Plasmid 58 (2007) 89–100 www.elsevier.com/locate/yplas Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae q K. Daniel Murray a,*, Katherine A. Aronstein a, Jesse H. de Leo´n b a USDA-ARS, Honey Bee Research Unit, Kika de la Garza Subtropical Agricultural Center, 2413 E. Hwy 83, Weslaco, TX 78596, USA b USDA-ARS, Beneficial Insects Research Unit, Kika de la Garza Subtropical Agricultural Center, 2413 E. Hwy 83, Weslaco, TX 78596, USA Received 25 October 2005, revised 31 January 2007 Available online 23 March 2007 Communicated by Manuel Espinosa Abstract This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae—a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling-cir- cle mechanism, and sequence comparisons place it in the pMV158 family of rolling-circle replicons. The plasmid contains predicted rep, cop, and rnaII genes for control of replication initiating at a predicted double-strand origin. The plasmid has an ssoT single-strand origin, which is efficient enough to allow only very small amounts of the single-stranded DNA inter- mediate to accumulate. The overall efficiency of replication is sufficient to render the plasmid segregationally stable without selection in P. larvae and in Bacillus megaterium, but not in Escherichia coli. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements. Published by Elsevier Inc. Keywords: Paenibacillus larvae; Rolling-circle replication; Plasmid; tetL; Tetracycline; American Foulbrood 1. Introduction q Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and Paenibacillus larvae is a Gram-positive bacterial does not imply recommendation or endorsement by the US pathogen which causes American Foulbrood, the Department of Agriculture. * Corresponding author. Fax: +1 956 969 5033. most serious infectious disease of honey bees. E-mail address: [email protected] (K.D. Commercial and hobbyist beekeepers have con- Murray). trolled this disease for decades with the antibiotic 0147-619X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.plasmid.2007.02.001 90 K.D. Murray et al. / Plasmid 58 (2007) 89–100 oxytetracycline (OTC). However, P. larvae resis- 2. Materials and methods tance to this antibiotic has become widespread in the past few years (Cox et al., 2005; Evans, 2003; Miyagi et al., 2000; Mussen, 2000). We recently 2.1. Southern hybridization discovered a plasmid in P. larvae conferring OTC- DNA was isolated from stationary phase cultures of P. resistance, which we named pMA67, and found that larvae strain 67E (pMA67-containing; Murray and Aron- among 36 strains tested from across North America, stein, 2006) by the method of O’Sullivan and Klaenham- there was a perfect correlation between the presence mer (1993). DNA samples were run on a 1% TAE of the plasmid and resistance to tetracyclines (Mur- agarose gel at 60 V for 90 min in duplicate lanes, which ray and Aronstein, 2006). The predicted OTC-resis- were then separated. One was kept in the neutralization tance gene on this plasmid is tetL, and it is the first buffer (1 M Tris, pH 7.4, 1.5 M NaCl) while the other representative of the tetracycline resistance genes to was treated with alkali (0.5 M NaOH, 1.5 M NaCl) for be found in the Paenibacillus genus. 45 min before neutralization. DNA was transferred by A preliminary analysis of pMA67 suggested that capillary action to a BrightStar Plus Nylon membrane the plasmid replicates by the rolling-circle mecha- (Ambion Inc., Austin, TX) in 10· SSC. The transfer pro- nism (Murray and Aronstein, 2006). Rolling circle cedure, hybridizations, and washes were done according to standard procedures (Sambrook and Russell, 2001). replication (RCR) plasmids have a double-strand The probe used was a digoxigenin-labeled fragment of replication origin (dso), which is nicked by a plas- pMA67 made by PCR using primers 1776-F (GTG mid-encoded Rep protein in order to initiate replica- TTGGAAGCAAAACAATAT) and 2371-R (GCTTTC tion. The leading strand is extended from that nick, CATATAGAGCTGTT) (Fig. 1), and detection was per- generating a full length single-stranded copy of the formed according to the manufacturer’s instructions plasmid, which can often be detected in cells con- (Roche Diagnostics, Mannheim, Germany). taining RCR plasmids. This single-stranded DNA (ssDNA) is then used as template in synthesis of 2.2. Segregational stability of pMA67 the lagging strand, beginning at the single-strand origin (sso) (reviewed in Khan, 2005). There are Serial cultures of P. larvae strain SD1 (pMA67-contain- no plasmid-encoded functions necessary for produc- ing; Murray and Aronstein, 2006) were grown for 24 h at tion of the lagging strand from the ssDNA 35 °C with shaking in liquid J media (St. Julian et al., intermediate. 