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Genetic Manipulation of Borrelia Curr. Issues Mol. Biol. 42: 307-332. caister.com/cimb Genetic Manipulation of Borrelia Patricia A. Rosa1* and Mollie W. Jewett2 1Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S 4th St. Hamilton, MT 59840 USA 2Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, 6900 Lake Nona Blvd, Orlando, FL 32827 USA *Corresponding author: [email protected] DOI: https://doi.org/10.21775/cimb.042.307 Abstract study the biology and pathogenic potential of these Genetic studies in Borrelia require special tick-borne spirochetes (Tilly et al., 2000; Cabello et consideration of the highly segmented genome, al., 2001; Rosa et al., 2005; Battisti et al., 2008; Fine complex growth requirements and evolutionary et al., 2011; Brisson et al., 2012; Raffel et al., 2014; distance of spirochetes from other genetically Krishnavajhala et al., 2017; Raffel et al., 2018; tractable bacteria. Despite these challenges, a robust Samuels et al., 2018). Both in vitro and in vivo molecular genetic toolbox has been constructed to analyses have thus been conducted to identify and investigate the biology and pathogenic potential of describe the basic functions of many Borrelia genes these important human pathogens. In this review we and gene products and their contributions to the summarize the tools and techniques that are relationship of these important pathogens with their currently available for the genetic manipulation of tick vectors and mammalian hosts. Direct Borrelia, including the relapsing fever spirochetes, applications of genetic tools developed for Gram- viewing them in the context of their utility and positive and Gram-negative bacteria have shortcomings. Our primary objective is to help encountered limitations, however, due to the genetic researchers discern what is feasible and what is not distance and distinct codon usage of Borrelia relative practical when thinking about potential genetic to these bacteria (Paster et al., 1984; Fraser et al., experiments in Borrelia. We have summarized 1997). Hence there is still a significant need to published methods and highlighted their critical develop additional and alternative genetic tools for elements, but we are not providing detailed protocols. Borrelia in particular, and for spirochetes in general. Although many advances have been made since B. burgdorferi was first transformed over 25 years ago, Over 30 years ago, Dr. Stanley Falkow, the father of some standard genetic tools remain elusive for the field of microbial pathogenesis, restated Koch's Borrelia. We mention these limitations and why they postulates as they could be applied in microbiology persist, if known. We hope to encourage using the tools of molecular genetics (Falkow, 1988). investigators to explore what might be possible, in In their simplest form, Falkow's molecular postulates addition to optimizing what currently can be entail the ability to create mutations, restore genes achieved, through genetic manipulation of Borrelia. and analyze the phenotypes of an otherwise isogenic set of microbes. However, their interpretation and Introduction application continue to evolve as new technologies Spirochetes in the genus Borrelia have been known emerge with which to investigate the complex biology to cause human disease for well over a century, but of pathogenic microbes (Falkow, 2004; Casadevall, in-depth investigations of these pathogenic bacteria 2017). awaited many developments, including the advent of molecular genetics. Since transformation of Borrelia Molecular genetic analyses have provided insight into burgdorferi was first described in 1994 (Samuels et a wide array of regulatory and structural elements of al., 1994b; Samuels, 1995), a molecular genetic Borrelia. However, limitations persist for genetic approach has been applied with growing success to studies of both the Lyme disease (LD) and relapsing caister.com/cimb 307 Curr. Issues Mol. Biol. Vol. 42 Genetic Manipulation of Borrelia Rosa and Jewett fever (RF) Borrelia, including low frequencies and than a short region at one telomere, whereas the efficiencies of transformation, complex and undefined plasmid component of the genome has undergone growth media, and a variable ability to quantify or substantial rearrangement among strains. However, clone spirochetes as colonies in solid medium. In the total gene content of B. burgdorferi ss is relatively addition, the generation of isogenic wild-type, mutant constant, with few absolute differences among the and complemented clones present unique challenges large number of isolates analyzed to date, despite in Borrelia due to the segmented and unstable nature widespread plasmid reorganization (Casjens et al., of their genomes, and