future potential for aiding phytoremediation in metal(loid)-contaminated soil. © 2012, American Society for Microbiology.
• Baltrus, D. A., Nishimura, M. T., Romanchuk, A., Chang, J. H., Mukhtar, M. S., Cherkis, K., Roach, J., Grant, S. R., Jones, C. D., & Dangl, J. L. (2011). Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 pseudomonas syringae isolates. PLoS Pathogens, 7(7).
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  PMID: 21799664;PMCID: PMC3136466;Abstract: Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. © 2011 Baltrus et al.
• Lin, Y., Hao, X., Johnstone, L., Miller, S. J., Baltrus, D. A., Rensing, C., & Wei, G. (2011). Draft genome of Streptomyces zinciresistens K42, a novel metal-resistant species isolated from copper-zinc mine tailings. Journal of Bacteriology, 193(22), 6408-6409.
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  PMID: 22038968;PMCID: PMC3209202;Abstract: A draft genome sequence of Streptomyces zinciresistens K42, a novel Streptomyces species displaying a high level of resistance to zinc and cadmium, is presented here. The genome contains a large number of genes encoding proteins predicted to be involved in conferring metal resistance. Many of these genes appear to have been acquired through horizontal gene transfer. © 2011, American Society for Microbiology.
• Baltrus, D. A., Amieva, M. R., Covacci, A., Lowe, T. M., Merrell, D. S., Ottemann, K. M., Stein, M., Salama, N. R., & Guillemin, K. (2009). The complete genome sequence of Helicobacter pylori strain G27. Journal of Bacteriology, 91(1), 447-448.
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  PMID: 18952803;PMCID: PMC2612421;Abstract: Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the world's population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Baltrus, D. A., Blaser, M. J., & Guillemin, K. (2009). Helicobacter pylori genome plasticity. Genome Dynamics, 6, 75-90.
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  PMID: 19696495;Abstract: Helicobacter pylori, a Gram-negative pathogen associated with ulcers, chronic gastritis, and gastric cancers, has been a resident of the human stomach since early human history [1]. This association has only recently begun to erode with the advent of antibiotics and modern lifestyles, but even today H. pylori colonizes approximately half the world's population. To have remained a successful colonizer of humans during thousands of years of association, populations of H. pylori must have been able to survive and adapt to countless evolutionary challenges within and between hosts. As a species, H. pylori possesses one of the most fluid genomes within the prokaryotic kingdom [2], a characteristic that has likely aided its continued success. H. pylori exhibits exceptionally high rates of DNA point mutations, intragenomic recombination (facilitated by repetitive elements common in H. pylori genomes), and intergenomic recombination (mediated by natural transformation), all of which contribute to the high genomic variability between isolates. Previous reviews have focused on these processes as agents of evolutionary change within H. pylori [2-8]. The mechanisms of both mutation and natural transformation, and the evolutionary processes that retain genetic variation generated by these mechanisms, dictate the extent to which each contributes to genomic diversity in the context of different bacterial population structures [9-13]. Unlike well-studied evolutionary systems, such as Salmonella and Escherichia coli, H. pylori is notable in its lack of an environmental reservoir outside of human and other primate stomachs, suggesting that between-host survival is a relatively weak determinant of selection pressures [14, 15]. Given that H. pylori exist largely as distinct host-associated populations, it is possible to begin to model the evolutionary mechanisms that affect the long-term persistence of this species. In this chapter, we consider how the attributes of H. pylori's natural history as a long-term resident of the human stomach and the specific mechanisms of mutation and genetic exchange in this organism have shaped the H. pylori genome. We begin with a survey of genome plasticity in H. pylori. We then discuss mechanisms of mutation and natural transformation in H. pylori and examine experimental evidence for the generation of genomic changes within populations. Finally, we consider how different models of H. pylori population structure affect the relative contributions of mutation and recombination to the evolutionary success of this organism. By bridging evolutionary studies with investigations of pathogenesis from a molecular perspective, we hope to shed new light on how H. pylori has and continues to evolve with its human hosts. Copyright © 2009 S. Karger AG, Basel.
• Reinhardt, J. A., Baltrus, D. A., Nishimura, M. T., Jeck, W. R., Jones, C. D., & Dangl, J. L. (2009). De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae. Genome Research, 19(2), 294-305.
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  PMID: 19015323;PMCID: PMC2652211;Abstract: We developed a novel approach for de novo genome assembly using only sequence data from high-throughput short read sequencing technologies. By combining data generated from 454 Life Sciences (Roche) and Illumina (formerly known as Solexa sequencing) sequencing platforms, we reliably assembled genomes into large scaffolds at a fraction of the traditional cost and without use of a reference sequence. We applied this method to two isolates of the phytopathogenic bacteria Pseudomonas syringae. Sequencing and reassembly of the well-studied tomato and Arabidopsis pathogen, PtoDC3000, facilitated development and testing of our method. Sequencing of a distantly related rice pathogen, Por1-6, demonstrated our method's efficacy for de novo assembly of novel genomes. Our assembly of Por1-6 yielded an N50 scaffold size of 531,821 bp with >75% of the predicted genome covered by scaffolds over 100,000 bp. One of the critical phenotypic differences between strains of P. syringae is the range of plant hosts they infect. This is largely determined by their complement of type III effector proteins. The genome of Por1-6 is the first sequenced for a P. syringae isolate that is a pathogen of monocots, and, as might be predicted, its complement of type III effectors differs substantially from the previously sequenced isolates of this species. The genome of Por1-6 helps to define an expansion of the P. syringae pan-genome, a corresponding contraction of the core genome, and a further diversification of the type III effector complement for this important plant pathogen species. © 2009 by Cold Spring Harbor Laboratory Press.
