Analysis of Pma67, a Predicted Rolling-Circle Replicating, Mobilizable, Tetracycline-Resistance Plasmid from the Honey Bee Pathogen, Paenibacillus Larvae Q

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-circle 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 intermediate 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 antibiotics 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. Con- followed by a final extension of 72 C for 10 min. Plasmid firmation of cloning of correct tetL constructs was done minipreps of potential pMA67-carrying B. megaterium by digestion of plasmid minipreps with BglII, and PCR transformants were done by the method of O’Sullivan amplification with primer pairs 1776-F/805-R (for 50 half and Klaenhammer (1993). Plasmid minipreps of E. coli of tetL) and 741-F/3664-R (for 30 half of tetL). The 741-F were done using an Eppendorf Fast Plasmid Mini kit primer sequence is ACAGAACCTTTTGTTGAACC. (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions. 2.5. Bioinformatics

2.4. Construction and cloning of tetL alleles derived from Genetic database searches for homologies to pMA67 pMA67 sequences were done using BLAST (Altschul et al., 1990). Accession numbers for sequences related to The full length tetL gene for cloning was generated by pMA67 used in this study are shown in Table 1. PCR with primers 1776-F and 3664-R (CACTTTT For the phylogenetic analysis, the alignment software AGCCGAAGAAAATAA), and the 30 truncated allele ClustalX (Thompson et al., 1997) and the phylogenetic with primers 1776-F and 805-R (CCACAA program PAUP version 4.0b10 for the Macintosh (PPC) AGGACACCAATTAT) (Fig. 1). The PCR cycling con- (Swofford, 2002) were utilized for alignment, bootstrap- ditions for the full length tetL were 94 C for 4 min, then ping (as percentage of 1000 replications) (Felsenstein, 35 cycles of 94 C 1 min, 60 C 1 min and 72 C for 2 min, 1995), and reconstruction of trees as described in de Leoń followed by a final extension of 72 C for 10 min, and et al. (2006). Phylogenetic trees were constructed using were the same for the truncated allele except that anneal- both distance and maximum parsimony methods. For ing temperature was 56 C and extension time was 1 min. the distance analysis, the neighbor-joining algorithmic 92 K.D. Murray et al. / Plasmid 58 (2007) 89–100

Table 1 Nucleotide sequence accession numbers Plasmid or organism Accession pAMa1 NC_005013 pBAA1 M24251 pBC16 X51366 pBMY1 AJ243967 pBMBt1 AY822042 pCCK3259 NC_006976 pGI2 X13481 pJB01 AY425961 pJH1 U17153 pJHI AY129652 pLC2 Z14234 pLS1 M29725 pLS141-1 AB109041 pMA67 DQ367664 pMV158 X15669 Fig. 2. Detection of ssDNA from a pMA67-containing P. larvae pNS1 M16217 strain. Molecular weight standard, ethidium bromide stained pNS1981 D00006 (lane M). DNA preparation from pMA67-containing P. larvae pPF107-3 Y12675 strain 67E, ethidium bromide stained (lane 1). Southern blot of pSTE1 X60828 same DNA preparation as lane 1, with denaturation of nucleic pTA1060 U32380 acids before transfer (lane 2). Southern blot of same DNA pTB19 M63891 preparation as lane 1, without denaturation before transfer (lane pTHT15 M11036 3). The probe was a DIG-labeled segment of pMA67 (see Section pTX14-3 NC_001446 2). No hybridization signal was detected in a plasmid-free pUIBI-1 AF516904 P. larvae strain (B2605) in multiple attempts (not shown). a Bacillus subtilis 168 X08034 Chrom, chromosomal DNA; OC, pMA67 open circle form; SC, a Bacillus subtilis R D12567 pMA67 supercoiled form; SS, pMA67 single stranded. Enterococcus faeciuma AY081910 a Chromosomal tetL sequences for Fig. 7. ethidium bromide (Fig. 2, lane 1), but is detected by hybridization (Fig. 2, lanes 2 and 3). When DNA is method was performed utilizing the uncorrected ‘p’ not denatured by alkali before transfer, only the genetic distance parameter (Saitou and Nei, 1987). For DNA which is already single-stranded binds to the the parsimony analysis, heuristic searches for the most parsimonious trees were conducted using closest step-wise membrane (Fig. 2, lane 3). addition and the branch swapping algorithm by tree bisec- RCR plasmids are classified on the basis of tion–reconnection. Gaps were treated as missing data and amino acid sequence homologies among their Rep constraints were not enforced. proteins. There are more than a dozen