poorly defined, multifactorial 2017). The variability in plasmid structure among barriers to the uptake and incorporation of strains suggests that the phenotype of a particular exogenous DNA. Polar effects following insertional mutant (role/contribution of a particular gene) could inactivation and allelic exchange can arise as a vary with the context (strain background) in which it is consequence of the compact chromosome of B. analyzed. Ideally, mutations would be introduced and burgdorferi, which contains many genes with analyzed in more than a single clone or strain (Gilbert overlapping transcriptional and termination signals et al., 2007; Lybecker and Samuels, 2007). However, (Fraser et al., 1997; Adams et al., 2017b). The recognizing that experimental outcome may differ presence of paralogous gene families (Casjens et al., between strains and interpreting results accordingly 2000) encoding related products with potentially is currently acceptable, given the limitations and redundant functions in B. burgdorferi can also challenges presented by genetic studies in B. complicate the interpretation of mutant phenotypes. burgdorferi, Establishing the connection between a mutation and a phenotype through complementation is essential, Strain choice by individual labs generally reflects but it can be particularly challenging. Isolation of what has been used in previous experiments to nutritional mutants in Borrelia is limited by the facilitate comparison with existing data. There are no complex and undefined nature of the growth media data indicating that a particular LD spirochete strain and a relatively long doubling time (>5 hours under is inherently more suitable than another. However, optimal conditions) (Barbour, 1984). Despite these the ease with which spirochetes can be grown and potential and real challenges, substantial progress plated, the efficiency at which they can be has been made in utilizing genetic approaches to transformed, and their phenotype in the mouse/tick study factors involved in the biology and physiology infectious cycle are relevant traits that vary of Borrelia and pathogenesis of the diseases they substantially among B. burgdorferi strains and cause, as illustrated elsewhere (Radolf and Samuels, between clones derived from a single strain (Elias et 2021). al., 2002). Hence a key consideration at the outset of any experiment is whether characteristics of the Clone and strain considerations clone/strain/genospecies being used, such as those Lyme disease spirochetes described above, are appropriate for the question(s) Most genetic studies to date have been conducted being asked. with B. burgdorferi sensu stricto (ss) strains, with only a few studies utilizing B. afzelii or B. garinii strains Although differences in plasmid content (and (Fingerle et al., 2007; Bontemps-Gallo et al., 2018; corresponding differences in plasmid-borne Cuellar et al., 2019; Strnad and Rego, 2020). Initially, restriction/modification systems) clearly contribute to the complete and annotated genomic sequence, the efficiency with which different LD strains and including all plasmids, was only available for B. clonal derivatives can be transformed (Lawrenz et al., burgdorferi ss type strain B31 (Fraser et al., 1997; 2002; Kawabata et al., 2004; Rego et al., 2011), Casjens et al., 2000), which made it the obvious additional traits also affect transformability. These choice for genetic studies. However, this limitation no include surface properties that permit or restrict longer exists, and the sequences for the complete resuspension in electroporation solution (Tilly et al., genomes, including plasmids, are either available or 2000; Tilly et al., 2016). The highly segmented and readily determined for virtually any Borrelia isolate potentially unstable structure of the B. burgdorferi (Casjens et al., 2017; Casjens et al., 2018). genome mandates that plasmid content be Sequencing advances that yield extremely long and continuously monitored to assure that mutant and accurate reads from single DNA molecules have wild-type clones remain isogenic except for directed been key to this development (Margos et al., 2017). genetic changes (Elias et al., 2002). Determining the The linear chromosome is highly conserved other plasmid content of strain B31 derivatives has been caister.com/cimb 308 Curr. Issues Mol. Biol. Vol. 42 Genetic Manipulation of Borrelia Rosa and Jewett greatly facilitated by development of multiplex PCR describes what fraction of the population that methods (Bunikis et al., 2011; Norris et al., 2011). survives electroporation (or other transformation Complementation also represents a critical method) has stably acquired the introduced DNA. component
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