1963) without antibiotics, except for the first culture in the Regulation of replication of RCR plasmids series, which contained 5 lg/ml tetracycline. Dilutions of occurs mainly at initiation of leading strand synthe- each serial culture were spread on J plates without antibiot- ics in order to generate colonies for tetracycline resistance sis at the dso, such that Rep protein concentration testing. After the J plates were incubated three days at controls plasmid replication. The Rep concentration 35 °Cin6%CO2, single colonies were picked and replicated is regulated by countertranscribed RNAs (ctRNA) onto J plates with and without tetracycline (10 lg/ml), and alone or in combination with a protein (del Solar these were also incubated under the same conditions. These et al., 1998). RCR plasmids seem to lack active par- plates were scored after three days incubation to obtain the titioning systems, so that segregational stability is fraction of tetracycline-resistant (Tetr) colonies. dependent on random distribution of plasmids to For Escherichia coli segregational stability experi- daughter cells. ments, serial cultures of strain YMC9 (Backman et al., In this work, we analyzed the DNA sequence of 1981) transformed with pMA67 (or pBR322) were grown plasmid pMA67, and identified conserved sequences for 9–16 h at 37 °C with shaking in LB media without and putative secondary structures suspected to be antibiotics, except for the first culture in the series, which contained 10 lg/ml tetracycline. Dilutions of each serial important for various pMA67 functions. All genes culture were spread on LB plates with or without tetracy- on pMA67 are predicted by sequence to be involved cline (10 lg/ml) and incubated at 37 °C. Plates were with either plasmid replication, plasmid mobiliza- scored after 24 h of growth to obtain the fraction of Tetr tion, or antibiotic resistance. In addition, we par- colonies. tially characterized the plasmid with respect to its sso function, physiological stability, and host range, 2.3. Bacterial transformations with pMA67 and screening and we demonstrated the functionality of the tetL gene. We also report the uniqueness of this tetL in Commercially prepared protoplasts of Bacillus megate- terms of its relatively ancient divergence from other rium strain WH320 (MoBiTech, Goettingen, Germany) plasmid-encoded tetL genes. were used in transformation experiments according to K.D. Murray et al. / Plasmid 58 (2007) 89–100 91 0 1.0 2.0 3.0 4.0 5.0 pMV158 dso copG rnaII repB tetL ssoU oriT mobM ssoA pMA67 dso cop rnaII rep tetL ssoT oriT mob 1776F 2371R 1776F 3664R 1776F 805R 741F 3664R Fig. 1. Comparison of overall genetic structure of pMV158 and pMA67 plasmids. Arrows show the direction of transcription. Ruler units are in kb. pMV158 has 5541 bp, and pMA67 has 5030 bp. Horizontal lines at the bottom of the figure represent products from PCR amplifications of pMA67 using the primers indicated. See text for further details. Part of this figure is reproduced by permission from an earlier publication in the Journal of Apicultural Research. the manufacturer’s instructions. The DNA used was 1 lg We gel purified the PCR products using a QIAquick Gel of pMA67 or pWH1520 (positive control; MoBiTech). Extraction Kit (Qiagen Inc., Valencia, CA) according to For E. coli, chemical transformation of strain YMC9 the manufacturer’s instructions, ligated these two frag- was done using a standard protocol (Sambrook and Rus- ments into the pCR2.1-TOPO vector (Invitrogen Corp., sell, 2001). The DNA used was 10 ng of pMA67 or Carlsbad, CA), and transformed TOP10 (Invitrogen) cells pBR322 (positive control). For either bacterium, selection according to the manufacturer’s instructions. Tetracycline was for tetracycline resistance at 10 lg/ml. Negative con- at 10 lg/ml was used for selection of transformants, and trol transformation attempts done without addition of for the truncated tetL construct, a separate selection for any DNA yielded no colonies. ampicillin (100 lg/ml) resistance was also done (vector Screening of potential pMA67-carrying transformants contains the bla gene). was done initially by colony PCR with primers 1776-F Colonies confirmed by PCR to have pMA67 tetL and 2371-R (Fig. 1). Cycling conditions were 95 °C for sequences (primers 1776-F/2371-R) were tested for resis- 5 min, then 35 cycles of 94, 60, and 72 °C for 1 min each, tance to 10 lg/ml tetracycline on LB plates at 37 °C.