Douglas W. Dingman

DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions.

Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

Comparative analysis of Paenibacillus larvae genotypes isolated in Connecticut

Ninety-six strains of Paenibacillus larvae, causative agent of American foulbrood in honey bee (Apis mellifera) larvae, collected from Connecticut, USA (CT), honey bees, and 12 P. larvae strains not from CT, were genotyped via ERIC-PCR and XbaI-RFLP analysis. All CT-isolates, five strains isolated in South America, three strains from North America (not CT), and one strain isolated in Australia grouped into the ERIC I genotype. Three P. larvae formerly subsp. pulvifaciens strains grouped into ERIC III and IV genotypes. XbaI-RFLP genotyping showed three genotypes within the CT-isolates, and two were identified as XbaI-RFLP Type I and III. The third XbaI-RFLP genotype (Type Ib) represented one of four new XbaI-RFLP genotypes identified. Comparison of genotype results for the P. larvae strains tested was used to develop a correlation between ERIC-PCR genotyping and XbaI-RFLP genotyping. Sixteen CT-isolates were tetracycline-resistant and demonstrated PCR amplification using oligonucleotide primers for tetL. All 16 isolates grouped within XbaI-RFLP Type Ib, suggesting limited introduction of a tetracycline-resistant strain into CT.

Inactivation of Paenibacillus larvae endospores by a hydrogen peroxide/peroxyacetic acid biocide

(2011). Inactivation of Paenibacillus larvae endospores by a hydrogen peroxide/peroxyacetic acid biocide. Journal of Apicultural Research: Vol. 50, No. 2, pp. 173-175.

The solute transport profile of two Aza-guanine transporters from the Honey bee pathogen Paenibacillus larvae

&NA; Two nucleobase transporters encoded in the genome of the Honey bee bacterial pathogen Paenibacillus larvae belong to the azaguanine‐like transporters and are referred to as PlAzg1 and PlAzg2. PlAzg1 and 2 display significant amino acid sequence similarity, and share predicted secondary structures and functional sequence motifs with two Escherichia coli nucleobase cation symporter 2 (NCS2) members: adenine permease (EcAdeP) and guanine‐hypoxanthine permease EcGhxP. However, similarity does not define function. Heterologous complementation and functional analysis using nucleobase transporter‐deficient Saccharomyces cerevisiae strains revealed that PlAzg1 transports adenine, hypoxanthine, xanthine and uracil, while PlAzg2 transports adenine, guanine, hypoxanthine, xanthine, cytosine and uracil. Both PlAzg1 and 2 display high affinity for adenine with Km of 2.95 ± 0.22 and 1.92 ± 0.22 &mgr;M, respectively. These broad nucleobase transport profiles are in stark contrast to the narrow transport range observed for EcAdeP (adenine) and EcGhxP (guanine and hypoxanthine). PlAzg1 and 2 are similar to eukaryotic Azg‐like transporters in that they share a broad solute transport profile, particularly the fungal Aspergillus nidulans AzgA (that transports adenine, guanine and hypoxanthine) and plant AzgA transporters from Arabidopsis thaliana and Zea mays (that collectively move adenine, guanine, hypoxanthine, xanthine, cytosine and uracil).

Functional characterization of the uracil transporter from honeybee pathogen Paenibacillus larvae

The genome of the Honeybee bacterial pathogen, Paenibacillus larvae, encodes for protein a with substantial amino acid sequence similarity to the canonical Escherichia coli uracil transporter UraA. P. larvae expresses the uracil permease (PlUP) locus, and is sensitive to the presence of the toxic uracil analog 5-fluorouracil under vegetative growth conditions. The solute transport and binding profile of PlUP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUP is specific for the transport of uracil and competitively binds xanthine and uric acid. Further biochemical characterization reveals that PlUP has a strong affinity for uracil with a Km 19.5 ± 1.6 μM. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae

Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease‐like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter‐deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine Km 7.04 ± 0.18 μm, and as with other bacterial and plant NCS2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m‐chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.

Functionality of Tn916 in Paenibacillus larvae

The conjugative transposon Tn916 was determined to be functional in Paenibacillus larvae in regard to expression of tetracycline resistance and conjugative transfer. Expression of erythromycin resistance, using Tn916ΔE, was also observed. Conjugative transfer experiments employing Paenibacillus popilliae strains Tc1001 and Em1001 as transposon donors and experiments using different P. larvae subspecies or different transposon-containing strains demonstrated interspecies and intraspecies transfer occurred for Tn916 and Tn916ΔE. Southern hybridization analysis of several Tn916-containing P. larvae isolates showed that the transposon randomly inserted into the bacterial chromosome with an indication that hot spot insertion had occurred. Hybridization analysis indicated single-copy insertion of Tn916 into the genome predominated. However, selection of multiple-resistant isolates (i.e., isolates containing Tn916 and Tn916ΔE) demonstrated that multiple copies of the transposon could coexist in the bacterial genome. Growth of transposon-containing isolates in broth medium in the absence of selective antibiotic pressure showed that Tn916 and Tn916ΔE were stably maintained in the bacterium.