Samantha Orchard

Interests

Teaching

Biotechnology, microbial molecular biology, enzyme discovery

Courses

Scholarly Contributions

Journals/Publications

• Orchard, S., Rostron, J., & Segall, A. (2012). Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution. Microbiology (UK), 158(2). doi:10.1099/mic.0.054361-0
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  The peptide wrwycr inhibits Holliday junction resolution and is a potent antimicrobial. To study the physiological effects of wrwycr treatment on Escherichia coli cells, we partially screened the Keio collection of knockout mutants for those with increased sensitivity to wrwycr. Strains lacking part of the ferric-enterobactin (iron-bound siderophore) uptake and utilization system, parts of the enterobactin synthesis pathway, TolC (an outer-membrane channel protein) or Fur (an ironresponsive regulator) were hypersensitive to wrwycr. We provide evidence that the DtolC mutant was hypersensitive to wrwycr due to its reduced ability to efflux wrwycr from the cell rather than due to its export of newly synthesized enterobactin. Deleting ryhB, which encodes a small RNA involved in iron regulation, mostly relieved the wrwycr hypersensitivity of the fur and ferricenterobactin uptake mutants, indicating that the altered regulation of a RyhB-controlled gene was at least partly responsible for the hypersensitivity of these strains. Chelatable iron in the cell, measured by electron paramagnetic resonance spectroscopy, increased dramatically following wrwycr treatment, as did expression of Fur-repressed genes and, to some extent, mutation frequency. These incongruous results suggest that while wrwycr treatment caused accumulation of chelatable iron in the cell, iron was not available to bind to Fur. This is corroborated by the observed induction of the suf system, which assembles iron-sulfur clusters in low-iron conditions. Disruption of iron metabolism by wrwycr, in addition to its effects on DNA repair, may make it a particularly effective antimicrobial in the context of the low-iron environment of a mammalian host. G 2012 SGM Printed in Great Britain.
• Chaston, J., Suen, G., Tucker, S., Andersen, A., Bhasin, A., Bode, E., Bode, H., Brachmann, A., Cowles, C., Cowles, K., Darby, C., Drace, K., Du, Z., Givaudan, A., Herbert Tran, E., Jewell, K., Knack, J., Krasomil-Osterfeld, K., Kukor, R., , Lanois, A., et al. (2011). The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes. PLoS One, 6(11). doi:10.1371/journal.pone.0027909
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  Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points. © 2011 Chaston et al.
• Peterson, S., Orchard, S., & Menon, S. (2011). Penicillium menonorum, a new species related to P. pimiteouiense. IMA Fungus, 2(2). doi:10.5598/imafungus.2011.02.02.02
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  Penicillium menonorum is described as a new monoverticillate, non-vesiculate species that resembles P. restrictum and P. pimiteouiense. On the basis of phylogenetic analysis of DNA sequences from four loci, P. menonorum occurs in a clade with P. pimiteouiense, P. vinaceum, P. guttulosum, P. rubidurum, and P. parvum. Genealogical concordance analysis was applied to P. pimiteouiense and P. parvum, substantiating the phenotypically defined species. The species P. rubidurum, P. guttulosum, and P. menonorum were on distinct branches statistically excluded from inclusion in other species and have distinct phenotypes.
• Park, Y., Herbert, E., Cowles, C., Cowles, K., Menard, M., Orchard, S., & Goodrich-Blair, H. (2007). Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity. Cellular Microbiology, 9(3). doi:10.1111/j.1462-5822.2006.00815.x
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  Virulence of the insect pathogen Xenorhabdus nematophila is attributed in part to its ability to suppress immunity. For example, X. nematophila suppresses transcripts encoding several antimicrobial proteins, even in the presence of Salmonella enterica, an inducer of these transcripts. We show here that virulence and immune suppression phenotypes can be lost in a subpopulation of X. nematophila. Cells that have undergone 'virulence modulation' (vmo) have attenuated virulence and fail to suppress antimicrobial transcript levels, haemocyte aggregation and nodulation in Manduca sexta insects. When plated on certain media, vmo cells have a higher proportion of translucent (versus opaque) colonies compared with non-vmo cells. Like vmo strains, translucent colony isolates are defective in virulence and immune suppression. The X. nematophila genome encodes two 'opacity' genes with similarity to the Ail/PagC/Rck family of outer membrane proteins involved in adherence, invasion and serum resistance. Quantitative polymerase chain reaction analysis shows that RNA levels of one of these opacity genes, opaB, are higher in opaque relative to translucent colonies. We propose that in X. nematophila opaB may be one of several factors involved in immune suppression during infection, and expression of these factors can be co-ordinately eliminated in a subpopulation, possibly through a phase variation mechanism. © 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd.
• Orchard, S., & Goodrich-Blair, H. (2005). An encoded N-terminal extension results in low levels of heterologous protein production in Escherichia coli. Microbial Cell Factories, 4. doi:10.1186/1475-2859-4-22
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  Background: The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form" (using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium. Results: Short-form Tdk complemented the E. coli tdk mutant strain, resulting in FUdR sensitivity of the strain. However, the E. coli tdk mutant expressing the long form of tdk remained FUdR resistant, indicating it did not have a functional deoxythymidine kinase enzyme. We report that long-form Tdk is at least 13-fold less abundant than short-form Tdk, the limited protein produced was as stable as short-form Tdk and the long-form transcript was 1.7-fold less abundant than short-form transcript. Additionally, we report that the long-form extension was sufficient to decrease heterologous production of a different X. nematophila protein, NilC. Conclusion: We conclude that the difference in the FUdR growth phenotype between the E. coli tdk mutant carrying the long-or short-form X. nematophila tdk is due to a difference in Tdk levels. The lower long-form protein level does not result from protein instability, but instead from reduced transcript levels possibly combined with reduced translation efficiency. Because the observed effect of the encoded N-terminal extension is not specific to Tdk production and can be overcome with induction of gene expression, these results may have particular relevance to researchers attempting to limit production of toxic proteins under non-inducing conditions. © 2005 Orchard and Goodrich-Blair; licensee BioMed Central Ltd.
• Orchard, S., & Goodrich-Blair, H. (2005). Pyrimidine nucleoside salvage confers an advantage to Xenorhabdus nematophila in its host interactions. Applied and Environmental Microbiology, 71(10). doi:10.1128/AEM.71.10.6254-6259.2005
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  Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Orchard, S., & Goodrich-Blair, H. (2004). Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease. Applied and Environmental Microbiology, 70(9). doi:10.1128/AEM.70.9.5621-5627.2004
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  The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA1, oppA2, of oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.

Presentations

• Orchard, S. (2022). Promoting Innovative Thinking in Biotechnology. American Society for Microbiology Conference on Undergraduate Education. Virtual: American Society for Microbiology.
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  "Microbrew" oral presentation. Abstract: In the coming decades, humans will face challenges related to climate change, the increasing human population, antimicrobial resistance, and more. Overcoming these challenges will require creative problem solving and innovation. To help guide students through the transition from being learners of existing knowledge to the creators of new solutions, I use an assignment in which I ask students to propose a biotechnology-related solution to a problem of their choice. This encourages them to think creatively and to see how they can apply their knowledge to tackle new challenges. Out of respect for Universal Design for Learning guidelines, I allow the students to submit their work in a variety of formats, including websites, podcasts, or videos.