• Baltrus, D. A., Guillemin, K., & Phillips, P. C. (2008). Natural transformation increases the rate of adaptation in the human pathogen Helicobacter pylori. Evolution, 62(1), 39-49.
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  PMID: 17976191;Abstract: Gene exchange between individuals can lead to profound evolutionary effects at both the genomic and population levels. These effects have sparked widespread interest in examining the specific adaptive benefits of recombination. Although this work has primarily focused on the benefits of sex in eukaryotes, it is assumed that similar benefits of genetic exchange apply across eukaryotes and prokaryotes. Here we report a direct test of this assumption using the naturally transformable human gastric pathogen Helicobacter pylori as a model organism. We show that genetic exchange accelerates adaptation to a novel laboratory environment within bacterial populations and that a general adaptive advantage exists for naturally transformable strains when transfer occurs among conspecific backgrounds. This finding demonstrates that there are generalized benefits to adaptation in both eukaryotes and prokaryotes even though the underlying processes are mechanistically different. © 2007 The Author(s).
• Jeck, W. R., Reinhardt, J. A., Baltrus, D. A., Hickenbotham, M. T., Magrini, V., Mardis, E. R., Dangl, J. L., & Jones, C. D. (2007). Extending assembly of short DNA sequences to handle error. Bioinformatics, 23(21), 2942-2944.
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  PMID: 17893086;Abstract: Inexpensive de novo genome sequencing, particularly in organisms with small genomes, is now possible using several new sequencing technologies. Some of these technologies such as that from Illumina's Solexa Sequencing, produce high genomic coverage by generating a very large number of small reads (∼30 bp). While prior work shows that partial assembly can be performed by k-mer extension in error-free reads, this algorithm is unsuccessful with the sequencing error rates found in practice. We present VCAKE (Verified Consensus Assembly by K-mer Extension), a modification of simple k-mer extension that overcomes error by using high depth coverage. Though it is a simple modification of a previous approach, we show significant improvements in assembly results on simulated and experimental datasets that include error. © 2007 The Author(s).
• Mole, B. M., Baltrus, D. A., Dangl, J. L., & Grant, S. R. (2007). Global virulence regulation networks in phytopathogenic bacteria. Trends in Microbiology, 15(8), 363-371.
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  PMID: 17627825;Abstract: Phytopathogens coordinate multifaceted life histories and deploy stratified virulence determinants via complex, global regulation networks. We dissect the global regulation of four distantly related model phytopathogens to evaluate large-scale events and mechanisms that determine successful pathogenesis. Overarching themes include dependence on centralized cell-to-cell communication systems, pervasive two-component signal-transduction systems, post-transcriptional regulation systems, AraC-like regulators and sigma factors. Although these common regulatory systems control virulence, each functions in different capacities, and to differing ends, in the diverse species. Hence, the virulence regulation network of each species determines its survival and success in various life histories and niches. © 2006 Elsevier Ltd. All rights reserved.
• Baltrus, D. A., & Guillemin, K. (2006). Multiple phases of competence occur during the Helicobacter pylori growth cycle. FEMS Microbiology Letters, 255(1), 148-155.
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  PMID: 16436074;Abstract: The gastric pathogen Helicobacter pylori undergoes genetic exchange at unusually high frequencies, primarily through natural transformation. Despite progress toward understanding the molecular mechanism of natural transformation in H. pylori, little is known about how competence is regulated or its relationship to DNA release. By measuring transformation incrementally throughout the growth curve, we show that H. pylori exhibits a novel pattern of competence with distinct peaks of transformation during both logarithmic and stationary growth phases. Furthermore, different H. pylori strains vary in the presence and timing of their competence peaks. We also examined the process of DNA release in relation to competence. Although extensive DNA release does not occur until late stationary phase, sufficient genomic DNA was present during the logarithmic phase to yield measurable transformants. These results demonstrate that the state of competence in H. pylori occurs in an unprecedented pattern during the growth curve with no clear relationship to DNA release. © 2005 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
• Cresko, W. A., Yan, Y., Baltrus, D. A., Amores, A., Singer, A., Rodríguez-Marí, A., & Postlethwait, J. H. (2003). Genome Duplication, Subfunction Partitioning, and Lineage Divergence: Sox9 in Stickleback and Zebrafish. Developmental Dynamics, 228(3), 480-489.
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  PMID: 14579386;Abstract: Teleosts are the most species-rich group of vertebrates, and a genome duplication (tetraploidization) event in ray-fin fish appears to have preceded this remarkable explosion of biodiversity. What is the relationship of the ray-fin genome duplication to the teleost radiation? Genome duplication may have facilitated lineage divergence by partitioning different ancestral gene subfunctions among co-orthologs of tetrapod genes in different teleost lineages. To test this hypothesis, we investigated gene expression patterns for Sox9 gene duplicates in stickleback and zebrafish, teleosts whose lineages diverged early in Euteleost evolution. Most expression domains appear to have been partitioned between Sox9a and Sox9b before the divergence of stickleback and zebrafish lineages, but some ancestral expression domains were distributed differentially in each lineage. We conclude that some gene subfunctions, as represented by lineage-specific expression domains, may have assorted differently in separate lineages and that these may have contributed to lineage diversification during teleost evolution. © 2003 Wiley-Liss, Inc.

