Paenibacillus larvae 16S–23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization

Abstract

Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S–23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3′ terminal nucleotides of the 16S rRNA gene, 5′ terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA^ala, and tRNA^ile genes. Gene organization within the three rrn operon types was 16S–23S, 16S–tRNA^ala–23S, and l6S–5S–tRNA^ile–tRNA^ala–23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S–23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions.

Introduction

Within bacteria, rRNA genes generally follow an organizational pattern of 16S–23S–5S rRNA in ribosomal RNA (rrn) operons. Also, copy number of rrn operons per bacterial genome is presently reported to vary from 1 to 15 (Klappenbach et al., 2000). For example, Escherichia coli contains seven copies of rrn operons whereas, Bacillus subtilis contains ten copies (Jinks-Robertson and Nomura, 1987, Jarvis et al., 1988). The segment of DNA sequence located between the 16S and 23S (16S–23S) and between the 23S and 5S (23S–5S) rRNA genes in rrn operons define intergenic transcribed spacer (ITS) regions. These regions are described as being involved in precursor rRNA maturation of the polycistronic RNA transcript produced from an rrn operon (Sogin et al., 1976, Sechi and Daneo-Moore, 1993, Luz et al., 1998, Condon et al., 2001).

The 16S–23S ITS region, most studied of the two ITS regions, varies significantly in nucleotide length and sequence between bacterial species. These regions are segments of variable DNA sequence located between the highly conserved rRNA genes. Also, length and/or sequence of 16S–23S rDNA ITS regions within a species are noted to vary; accounting for different types of rrn operons within the bacterium. Thus, variability of 16S–23S rDNA ITS regions is species-specific and multiple copies of any one type of ITS region (establishing different rrn operon types) may occur within a bacterium.

Due to species-specificity, 16S–23S ITS regions have proven to be a powerful research tool for various bacterial investigations. Comparative analyses of ITS region DNA sequences, nucleotide lengths, and numbers present in a chromosome have been useful for phylogenetic grouping of bacteria (Martínez-Murcia et al., 1999, Daffonchio et al., 2000, Cherif et al., 2003). Also, these comparisons have helped to identify polymorphic rrn operon loci in the same chromosome and, thus characterize closely related strains and species of bacteria (García-Martínez et al., 1996, Gürtler and Stanisich, 1996, Chun et al., 1999, Gürtler et al., 1999).

PCR amplification of ITS DNA has been successfully used for rapid and accurate bacterial identification. Employing oligonucleotide primers directed to the universally conserved DNA sequences within bacterial 16S and 23S rDNA (i.e., 16S rDNA region 2 and 23S rDNA region 10; Gürtler and Stanisich, 1996), all 16S–23S rDNA ITS regions within any bacterial isolate can be PCR amplified. Species-specificity of ITS sequence lengths, and presence of multiple copies of rrn operons (i.e., different ITS regions), will produce a migration pattern of DNA bands (i.e., ITS-PCR fingerprint) that is unique to species following agarose gel electrophoresis of PCR-amplified ITS region DNA (Gürtler and Stanisich, 1996, Chun et al., 1999, Roth et al., 2000). Jensen and associates (1993) used ITS-PCR fingerprinting to identify over 300 strains of bacteria comprising 28 species and eight genera. ITS-PCR DNA fingerprinting has been applied to the identification of Paenibacillus popilliae/Paenibacillus lentimorbus and has been suggested as useful for identification of other paenibacilli (Dingman, 2009).

Efforts to sequence the complete genome of Paenibacillus larvae, causative agent of American foulbrood in honey bee (Apis mellifera L.) larvae, are underway for strains BRL 230010 and NRRL B-3650 (GenBank accession numbers; AARF00000000 and ADZY00000000). Both projects, demonstrating genome sizes of 4.0 and 4.4 MB, respectively, are providing information to understand genetic structure of this insect pathogen, an ability to perform comparative analysis between bacterial genomes, and helping investigators discern molecular biological and pathological properties of this microbe. In each project, difficulty encountered in assembly of the genomic sequence exists at the rrn operons. A comprehensive identification and characterization of the various rrn operons in P. larvae has not been reported. Also, rrn operon copy number in P. larvae has not been determined.

Based on ITS variability, three rRNA operon types (represented by; rrnA, rrnF, and rrnG), comprising eight operon copies, were reported for P. popilliae (Dingman, 2004). In Paenibacillus polymyxa SC2, 14 rrn operon copies are reported (Ma et al., 2011). However, no distinction of rrn operon types has been provided. An initial investigation of ITS regions in other paenibacilli (including identification of a single ITS region in P. larvae) has been reported (Xu and Côté, 2003). In silico examination of sequence datum shows two different types of ITS regions in the two rrn operons contained in P. larvae B-3650 Contig368 (GenBank accession number; ADZY01000002). Knowing copy number, organizational structure, and sequences for the various rrn operons contained in the P. larvae genome will aid assembly of the genomic sequence. Understanding ITS region structure and chromosomal distribution may also provide insight into phylogenic investigations of this bacterium.

This paper presents a unique ITS-PCR fingerprint profile for P. larvae isolates. The DNA fingerprint produced is applicable for use as a simple, rapid, and accurate identification protocol for this organism. Also presented is a characterization of the various ITS regions in P. larvae. Three ITS region sequence patterns, defining three types of rrn operons, were determined and, for seven different strains, seven copies of 23S rDNA (i.e., rrn operons) were identified.