The Way Forward: Improving Genetic Systems

Ulrike G. Munderloh; Roderick F. Felsheim; Nicole Y. Burkhardt; Michael J. Herron; Adela S. Oliva Chávez; Curtis M. Nelson; Timothy J. Kurtti

doi:10.1128/9781555817336.ch14

Chapter 14 : The Way Forward: Improving Genetic Systems

Authors: Ulrike G. Munderloh¹, Roderick F. Felsheim¹, Nicole Y. Burkhardt¹, Michael J. Herron¹, Adela S. Oliva Chávez¹, Curtis M. Nelson¹, Timothy J. Kurtti¹

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Affiliations: 1: Department of Entomology, University of Minnesota, St. Paul, MN 55108

Content Type: Monograph

Publication Year: 2012

Category: Bacterial Pathogenesis

Book DOI: 10.1128/9781555817336

Chapter DOI: 10.1128/9781555817336.ch14

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Abstract:

This chapter reviews the research that has led to the first successes in genetic transformation of arthropod-borne bacteria belonging to the order Rickettsiales in the Alphaproteobacteria subdivision. Seminal results provided the very first evidence that direct genetic manipulation of rickettsiae was achievable, and that the bacteria were able to maintain and express foreign genetic sequences inserted via allelic exchange/homologous recombination under control of rickettsial or Escherichia coli promoters. This work set the stage for all subsequent research efforts aspiring to manipulate and analyze rickettsiae in a manner that is nearly commonplace in extracellular bacteria. The Himar1 transposase allele A7 has been successfully used for mutagenesis of A.phagocytophilum and fever groups of rickettsial tick symbionts have been spotted using fluorescent markers and antibiotic resistance. Selection of mutants using growth inhibitors is an indispensible strategy to recover mutants from the background of nontransformed bacteria. The most common strategy is incorporation of an antibiotic resistance gene in the transformation cassette. When working with human or animal pathogens, this is a sensitive issue, as introduction of resistance to antibiotics used to treat disease induced by the pathogen to be transformed is not encouraged. This poses a dilemma, as the most effective selection is likely achieved by using the clinically most effective antibiotics. The authors suggests that shuttle vectors would be useful for testing the function of genes that are naturally defective in certain Rickettsia spp., in complementation assays, overexpression of native or foreign genes, testing gene regulation, and other applications carried out in E.coli.

Citation: Munderloh U, Felsheim R, Burkhardt N, Herron M, Oliva Chávez A, Nelson C, Kurtti T. 2012. The Way Forward: Improving Genetic Systems, p 416-432. In Palmer G, Azad A (ed), Intracellular Pathogens II: Rickettsiales. ASM Press, Washington, DC. doi: 10.1128/9781555817336.ch14

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The Way Forward: Improving Genetic Systems

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/content/10.1128/9781555817336.chap14.fig14-1

FIGURE 1

General features of plasmid constructs being developed for use in Rickettsiales genetics. (A) Construct for transposon-mediated mutagenesis of Anaplasma. Depicted is a cis-plasmid construct: both transposase (Himar1) and a transposon containing a bar (spectinomycin resistance) gene and GFPuv are encoded on a single plasmid (pCis GFPuv Himar1 A7) in order to improve efficiency. In this construct two A. marginale Am tr promoters are used to drive expression of Himar1 and the reporter and resistance genes. (B) Confocal fluorescent microscopic image of GFPuv-expressing A. phagocytophilum infecting a rhesus cell (RF/6A) expressing DsRed2, a red fluorescent protein. (C) Shuttle vector for transformation of rickettsiae. Depicted is a generalized example of the essential elements of shuttle vectors based on pRAM18 from R. amblyommii. A fragment of pRAM18 containing parA and the DnaA-likeprotein gene was ligated into an E. coli cloning vector containing genes for rifampin resistance and GFPuv. (D) Fluorescent microscopic image of pRAM18dRGA-transformed R. bellii strain 369 in an I. scapularis cell (cell line ISE6). Bars (B and D), 5 µm. doi:10.1128/9781555817336.ch14.f1

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FIGURE 1

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Tables

The Way Forward: Improving Genetic Systems

/content/10.1128/9781555817336.chap14.tab14-1

TABLE 1a

Rickettsiales genetic transformation systems

a Rifampin-resistant mutant gene of R. prowazekii Madrid E.

b E. coli ereB gene.

c E. coli srp promoter.

d R. prowazekii-adapted rifampin resistance arr-2 gene.

e CAT, chloramphenicol acetyltransferase.

10.1128/9781555817336/tab14-1a_thmb.gif

10.1128/9781555817336/tab14-1a.gif

TABLE 1a

Rickettsiales genetic transformation systems

a Rifampin-resistant mutant gene of R. prowazekii Madrid E.

b E. coli ereB gene.

c E. coli srp promoter.

d R. prowazekii-adapted rifampin resistance arr-2 gene.

e CAT, chloramphenicol acetyltransferase.

/content/10.1128/9781555817336.chap14.tab14-1b

TABLE 1b

Rickettsiales genetic transformation systems

a Rifampin-resistant mutant gene of R. prowazekii Madrid E.

b E. coli ereB gene.

c E. coli srp promoter.

d R. prowazekii-adapted rifampin resistance arr-2 gene.

e CAT, chloramphenicol acetyltransferase.

10.1128/9781555817336/tab14-1b_thmb.gif

10.1128/9781555817336/tab14-1b.gif

TABLE 1b

Rickettsiales genetic transformation systems

a Rifampin-resistant mutant gene of R. prowazekii Madrid E.

b E. coli ereB gene.

c E. coli srp promoter.

d R. prowazekii-adapted rifampin resistance arr-2 gene.

e CAT, chloramphenicol acetyltransferase.