groups (or families) recognized, with four major groups called I–IV (www.essex.ac.uk/bs/staff/osborn/DPR_ho- 3. Results and discussion me.htm). Using this system, pMA67 is classified as a Group II (also called the pMV158 family) plas- 3.1. pMA67 is a Group II RCR plasmid mid. Indeed, based on nucleotide and amino acid sequence analyses of the entire plasmid, pMA67 is The definitive result that confirms a plasmid rep- quite similar in size and overall organization to licates by the RCR mechanism is the presence of the archetypal Group II plasmid, pMV158 from ssDNA in cells that harbor the plasmid. However, Streptococcus agalactiae, the main difference being several RCR plasmids do not produce detectable the presence of a second sso in pMV158, which ssDNA (discussed below). We were able to detect pMA67 lacks (Fig. 1). a small amount of ssDNA by Southern hybridization from cultures of pMA67-containing P. larvae 3.2. Rep protein and double-strand replication origin (Fig. 2). The 5.0 kb pMA67 supercoiled monomer migrates at about the rate of a 3 kb linear standard, The best match in NCBI databases to the pre- as has been seen previously (Murray and Aronstein, dicted Rep protein of pMA67 is the Rep protein 2006). The ssDNA does not significantly stain with from Lactobacillus sakei plasmid pLS141-1 with K.D. Murray et al. / Plasmid 58 (2007) 89–100 93 which it shares 80% amino acid identity. Rep pro- with the Rep protein of an RCR plasmid (Chang teins across the four major RCR plasmid groups et al., 2002). PcrA helicases may play a role in deter- have conserved motifs. Two of the more important mining RCR plasmid host range (Ruiz-Maso´ et al., ones are called motif 2 (or HUH motif), thought to 2006). The P. larvae genome has recently been be a part of a metal binding domain, and motif 3, sequenced (Qin et al., 2006), and it contains a likely part of the catalytic (nicking) domain (del Solar pcrA gene (accession CH981424; ORF is at coordi- et al., 1998). Motifs 2 and 3 have consensus nates 16,906–19,220) whose predicted protein is sequences of KkxHYHUUU and gxUxYUtHxxxD more than 50% identical in amino acid sequence respectively in Group II plasmids (del Solar et al., to PcrA proteins from several Bacillus spp. (not 1998), where ‘x’ is any amino acid, ‘U’ is a hydro- shown). phobic residue, and uppercase and lowercase letters The dsos of RCR plasmids contain a Rep protein represent strongly- and weakly-conserved residues, binding site and nick site. Typically, the sequence of respectively. Both of these motifs are present in the nick site is well conserved in all plasmids belong- the predicted pMA67 Rep protein, with sequences ing to the same RCR group, but Rep binding site KKAHYHVIY and NIYLYLTHESKD (corre- sequences are not as well conserved (Khan, 2005). sponding to nucleotide positions 653–679 and 785– However, Fig. 3 shows that there is a high degree 820, respectively in the GenBank sequence) being of sequence identity between both the nick and perfect matches to consensus with the exception of Rep binding site regions of the dsos of pMA67 the first weakly conserved glycine residue of the and pLS1—a D(ssoU–mobM) derivative of motif 3 consensus. The tyrosine residue in the motif pMV158 (Lacks et al., 1986). pMV158/pLS1 must 3 consensus is required for nicking the dso nick site be supercoiled in order for in vitro replication to ini- (del Solar et al., 1998), and this tyrosine is also pres- tiate, suggesting that hairpin formation at the dso ent in the predicted pMA67 Rep protein. nick site is required for nicking by Rep protein A host encoded helicase called PcrA, apparently (Puyet et al., 1988). The actual site of nicking is in ubiquitous in Gram-positive bacteria has been the loop of the hairpin (Puyet et al., 1988), and shown to be required for replication of a Group I the 9 bp sequence in the loop is identical between RCR plasmid (Iordanescu, 1993; Iordanescu and pMV158/pLS1 and pMA67 (Fig. 3). The Rep bind- Basheer, 1991), and is considered likely to be ing site in Group II plasmids is associated with two involved in replication of RCR plasmids of all or three direct repeats (del Solar et al., 1998). In groups (Khan, 2005). The PcrA helicase of Staphy- pMV158/pLS1 and pMA67, there are three 11 bp lococcus aureus was shown to physically interact direct repeats (Fig. 3).