Presentations

• Baltrus, D. A., & Baltrus, D. A. (2022, Fall). Developing phage-derived bacteriocins as strain specific antibacterials. Invited SeminarUniversity of Illinois Urbana Champaign.
• Baltrus, D. A. (2022, Spring). Developing phage-derived bacteriocins as strain specific antibacterials. Invited SeminarUniversity of Delaware.
• Baltrus, D. A. (2020, Jan/Spring). Are all Pseudomonas syringae infections the same? How modern sequencing tech enables investigation of genomic diversity within a single microbial "species". Plant and Animal Genome. San Diego, California: Scherago Internatinal.
• Baltrus, D. A. (2019, April/Spring). Harnessing Inter-microbial Interactions to Shape Evolutionary and Ecological Dynamics in Host Associated Communities. Faculty Seminar. Davis, California: UC Davis Plant Pathology.
• Baltrus, D. A. (2019, April/Spring). Opportunities and Challenges in Real Time Sequence Based Identification of Phytopathogens. National Plant Diagnostic Network Meeting. Indianapolis, Indiana: NPDN.
• Baltrus, D. A. (2019, July/Summer). The Evolution of Strain Specificity in Pseudomonas Tailocins. Pseudomonas 2019. Kuala Lumpur, Malaysia: Pseudomonas.
• Baltrus, D. A. (2019, June/Summer). How Microbial (and Host) Biogeography Affects Phytobiome Composition and Assembly. ASM Microbe. San Francisco, California: American Society for Microbiology.
• Baltrus, D. A. (2019, Sept/Fall). Harnessing inter-microbial interactions to shape evolutionary and ecological dynamics in host-associated communities. UM Microbiome Madness Symposium. Minneapolis, Minnesota: University of Minnesota.
• Baltrus, D. A. (2019, Sept/Fall). How Biogeography Shapes Microbiome Structure. Ecosystem Genomics Class at UA. University of Arizona: University of Arizona Ecosystem Genomics.
• Baltrus, D. A., Clark, M., & Hockett, K. L. (2017, July). Conditional Targeting of Phage Derived Tailocins in Pseudomonas syringae. GRC Microbial Populations. Andover, NH: Gordon Research Conferences.
• Baltrus, D. A. (2015, June). Microbiomes inside of microbes. ASM Annual Meeting. New Orleans, LA: American Society for Microbiology.
• Baltrus, D. A. (2014, Dec). A Microbial Matryoshka Principle: Facultative Bacterial Symbionts of Fungi Shape Phyllosphere Diversity and Functions. Invited Seminar Speaker. Provo, Utah: Brigham Young University.
• Baltrus, D. A. (2014, Feb). How comparative genomics breathes new life into studies of bacterial pathogenesis and death. Invited Seminar Speaker. Knoxville Tennessee: University of Tennessee Knoxville.
• Baltrus, D. A., Arendt, K., & Arnold, A. E. (2014, June). Phenotypic Effects and Host Specificity of Endohyphal Bacteria. Evolution 2014.
• Baltrus, D. A. (2013, July). Exploring the cost of horizontal gene transfer. Evolution 2013. Snowbird, Utah: International Society for the Study of Evolution.
• Baltrus, D. A. (2013, Summer). Two steps forward, one back: How side effects of HGT shift adaptive and phenotypic landscapes. Gordon Research Conference on Microbial Population Biology. Andover, New Hampshire: Gordon Research Conferences.
• Baltrus, D. A. (2012, August). Research Talk to Incoming ABBS Students at UA. Tucson, Arizona.
• Baltrus, D. A. (2012, July). Bacterial Genomics and the Rise of Microbial GWAS and Reverse Ecology. Molecular Plant Microbe Interactions (MPMI) XV. Kyoto, Japan.
• Baltrus, D. A. (2012, June). The Cost of Horizontal Gene Transfer. ASM Annual Meeting. San Francisco, CA: American Society for Microbiology.

