University at Buffalo - The State University of New York
Skip to Content
A simplified erythromycin resistance cassette for Treponema denticola mutagenesis

Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Microbiol Methods. Author manuscript; available in PMC 2011 October 1.
Published in final edited form as:
PMCID: PMC2953711

A simplified erythromycin resistance cassette for Treponema denticola mutagenesis


The primary selectable marker for genetic studies of Treponema denticola is a hybrid gene cassette containing both ermF and ermAM (ermB) genes. ErmB functions in Escherichia coli, while ErmF has been assumed to confer resistance in T. denticola. We demonstrate here that ErmB is sufficient for erythromycin selection in T. denticola and that the native ermB promoter drives ErmB expression.

Keywords: spirochete, selectable marker, mutagenesis

Progress in molecular analysis of the anaerobic oral spirochete Treponema denticola has been limited by factors including fastidious nutrient requirements, slow growth rate and poor plating efficiency. In addition, genetic analysis of T. denticola is limited by its extremely low transformation efficiency and strain-limited shuttle plasmid as well as by the small number of validated selectable markers. Erythromycin has been the most reliable and widely used selectable marker; chloramphenicol, gentamicin and coumermycin have also been reported. However, some Treponema strains are relatively resistant to chloramphenicol ( and J.C. Fenno, data not shown), and coumermycin is generally regarded as unsuitable due to pleiotropic phenotypes of gyrB mutants.

The ermF gene isolated from the Bacteroidesplasmid pBF4 is used widely as a selectable marker for mutagenesis studies in organisms in the Cytophaga-Flavobacterium-Bacteroides group. The ermAM gene (hereinafter designated ermB in agreement with current suggested taxonomy ), was first isolated from enterococcal conjugative plasmid pAMβ1 and subsequently developed as part of a shuttle vector for other streptococci. The transmethylase encoded by ermB confers erythromycin resistance in a wide range of Gram-positive organisms and also confers high-level resistance in E. coli.

Fletcher et al. designed a hybrid ermF-ermB gene cassette for use as a selectable marker for Porphyromonas gingivalis allelic replacement mutagenesis. The cassette structure permits selection in both E. coli (ermB) and Bacteroides-family organisms (ermF), thus facilitating intermediate cloning steps. In addition to its use in numerous studies in P. gingivalis, it has become a standard selectable marker in Treponema denticola, Fusobacterium nucleatum and Tannerella forsythia. While spirochetes (including T. denticola) are classified as Gram-negative due to the presence of an outer membrane and thin peptidoglycan layer, they carry large numbers of genes having closest homologies with Gram-positive organisms, particularly genes encoding various membrane proteins, transcriptional regulators and membrane trafficking functions. We thus hypothesized that ermB, primarily characterized as active in Gram-positive organisms, might be sufficient for erythromycin resistance in T. denticola. This had the potential to substantially simplify construction of defined isogenic mutants in T. denticola.

To characterize activity of ermB in T. denticola, we constructed two isogenic mutant strains in the dentilisin protease locus: one each in prcA and prcB, the first two genes in the prcB-prcA-prtP operon (Figure 1). A 1142 bp BstZ17I-PmeI fragment of pVA2198 including the ermB coding region and putative promoter originally derived from pAMβ1 was cloned in plasmids carrying the T. denticola target gene sequences such that each end of the ermB cassette was flanked by at least 300 bp of target gene DNA. The resulting plasmids, based on plasmid vector pSTBlue-1 (Novagen, Inc., Madison, WI, USA)), conferred both kanamycin resistance (30 μg ml−1) and erythromycin (300 μg ml−1) in the E. coli JM109 host. Plasmids carrying the mutant constructs were linearized to facilitate allelic replacement by homologous recombination. T. denticola ATCC 35405 (the Type strain, which has been passaged extensively in various laboratories) was electroporated with the resulting linear DNA s and plated in NOS-GN soft agar medium containing erythromycin (40 μg ml−1, Sigma Chemical Co., St. Louis, MO) as described previously. Resulting erythromycin-resistant colonies growing under the agar surface were picked using Pasteur pipets, grown in NOS broth medium containing erythromycin (40 μg ml−1) and screened for the expected mutations by PCR and DNA sequencing. The mutation constructed in prcB is identical to that in prcB mutantstrain T. denticola P0760, except that P0760 carries the ermF-ermB cassette. The mutation constructed in prcA was constructed by replacing a 287 bp StuI fragment with ermB yielding a mutant essentially identical to prcA mutant strain T. denticola PNE, which carries the ermF-ermB cassette. T. denticola parent and mutant strains were tested for expression of PrcA and PrtP proteins, using FlaA expression as a positive control. Immunoblots of T. denticola cell lysates were probed with rabbit polyclonal antibodies raised against PrtP, PrcA or FlaA and immunoreactive bands were detected using horseradish peroxidase-conjugated goat anti-rabbit antibodies and a chemiluminescent detection as described previously. As shown in Figure 2, neither mutant produced PrcA or PrtP, while all strains expressed wildtype levels of FlaA. The ermB cassette is in opposite transcriptional orientation relative to the target gene in both mutant strains (Figure 1). These results indicate that ermB transcription is driven by a promoter sequence present on the ermB cassette, presumably the native promoter carried on pAMβ1.