Fig. 3. Double strand origins and Pcr promoters known in pMV158/pLS1, and predicted in pMA67. Bases in common are shaded. Solid arrows indicate inverted repeats (IR). Dashed arrows indicate an IR present in pMV158/pLS1 but absent in pMA67. The site of nicking by the Rep protein in pMV158/pLS1, and the Rep protein binding site (dotted underline) are identiﬁed in del Solar et al. (1993a). The Pcr promoter -35 and -10 sequences of pMV158/pLS1 as well as potential promoter hexamers in pMA67 are solid underlined. The bold nucleotides are short IRs. The +1 nucleotide for the pMV158/pLS1 copG–repB transcript is shown. Numbered bases at beginning and end of sequence refer to base numbers in the GenBank sequences. (Sequences shown are of pLS1; the ﬁrst base of the -35 hexamer is an ‘‘A’’ in the pMV158 GenBank entry.) 94 K.D. Murray et al. / Plasmid 58 (2007) 89–100

3.3. Cop and RNAII regulators of rep expression 69% to 73% similarity when conserved substitutions are included. The homolog from pJB01 was recently In Group II plasmids, the cop and rnaII gene tentatively identified as a Cop protein (Kim et al., products are negative regulators of rep expression 2006). at the transcriptional and translational levels, A thorough structural analysis of the pMV158/ respectively. Cop is a repressor of transcription pLS1 Cop protein revealed that the protein forms from Pcr, the promoter for rep, and RNAII is a a ribbon–helix–helix structure (Gomis-Ru¨th et al., ctRNA which is thought to interfere with transla- 1998). An amino acid sequence alignment of this tion initiation from the rep mRNA (del Solar protein to many known and putative Cop proteins, et al., 1995). For Group II plasmids, the regulation including those unknown proteins from pLC2 and of plasmid replication has been best characterized pPF107-3, revealed several conserved amino acids for plasmid pLS1 (del Solar et al., 1993a). The in crucial positions throughout the group, including pMV158/pLS1 copG and repB genes are cotran- a highly conserved glycine mediating a turn between scribed onto a single polycistronic mRNA from two a-helices, a serine or threonine residue also in promoter Pcr (Fig. 3). There is a putative cop gene the turn, and conservation of hydrophobicity at in pMA67 upstream of rep (Fig. 1), as well as two seven isolated residues necessary for maintaining possible promoters which may correspond to the the hydrophobic core of the dimer protein Pcr promoter of pLS1, and there is a short IR over- (Gomis-Ru¨th et al., 1998). The six most highly con- lapping each of the potential pMA67 Pcr promoters served hydrophobic amino acid positions in that (Fig. 3). A similar IR in pMV158/pLS1 constitutes analysis are also present in pMA67, as well as the the central element of the binding site for the conserved glycine in the turn (Fig. 4a). Interestingly, dimeric CopG protein (del Solar et al., 1990). in the pMA67-encoded Cop protein, as well as in The predicted pMA67 cop gene would produce a each of those pMA67-like predicted Cop proteins protein of 56 amino acids, similar in length to the from the four Lactobacillus and Enterococcus plas- pMV158/pLS1 CopG protein of 45 amino acids. mids mentioned above, an isoleucine residue occu- The predicted Cop protein of pMA67 has little pies the position in the turn of the conserved amino acid homology to known Cop proteins pres- serine/threonine. ent in NCBI databases. It does have significant The sequence downstream of cop in pMA67 pre- homology to unidentified proteins predicted from dicts that an RNAII is produced by this plasmid, as four other RCR plasmids, pLS141-1 from L. sakei, in pMV158/pLS1. The promoter and terminator of pLC2 from Lactobacillus curvatus, pPF107-3 from the rnaII gene from pLS1 (del Solar and Espinosa, Lactobacillus lactis, and pJB01 from Enterococcus 1992), and the corresponding hypothetical struc- faecium, ranging from 50% to 57% identity, and tures in pMA67 are shown in Fig. 4b. Like most