Poster Presentations

• Baltrus, D. A. (2013, July). The cost of horizontal gene transfer. American Society for Microbiology annual meeting. Denver, Colorado: ASM.
• Garcia, K., Schaffer, J., Sarmiento, C., Zalamea, C., Dalling, J., Davis, A., Baltrus, D. A., Gallery, R. E., & Arnold, A. E. (2013, August). Diversity and evolutionary relationships of bacteria affiliated with tropical seeds and seed-associated fungi. Mycological Society of America (MSA). Austin, TX: Mycological Society of America (MSA).
• Schaffer, J., Gallery, R. E., Baltrus, D. A., & Arnold, A. E. (2013, August). Phylogenetic relationships and diversity of endohyphal bacteria of plant-associated Pezizomycotina. Mycological Society of America (MSA). Austin, TX: Mycological Society of America (MSA).

Reviews

• Baltrus, D. A. (2017. Adaptation, specialization, and coevolution within phytobiomes(pp 109-116).
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  Growth patterns of individual plants and evolutionary trajectories of plant communities are intimately linked with and are critically affected by host-associated microbiomes. Research across systems has begun to shed light on how these phytobiomes are established under laboratory and natural conditions, and have cultivated hope that a better understanding of the governing principles for host-microbe interactions can guide attempts to engineer microbiomes to boost agricultural yields. One important, yet relatively understudied, parameter in regards to phytobiome membership is the degree to which specialization and coevolution between plant species and microbes provides structure to these communities. In this article, I provide an overview of mechanisms enabling adaptation and specialization of phytobiome communities to host plants as well as the potential for plants themselves to recruit and cultivate beneficial interactions. I further explore the possibility of host-beneficial microbe coevolution and suggest particular situations that could promote the evolution of such close-knit partnerships. It is my hope that this overview will encourage future experiments that can begin to fill in this black box of ecological and evolutionary interactions across phytobiomes.
• Baltrus, D. A., McCann, H. C., & Guttman, D. S. (2017. Evolution, genomics and epidemiology of Pseudomonas syringae: Challenges in Bacterial Molecular Plant Pathology(pp 152-168).
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  A remarkable shift in our understanding of plant-pathogenic bacteria is underway. Until recently, nearly all research on phytopathogenic bacteria was focused on a small number of model strains, which provided a deep, but narrow, perspective on plant-microbe interactions. Advances in genome sequencing technologies have changed this by enabling the incorporation of much greater diversity into comparative and functional research. We are now moving beyond a typological understanding of a select collection of strains to a more generalized appreciation of the breadth and scope of plant-microbe interactions. The study of natural populations and evolution has particularly benefited from the expansion of genomic data. We are beginning to have a much deeper understanding of the natural genetic diversity, niche breadth, ecological constraints and defining characteristics of phytopathogenic species. Given this expanding genomic and ecological knowledge, we believe the time is ripe to evaluate what we know about the evolutionary dynamics of plant pathogens.

Others

• Baltrus, D. A. (2010, Fall). Blog Post on My Research at "Tree of Life".