Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae

doi:10.1016/j.plasmid.2007.02.001

Plasmid

Volume 58, Issue 2, September 2007, Pages 89-100

https://doi.org/10.1016/j.plasmid.2007.02.001 Get rights and content

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.

Introduction

Paenibacillus larvae is a Gram-positive bacterial pathogen which causes American Foulbrood, the most serious infectious disease of honey bees. Commercial and hobbyist beekeepers have controlled this disease for decades with the antibiotic oxytetracycline (OTC). However, P. larvae resistance 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 discovered a plasmid in P. larvae conferring OTC-resistance, which we named pMA67, and found that among 36 strains tested from across North America, there was a perfect correlation between the presence of the plasmid and resistance to tetracyclines (Murray and Aronstein, 2006). The predicted OTC-resistance gene on this plasmid is tetL, and it is the first representative of the tetracycline resistance genes to be found in the Paenibacillus genus.

A preliminary analysis of pMA67 suggested that the plasmid replicates by the rolling-circle mechanism (Murray and Aronstein, 2006). Rolling circle replication (RCR) plasmids have a double-strand replication origin (dso), which is nicked by a plasmid-encoded Rep protein in order to initiate replication. The leading strand is extended from that nick, generating a full length single-stranded copy of the plasmid, which can often be detected in cells containing RCR plasmids. This single-stranded DNA (ssDNA) is then used as template in synthesis of the lagging strand, beginning at the single-strand origin (sso) (reviewed in Khan, 2005). There are no plasmid-encoded functions necessary for production of the lagging strand from the ssDNA intermediate.

Regulation of replication of RCR plasmids occurs mainly at initiation of leading strand synthesis at the dso, such that Rep protein concentration controls plasmid replication. The Rep concentration is regulated by countertranscribed RNAs (ctRNA) alone or in combination with a protein (del Solar et al., 1998). RCR plasmids seem to lack active partitioning systems, so that segregational stability is dependent on random distribution of plasmids to daughter cells.

In this work, we analyzed the DNA sequence of plasmid pMA67, and identified conserved sequences and putative secondary structures suspected to be important for various pMA67 functions. All genes on pMA67 are predicted by sequence to be involved with either plasmid replication, plasmid mobilization, or antibiotic resistance. In addition, we partially characterized the plasmid with respect to its sso function, physiological stability, and host range, and we demonstrated the functionality of the tetL gene. We also report the uniqueness of this tetL in terms of its relatively ancient divergence from other plasmid-encoded tetL genes.

Section snippets

Southern hybridization

DNA was isolated from stationary phase cultures of P. larvae strain 67E (pMA67-containing; Murray and Aronstein, 2006) by the method of O’Sullivan and Klaenhammer (1993). DNA samples were run on a 1% TAE agarose gel at 60 V for 90 min in duplicate lanes, which were then separated. One was kept in the neutralization buffer (1 M Tris, pH 7.4, 1.5 M NaCl) while the other was treated with alkali (0.5 M NaOH, 1.5 M NaCl) for 45 min before neutralization. DNA was transferred by capillary action to a

pMA67 is a Group II RCR plasmid

The definitive result that confirms a plasmid replicates by the RCR mechanism is the presence of ssDNA in cells that harbor the plasmid. However, several RCR plasmids do not produce detectable ssDNA (discussed below). We were able to detect a small amount of ssDNA by Southern hybridization from cultures of pMA67-containing P. larvae (Fig. 2). The 5.0 kb pMA67 supercoiled monomer migrates at about the rate of a 3 kb linear standard, as has been seen previously (Murray and Aronstein, 2006). The

Acknowledgments

We thank Anita Davelos Baines (University of Texas-Pan American, Edinburg, TX), Jay Evans (USDA-ARS, Beltsville, MD), and Daniele Provenzano (University of Texas at Brownsville, Brownsville, TX) for their helpful critical reviews of the manuscript.

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Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae☆

Abstract

Introduction

Section snippets

Southern hybridization

pMA67 is a Group II RCR plasmid

Acknowledgments

J. Mol. Biol.

Plasmid

Plasmid

J. Biol. Chem.

Biol. Control

Plasmid

J. Biol. Chem.

J. Invertebr. Pathol.

FEMS Microbiol. Rev.

J. Mol. Biol.

Plasmid

J. Mol. Biol.

Plasmid

Plasmid

J. Mol. Biol.

Plasmid

FEMS Microbiol Rev.

J. Invertebr. Pathol.

J. Invertebr. Pathol.

FEMS Microbiol. Lett.

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus

J. Bacteriol.

Physical and genetic characterization of the ginA-ginG region of the Escherichia coli chromosome

Proc. Nati. Acad. Sci. USA

Plasmid DNA in Bacillus larvae

J. Apicult. Res.

Largescale isolation and partial characterization of plasmid DNA from B. larvae

Folia Microbiologica

UvrD-dependent replication of rolling-circle plasmids in Escherichia coli

Mol. Microbiol.

Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance

Microbiol. Mol. Biol. Rev.

American foulbrood survey in honey bees pollinating California almonds: III

Resistance to antibiotics. Am. Bee J.

RNA polymerase-promoter interactions: The comings and goings of RNA polymerase

J. Bacteriol.180