Fig. 4. (a) Alignment of the entire pMV158/pLS1 Cop amino acid sequence with corresponding sequence predicted from pMA67. Conserved hydrophobicity of Cop amino acids is indicated at the shaded positions, the highly conserved glycine is in bold, and the conserved serine/threonine position is indicated by an underline. The numbers in brackets indicate additional residues not shown in the alignment. (b) rnaII gene promoters and terminators. For pLS1, promoter hexamers are underlined, the +1 nucleotide is shown, and dotted underlined bases are an imperfect IR that forms the transcription terminator in the RNA. Predicted features for pMA67 are similarly indicated. The ‘‘C’’ at position 769 (adjacent to the loop) in pLS1 is a ‘‘G’’ in the GenBank pMV158 sequence. K.D. Murray et al. / Plasmid 58 (2007) 89–100 95

Fig. 5. Alignment of ssoT sequences of several plasmids. IRs are underlined in the individual sequences and shown by numbered arrows below the sequences. Dashed arrows indicate an IR unique to pGI2. Consensus motif sequences are based on the analysis of Andrup et al. (2003). Bases matching consensus in the motifs are shaded. Sequence alignments were initially performed with MegAlign software (DNASTAR Inc., Madison, WI), followed by manual editing to align the motifs and IRs.

Group II plasmids (but unlike pMV158 itself), the (Fig. 2), with those four plasmids with apparently promoter for the pMA67 ctRNA has a so-called highly efficient ssoTs, and the plasmid in NCBI dat- ‘‘extended 10’’ motif (del Solar and Espinosa, abases with the highest sso sequence identity to 2001). These motifs have a consensus of TGNTA pMA67, pUIBI-1 (78% identity over the 170 bp TAAT, and are associated with strong promoters, region encompassing pMA67 nucleotides 3185- at least in E. coli (deHaseth et al., 1998). The stretch 3348), which produces abundant ssDNA that can of T’s after the stem-loops of the terminators sug- be detected simply by ethidium bromide staining gest that they are rho-independent. of DNA preparations in agarose gels (Lopez-Meza et al., 2003). 3.4. Single strand origin The alignments suggest that a possible reason for the relatively poor function of the pMA67 and espe- Most research groups recognize four major types cially the pUIBI-1 ssoTs is the makeup of the ele- of ssos—ssoA, ssoT, ssoU, and ssoW (Andrup et al., ments of the first stem-loop, i.e., motifs I and II, 2003; Khan, 2005). The ssos of a particular type have and IR1. In motif I of pMA67 or pUIBI-1, neither only minimal sequence homology, but instead are the spacing between conserved bases nor the bases recognized mainly by their common secondary struc- themselves match consensus. For motif II, there is tures (Khan, 1997), and for some types of ssos, con- a suboptimal spacing between conserved bases for sensus sequences within motifs (Andrup et al., 2003). pMA67, and the consensus bases are not all present The pMA67 sso contains all the motifs and pre- in pUIBI-1. Perhaps most important for sso effi- dicted secondary structures that are typical of ssoT ciency, the bases making up the conserved stem of origins (Fig. 5). An efficient sso is expected to result IR1, which are immediately adjacent to motifs I in little accumulation of the ssDNA intermediate in and II in the other plasmids, are a few bases the cell. The inability to detect ssDNA in bacteria removed from those motifs in pUIBI-1. Given that containing RCR plasmids with ssoT origins is quite the first stem-loop has been shown to be important common, as seen for plasmids pBAA1 (Seery and for ssoT function in pBAA1 (Seery and Devine, Devine, 1993), pGI2 (Hoflack et al., 1999), 1993), pTX14-3 (Madsen et al., 1993), and pTX14-3 (Boe et al., 1991), and pTA1060 (Meijer pTA1060 (Meijer et al., 1998), our speculation is et al., 1995). Fig. 5 compares the ssoT regions of that the non-consensus elements of that stem-loop pMA67, which produces a small amount of ssDNA in pMA67 and pUIBI-1 contribute to poorer 96 K.D. Murray et al. / Plasmid 58 (2007) 89–100 function for the sso, and subsequently the accumu- Ruiz-Maso´ et al., 2006). For lagging strand replica- lation of ssDNA. tion, host RNA polymerase recognition (or not) of the plasmid sso may play a role