Figure 1
Construction of T. denticola mutants using ermF-ermB and ermB cassettes. The protease operon (prcB-prcA-prtP) and adjacent gene TDE0759 in wildtype parent strain ATCC 35405 and isogenic strains mutated in prcB (P0760 and CF548) and prcA (PNE and CF547) ...
Figure 2
Western immunoblots of T. denticola parent and mutant strains, probed with antibodies raised against T. denticola PrcA, PrtP or FlaA. Strains are as in Figure 1. Lanes: 1, 35405 (parent); 2, P0760; 3, CF547; 4, CF548.

We previously demonstrated that insertion of the ermF-ermB cassette results in interruption of transcription downstream of the insertion site in the target gene or operon. Furthermore, the ermF-ermB cassette contains a promoter that is active in T. denticola, since its function is not dependent on its orientation relative to the target gene. While we have not addressed transcription or activity of ermF in T. denticola, we demonstrate that the ermB locus from pAMβ1 contains a promoter that is functional in T. denticola. We recently reported that nonpolar deletion of prcB does not block expression of PrcA protein. The polar effects on expression downstream of ermB insertion in prcB or prcA (Figure 2) indicate that the ermB cassette contains a transcription termination signal.

To date, well over 40 T. denticola mutants have been constructed using the ermF-ermB cassette, most of which have been reported in the literature. It is our intention here to provide improved characterization of this extremely useful antibiotic resistance cassette so that its utility can be expanded. A single gene cassette known to contain its own functional promoter is generally preferable to a cassette with two genes encoding the same activity driven by uncharacterized promoter(s). Furthermore, reducing the size of the cassette by fifty percent can be expected to facilitate the required preliminary cloning steps and may also increase transformation efficiency in this recalcitrant organism.


This work was supported by United States Public Health Service grants DE018221 (National Institute of Dental and Craniofacial Research Bethesda, MD) and AI079325 (National Institute of Allergy and Infectious Diseases, Bethesda, MD).


