Elsevier

Gene

Volume 303, 16 January 2003, Pages 131-137
Gene

Novel antibiotic-resistance markers in pGK12-derived vectors for Borrelia burgdorferi

https://doi.org/10.1016/S0378-1119(02)01146-0Get rights and content

Abstract

Extension of molecular genetics studies in Borrelia burgdorferi has been hampered by a lack of a variety of antibiotic resistance selective markers. Such markers are critical for isolation of B. burgdorferi strains with multiple mutants, for complementation with different cloning vectors, and for methods such as negative selection and reporter genes. To remedy this lack, resistance to various antibiotics of non-infectious (B31, 297) and infectious (N40) B. burgdorferi strains was examined and vectors incorporating appropriate antibiotic resistance genes as selective markers were developed. Minimal inhibitory concentrations for growth of B. burgdorferi on plates and in liquid media for aminoglycosides (kanamycin, gentamycin, sisomycin, amikacin, spectinomycin, neomycin), macrolides-lincosamids (erythromycin, lincomycin), coumarin derivatives (coumermycin A1, novobiocin), glycopeptides (vancomycin, ristocetin), peptides (bacitracin, cycloserine), and chloramphenicol were found to differ significantly. There were also striking differences in resistance to these antibiotics between non-infectious and infectious B. burgdorferi strains. Antibiotic-resistance genes aph(3′)-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA′ from Staphylococcus aureus, and aac(3)-VIa from Enterobacter cloacae (conferring resistance to kanamycin, spectinomycin, lincomycin, and gentamycin/sisomycin, respectively) were subcloned either with their own promoters or under the control of the B. burgdorferi flaB promoter into pGK12 or its derivative pED1 to develop new cloning vectors for B. burgdorferi with the rationale that the absence of homologous regions between derived recombinant plasmids lacking the flaB promoter and the B. burgdorferi genome would permit avoidance of possible recombination with target DNA. Resistance to the corresponding antibiotic was conferred by vectors containing aph(3′)-IIIa, aad9, linA′ or aac(3)-VIa whether under the control of their own promoters or under the control of the flaB promoter. We conclude that these markers can be used for genetic study of B. burgdorferi and suggest they will be an important addition to the previously used coumermycin A1, erythromycin and kanamycin in these studies.

Introduction

Development and application of molecular genetic systems have been crucial tools to unraveling bacterial biology (Cabello et al., 2001). Despite the availability of the Borrelia burgdorferi complete genome sequence for some time (Fraser et al., 1997), advances in our understanding of its biology and pathogenesis have been delayed at least in part by the lack of well-developed genetic systems to permit manipulation of its genome. Until recently, this dearth has been characterized by the lack of efficient methods for introducing in vitro manipulated DNA molecules into B. burgdorferi, multi-purpose cloning vectors and a wide variety of selectable genetic markers and reporter genes to monitor gene expression, as well as constraints in recombination and complementation, and the inability to generate comprehensive libraries of chromosomal recombinant and null mutants (Samuels et al., 1994). In choosing selectable antibiotic markers for B. burgdorferi, an important additional constraint is that they should not be used in the treatment of Lyme disease (Steere, 2001).

The introduction of coumermycin A1, erythromycin and kanamycin resistance as selective markers for genetic study of B. burgdorferi has been a key step in developing borrelial genetic systems. Coumermycin A1 resistance permitted initial development of the electroporation protocol and exchange between wild-type chromosomal alleles and in vitro mutagenized B. burgdorferi DNA (Samuels et al., 1994, Samuels, 1995). Kanamycin and erythromycin resistance were subsequently widely used in development of extrachromosomal vectors for B. burgdorferi and for insertional inactivation mutagenesis of several plasmid and chromosomal genes in B. burgdorferi (Bono et al., 2000, Sartakova et al., 2000, Cabello et al., 2001). Unfortunately, there are several important limitations to the widespread use of these three antibiotic resistance genes. The coumermycin A1 resistance gene can recombine with the wild-type allele in the chromosome (Samuels et al., 1994, Stevenson et al., 1998). The aphI kanamycin resistance gene from Tn903 is not well expressed in B. burgdorferi if it is cloned under the control of its own promoter (Stevenson et al., 1998) and when it is cloned under the control of the B. burgdorferi flaB promoter it may recombine into the flaB region (Morozova and Cabello, unpublished). B. burgdorferi erythromycin susceptibility is variable in different strains both clinically (Moody et al., 1994, Cinco et al., 1995, Hunfeld et al., 2000) and in vitro (Terekhova et al., 2002). In addition, it is frequently necessary to be able to use more than one selective marker in performing genetic experiments in bacteria, for example, in allelic exchange experiments generated by positive and negative selection (Rubin et al., 1999, Sartakova et al., 2001).

For these reasons and because our preliminary observations suggested that B. burgdorferi strains differed in their susceptibility to antibiotics (Terekhova et al., 2002), antibiotic susceptibility of well-characterized non-infectious B. burgdorferi B31 and 297 strains and the infectious B. burgdorferi N40 strain was examined to determine if new antibiotic resistance markers for B. burgdorferi could be identified. We have cloned four antibiotic resistance genes into the wide host range plasmid pGK12 and its derivative, pED1, and have examined their ability to be propagated in B. burgdorferi B31 after electroporation and their suitability for use as selective markers.

Section snippets

Bacterial strains and media

B31 and 297, high-passage, non-infectious B. burgdorferi strains and N40, an infectious B. burgdorferi strain, were grown at 32 °C in Barbour–Stoenner–Kelly (BSK)-H medium (Sigma Chemical Co., St. Louis, MO) supplemented with 6% rabbit serum (Sigma). Infectious B. burgdorferi N40 was isolated from infected mice and cultured no more than two passages in BSK-H medium before MIC testing.

Determination of minimal inhibitory concentrations (MIC) of antibiotics for B. burgdorferi on solid media

MIC was determined by plating 1×108 cells on the surface of agar plates of BSK-H medium supplemented with 6%

Antibiotic susceptibility of non-infectious and infectious B. burgdorferi strains

Antibiotic susceptibility studies of B. burgdorferi are scarce and usually directed at determining the susceptibility of this bacterium to antibiotics used in therapy for Lyme disease (Hunfeld et al., 2000). Because the only requirement for use of an antibiotic resistance genetic marker in genetic selection is its ability to differentiate between cells expressing and not expressing the resistance, susceptibility testing for this purpose was focused on antibiotics other than those used

Acknowledgements

We thank Dr. P. Trieu-Cuot, Dr. B. Stevenson and Dr. P.J. Piggot for providing us with plasmids pAT112, pBLS500 and pVK61, respectively, Dr. P.A. Mann (Schering-Plough Research Institute, Kenilworth, NJ, USA) for pSCH4101, and Dr. P. Courvalin for S. aureus BM4611. We also thank Dr. Julian Davies for discussions. This study was supported by grant R01 AI48856 from the U.S. National Institute of Allergy and Infectious Diseases to F.C.C.

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