in plasmid host 3.5. Segregational stability of pMA67 in P. larvae range. There is evidence that ssoA and ssoW are host-specific, while ssoT and ssoU are thought to As seen, each of the genetic elements—dso, rep, be able to be used by a number of Gram-positive cop, and rnaII—involved in leading strand initiation hosts (Khan, 2005). in pMV158/pLS1 has also been predicted by To determine whether pMA67 replication is pos- sequence in pMA67. The functionality of the repli- sible in another Gram-positive species, we cation initiation system can be partially assessed attempted transformations of the plasmid into by determining the segregational stability of a plas- B. megaterium, and were successful (data not mid. Therefore, we sought to investigate mainte- shown). Growth rates of the B. megaterium trans- nance of the pMA67 plasmid in P. larvae over formants in rich media were almost the same in many generations in the absence of selection. This the presence or absence of tetracycline (not shown), effort was somewhat hindered by the difficulty in implying that plasmid replication is able to keep maintaining viable P. larvae during serial subcultur- pace with cell division, and therefore that the B. ing, a phenomenon which has been previously megaterium PcrA helicase interacts well with the reported for P. larvae (Heyndrickx et al., 1996; Pic- pMA67 Rep protein, and that the B. megaterium cini and Zunino, 2001). We determined the fraction RNA polymerase recognizes the pMA67 ssoT. of cells maintaining pMA67 without selection in a Natural RCR plasmids are also known in Gram- liquid culture series by daily plating without selec- negative hosts (www.essex.ac.uk/bs/staff/osborn/ tion to generate colonies, then testing these colonies DPR_home.htm). In addition, although isolated (typically 100 each day) for tetracycline resistance from a Gram-positive host, pMV158 is able to rep- along with tetracycline-resistant and sensitive con- licate in some Gram-negative bacteria (del Solar trol strains. The longest growing culture series et al., 1993b; Hernandez-Arriaga et al., 2000; Lacks lasted 66 generations (10 days), and every colony et al., 1986). We attempted transformation of E. coli tested over the entire period was found to be tetra- with pMA67, and found this also to be successful. cycline resistant. Other attempts which resulted in Transformants of both B. megaterium and E. coli fewer generations before culture viability was lost, selected with tetracycline were confirmed as authen- nevertheless had similar results with respect to plas- tic both by pMA67-specific colony PCR and plasmid maintenance. These results indicate that the mid minipreps (not shown). replication initiation mechanisms encoded in Unlike the B. megaterium transformants, our pMA67 function quite efficiently in P. larvae, pro- E. coli pMA67 transformants grew very slowly in moting a high enough copy number to ensure distri- rich media in the presence of tetracycline (not bution of plasmids to daughter cells. We suspect shown). To determine if this was due to poor repli- that the segregational stability of pMA67 in P. lar- cation of pMA67, we spread dilutions of an E. coli vae may also be facilitated by the relatively slow pMA67 transformant culture on plates with and growth rate of P. larvae, since it has been shown without tetracycline at various intervals during that a plasmid with a negative replicon control sys- serial growth in liquid cultures in the absence of tem and without an active partitioning system is selection (LB media at 37 C), and found that the more segregationally stable at slower bacterial fraction of pMA67-containing cells dropped rapidly growth rates (Lin-Chao and Bremer, 1986). (Fig. 6). These results are similar to those seen in Hernandez-Arriaga et al. (2000) in experiments with 3.6. Establishment of pMA67 in alternate hosts pMV158 and pLS1 in E. coli. The instability of pMA67 in E. coli indicates that the slow growth pre- The host range of an RCR plasmid may be deter- viously observed in the presence of tetracycline is mined by the host’s ability to