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Abramson IJ, Smibert RM. Inhibition of growth of treponemes by antimicrobial agents. Br J Vener Dis. 1971;47:407–412. [PMC free article] [PubMed]
  • Alvarez B, Secades P, McBride MJ, Guijarro JA. Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol. 2004;70:581–587. [PMC free article] [PubMed]
  • Bian XL, Wang HT, Ning Y, Lee SY, Fenno JC. Mutagenesis of a novel gene in the prcA-prtP protease locus affects expression of Treponema denticola membrane complexes. Infect Immun. 2005;73:1252–1255. [PMC free article] [PubMed]
  • Brisson-Noel A, Arthur M, Courvalin P. Evidence for natural gene transfer from gram-positive cocci to Escherichia coli. J Bacteriol. 1988;170:1739–1745. [PMC free article] [PubMed]
  • Chan EC, Siboo R, Keng T, Psarra N, Hurley R, Cheng SL, Iugovaz I. Treponema denticola (ex Brumpt 1925) sp nov., nom rev., and identification of new spirochete isolates from periodontal pockets. Int J Syst Bacteriol. 1993;43:196–203. [PubMed]
  • Chan ECS, DeCiccio A, McLaughlin R, Klitorinos A, Siboo R. An inexpensive solid medium for obtaining colony-forming units of oral spirochetes. Oral Microbiol Immunol. 1997;12:372–376. [PubMed]
  • Chi B, Chauhan S, Kuramitsu HK. Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidumflaA gene. Infect Immun. 1999;67:3653–3656. [PMC free article] [PubMed]
  • Chi B, Limberger RJ, Kuramitsu HK. Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system. Infect Immun. 2002;70:2233–2237. [PMC free article] [PubMed]
  • Clewell DB, Yagi Y, Dunny GM, Schultz SK. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J Bacteriol. 1974;117:283–289. [PMC free article] [PubMed]
  • Fenno JC, Wong GWK, Hannam PM, McBride BC. Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola. FEMS Microbiol Lett. 1998;163:209–215. [PubMed]
  • Fletcher HM, Schenkein HA, Morgan RM, Bailey KA, Berry CR, Macrina FL. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene. Infect Immun. 1995;63:1521–1528. [PMC free article] [PubMed]
  • Godovikova V, Wang HT, Goetting-Minesky MP, Ning Y, Capone RF, Slater CK, Fenno JC. Treponema denticola PrcB is required for expression and activity of the PrcA-PrtP (dentilisin) complex. J Bacteriol. 2010;192:3337–3334. [PMC free article] [PubMed]
  • Haake SK, Yoder SC, Attarian G, Podkaminer K. Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems. J Bacteriol. 2000;182:1176–1180. [PMC free article] [PubMed]
  • Han YW, Ikegami A, Chung P, Zhang L, Deng CX. Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum. Appl Environ Microbiol. 2007;73:3677–3683. [PMC free article] [PubMed]
  • Honma K, Inagaki S, Okuda K, Kuramitsu HK, Sharma A. Role of a Tannerella forsythia exopolysaccharide synthesis operon in biofilm development. Microb Pathog. 2007;42:156–166. [PubMed]
  • Lee SY, Bian XL, Wong GW, Hannam PM, McBride BC, Fenno JC. Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins PrcA1 and PrcA2 is dependent on PrtP. J Bacteriol. 2002;184:3864–3870. [PMC free article] [PubMed]
  • Li H, Ruby J, Charon N, Kuramitsu H. Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant. J Bacteriol. 1996;178:3664–3667. [PMC free article] [PubMed]
  • Macrina FL, Tobian JA, Jones KR, Evans RP. Molecualr cloning in the streptoocci. In: Hollander A, DeMoss R, Kaplan S, Konisky J, Savage D, Wolfe R, editors. Genetic Engineering of Micro-organisms for Chemicals. Plenum; New York: 1982. pp. 195–210.
  • Macrina FL, Tobian JA, Jones KR, Evans RP, Clewell DB. A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene. 1982;19:345–353. [PubMed]
  • Mally M, Cornelis GR. Genetic tools for studying Capnocytophaga canimorsus. Appl Environ Microbiol. 2008;74:6369–6377. [PMC free article] [PubMed]
  • Mays TD, Smith CJ, Welch RA, Delfini C, Macrina FL. Novel antibiotic resistance transfer in Bacteroides. Antimicrob Agents Chemother. 1982;21:110–118. [PMC free article] [PubMed]
  • McBride MJ, Braun TF, Brust JL. Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization. J Bacteriol. 2003;185:6648–6657. [PMC free article] [PubMed]
  • Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, Rood J, Seppala H. Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob Agents Chemother. 1999;43:2823–2830. [PMC free article] [PubMed]
  • Seshadri R, Myers GS, Tettelin H, Eisen JA, Heidelberg JF, Dodson RJ, Davidsen TM, DeBoy RT, Fouts DE, Haft DH, Selengut J, Ren Q, Brinkac LM, Madupu R, Kolonay J, Durkin SA, Daugherty SC, Shetty J, Shvartsbeyn A, Gebregeorgis E, Geer K, Tsegaye G, Malek J, Ayodeji B, Shatsman S, McLeod MP, Smajs D, Howell JK, Pal S, Amin A, Vashisth P, McNeill TZ, Xiang Q, Sodergren E, Baca E, Weinstock GM, Norris SJ, Fraser CM, Paulsen IT. Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc Natl Acad Sci U S A. 2004;101:5646–5651. [PubMed]
  • Slivienski-Gebhardt LL, Izard J, Samsonoff WA, Limberger RJ. Development of a novel chloramphenicol resistance expression plasmid used for genetic complementation of a fliG deletion mutant in Treponema denticola. Infect Immun. 2004;72:5493–5497. [PMC free article] [PubMed]
  • Steck TR, Pruss GJ, Manes SH, Burg L, Drlica K. DNA supercoiling in gyrase mutants. J Bacteriol. 1984;158:397–403. [PMC free article] [PubMed]
  • Teuber M, Meile L, Schwarz F. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie Van Leeuwenhoek. 1999;76:115–137. [PubMed]
  • Woolley RC, Pennock A, Ashton RJ, Davies A, Young M. Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum. Plasmid. 1989;22:169–174. [PubMed]
  • Yang Y, Stewart PE, Shi X, Li C. Development of a transposon mutagenesis system in the oral spirochete Treponema denticola. Appl Environ Microbiol. 2008;74:6461–6464. [PMC free article] [PubMed]