replicate either the due to poor pMA67 replication, and suggests that leading or the lagging strand (Khan, 2005). For either leading or lagging strand synthesis (or both) leading strand replication, the interaction (or lack of pMA67 in E. coli is partially defective. Leading thereof) between the host PcrA helicase and the strand synthesis of pMV158 and pMA67 in E. coli plasmid Rep protein can affect the maintenance of probably requires interaction between the Rep pro- an RCR plasmid in the host (Anand et al., 2004; tein and the host UvrD helicase. This helicase has K.D. Murray et al. / Plasmid 58 (2007) 89–100 97

preparation for conjugation (Francia et al., 2004). BLAST searches indicated that the predicted pMA67 Mob protein is most closely related to the Mob proteins from plasmids pUIBI-1 and pBMBt1 from Bacillus thuringiensis, and pBMY1 from Bacil- lus mycoides, with 50–55% identities and up to 73% similarity when conserved substitutions are included. A classification scheme for mobilization regions of plasmids proposed by Francia et al. (2004) identifies four main superfamilies of mobilizable plasmids. Plasmids pUIBI-1 and pBMY1 are included in that analysis, and they are placed in the pMV158 Mob superfamily (these are different plasmid groupings than those based on the Rep pro- Fig. 6. Segregational stability of pMA67 in Escherichia coli. teins), indicating that pMA67 would belong in that Percent of YMC9 cells with plasmid during growth without family also. The superfamily is characterized by an selection was determined by plating dilutions of serial cultures N-terminus proximal motif with consensus ‘HxxR’, and counting colonies arising on plates with or without tetracy- followed by a putative catalytic NY(D/E)L motif, cline. n, pMA67; s, pBR322 (control). No Tetr colonies from pMA67 cultures were obtained at 31 and 42 generations. and a downstream ‘HxDExxPhxh’ motif (Francia et al., 2004; Guzmań and Espinosa, 1997). Each of significant sequence homology to PcrA helicases these is present in the predicted amino acid sequence from Gram-positive bacteria, and was shown to be of the pMA67 Mob protein—HLDR, NYDL, and required for in vivo leading strand synthesis of HNDETTPHMH (corresponding to nucleotide Group II plasmid pE194 in E. coli (Bruand and Ehr- positions 3567–3578, 3630–3641, and 3864–3893, lich, 2000). Hernandez-Arriaga et al. (2000) showed respectively, in the GenBank sequence). that lagging strand synthesis of pMV158 in E. coli Guzmań and Espinosa (1997) identified the oriT initiated at the ssoU, which had a low, but signifi- in pMV158. It contained two overlapping IRs, cant functionality. Since pMA67 can replicate in and had a GTGTGT loop in hairpin 2 (Fig. 7). E. coli, presumably the E. coli RNA polymerase is An analysis of plasmids in the pMV158 Mob super- also able to utilize the pMA67 ssoT to some degree. family has shown that there is a predicted 7–10 bp We have not performed experiments to determine stem and usually a 6 bp loop at the presumed oriT whether leading or lagging strand synthesis is limit- sites (Francia et al., 2004). The overlapping IRs, ing for overall pMA67 replication in E. coli. The the 7–10 bp stems, and the 6 bp loop are all present instability of pMA67 in E. coli does not necessarily in pMA67 (Fig. 7). The first nucleotide of the hair- imply that pMA67 or closely-related plasmids could pin loop in oriT sites ranges from 47 to 140 bp have no significant role in Gram-negative species in upstream of mob gene start codons in RCR plas- natural environments, where the growth rates would mids from various superfamilies (Meijer et al., typically be slower than those afforded by optimal 1998), and this distance is 71 bp in pMA67. laboratory growth conditions, and therefore plasmids would be more segregationally stable. Attempts to transform an E. coli recA mutant strain (TOP10) with pMA67 was unsuccessful in multiple trials (data not shown), consistent with the result from Lacks et al. (1986) in which they showed that E. coli strains must be recA+ for successful transformation with plasmids pMV158 and pLS1.

3.7. Mob protein and oriT site Fig. 7. oriT regions of pMV158 and pMA67. The IRs are shown by numbered arrows. The nicking site in pMV158 and presumed The mob genes of mobilizable plasmids encode nicking site in pMA67 are shown. The underlined ‘‘C’’ common DNA relaxases which nick supercoiled DNA in a to the stems of both pMA67 IRs would be unpaired in IR2, but site- and strand-speciﬁc fashion at the oriT site in not in IR1. 98 K.D. Murray et al. / Plasmid 58 (2007) 89–100

3.8. TetL protein most parsimonious tree with 87 parsimony-informa- tive characters with a length of 299 steps, consis- Currently in the NCBI databases, there are tetL tency index (CI) of 0.977, and a retention index genes from ten different natural plasmids. These, (RI) of 0.946, which showed the same topology as as well as one chromosomal tetL from E. faecium, the neighbor-joining tree with strong bootstrap val- are predicted to produce TetL proteins with 98% ues (data not shown). or higher amino acid sequence identity. These are In order to show that it is in fact the tetL gene of seen in the neighbor-joining phylogenetic tree of pMA67, which was identified only using genetic dat- Fig. 8 as clade ‘‘A’’, supported by a very strong abases, that is responsible for conferring tetracy- bootstrap value. By contrast, the predicted cline resistance to the pMA67-containing bacteria pMA67 TetL was found to be unique among plas- in this study, we cloned in isolation the entire tetL mid-encoded TetL proteins in that it has only 86– gene of pMA67, as well as a mutated tetL (30 trun- 88% amino acid sequence identity to the others. cated). The two tetL alleles were generated by PCR As seen from the phylogram, pMA67 forms its from the pMA67 regions indicated in Fig. 1 using own clade with strong support. Thus, the pMA67 primer pairs 1776-F/3664-R and 1776-F/805-R. tetL appears to have arisen from a relatively ancient We ligated each of the PCR products to a plasmid divergence from the other plasmid encoded tetL vector, and transformed E. coli. All transformants genes. Two chromosomal tetL genes from different with the full-length tetL were found to be tetracy- strains of Bacillus subtilis produce almost identical cline resistant, and all with the truncated tetL were TetL proteins, which form another clade. The found to be tetracycline sensitive, as expected. This Fig. 8 results were further supported by a single confirms that it is the tetL gene of pMA67 that con- fers the tetracycline-resistance phenotype. The tetL gene of pMA67 possesses translational 100 B. subtilis R attenuation sequences (GenBank nucleotide posi- 0.097 B. subtilis 168 tions 1450–1602) upstream of the coding region, containing the IRs and leader peptide ORF, which are pLS1 typical of inducible tet genes (Hoshino et al., 1985). pJHI These intact regulatory sequences further support that the pMA67 tetL gene is expressed in P. larvae. α pAM 1 Although there have been plasmids found in pTB19 P. larvae before (Benada et al., 1988; Bodorova- Urgosikova et al., 1992; Drobnı´kova´ et al., 1994; pCCK3259 Neuendorf et al., 2004) no phenotype was associ- pJH1 A ated with any of them, no genes were identified, 65 pNS1981 and no sequencing was done on them. Therefore, pMA67 is the first well characterized plasmid from 62 pBC16 P. larvae. Plasmid pMA67 also provides the first pTHT15 record of any tetracycline-resistance gene found in 0.317 100 the entire Paenibacillus genus (Chopra and Roberts, 0.061 E. faecium 98 2001; Roberts, 2005). Since pMA67 is predicted to pSTE1 be mobilizable, then this plasmid, or a close relative, 0.023 may be present in other bacteria sharing the same pMA67 0.057 natural environment as P. larvae. We have, in fact, pNS1 TetK detected tetL by PCR in Paenibacillus alvei— 0.01 changes another species which is closely associated with Fig. 8. Phylogram of tetL genes inferred by the predicted amino honey bees—so it may be that pMA67 or a closely acid sequences using a neighbor-joining algorithm. Bootstrap related plasmid will be found in that species also. support values are indicated at the nodes, and the branch lengths are underlined. The amino acid sequence of the Staphylococcus Acknowledgments aureus pNS1-borne tetK gene is provided as an outgroup. For plasmid-borne tetL genes, the plasmid name is shown. For chromosomal tetL genes, names of the organisms (and strain We thank Anita Davelos Baines (University of when applicable) are shown. Texas-Pan American, Edinburg, TX), Jay Evans K.D. Murray et al. / Plasmid 58 (2007) 89–100 99

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