A specific PCR system based on the gene encoding the RNA polymerase beta subunit, rpoB, was developed for amplification and denaturing gradient gel electrophoresis (DGGE) fingerprinting of Paenibacillus communities in environmental samples. This gene has been previously proven to be a powerful identification tool for the discrimination of species within the genus Paenibacillus and could avoid the limitations of 16S rRNA-based phylogenetic analysis. Initially, the PCR system based on universal rpoB primers were used to amplify DNAs of different Paenibacillus species. A new reverse primer (rpoBPAEN) was further designed based on an insertion of six nucleotides in the Paenibacillus sequences analyzed. This semi-nested PCR system was evaluated for specificity using DNAs isolated from 27 Paenibacillus species belonging to different 16S rRNA-based phylogenetic groups and seven non-Paenibacillus species. The non-Paenibacillus species were not amplified using this PCR approach and one group of Paenibacillus species consisting of strains without the six-base insert also were not amplified; these latter strains were found to be distinct based on 16S rRNA gene phylogeny. In addition, a clone library was generated from the rpoB fragments amplified from two Brazilian soil types (Cerrado and Forest) and all 62 clones sequenced were closely related to one of the 22 sequences from Paenibacillus previously obtained in this study. To assess the diversity of Paenibacillus species in Cerrado and Forest soils and in the rhizosphere of different cultivars of maize, a PCR-DGGE system was used. The Paenibacillus DGGE fingerprints showed a clear distinction between communities of Paenibacillus in Forest and Cerrado soils and rhizosphere samples clustered along Cerrado soil. Profiles of cultivars CMS22 and CMS36 clustered together, with only 53% of similarity to CMS11 and CMS04. The results presented here demonstrate the potential use of the rpoB-based Paenibacillus-specific PCR-DGGE method for studying the diversity of Paenibacillus populations in natural environments.

Among a large collection of Taiwanese soil isolates, a novel Gram-variable, rod-shaped, motile, endospore-forming bacterial strain, strain V10537(T), was subjected to a polyphasic study including 16S rRNA and gyrB gene sequence analysis, DNA-DNA hybridization experiments, cell wall peptidoglycan type, cellular fatty acid composition analysis and comparative phenotypic characterization. 16S rRNA gene sequence analysis indicated that the organism belonged to the genus Paenibacillus. Strain V10537(T) possessed meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. It contained menaquinone MK-7 as the predominant isoprenoid quinone and anteiso-C(15 : 0) (53.6 %) and C(16 : 0) (19.0 %) as the major fatty acids. Phylogenetically, the most closely related species to strain V10537(T) were Paenibacillus pabuli, Paenibacillus xylanilyticus, Paenibacillus amylolyticus, Paenibacillus barcinonensis and Paenibacillus illinoisensis, with 16S rRNA gene sequence similarities of 99.5, 98.8, 98.3, 98.2 and 98.1 % to the respective type strains. The gyrB gene sequence similarities between strain V10537(T) and these strains were 76.9-85.0 %. DNA-DNA hybridization experiments showed levels of relatedness of 8.5-45.6 % between strain V10537(T) and these strains. The DNA G+C content of strain V10537(T) was 46.7 mol%. Strain V10537(T) was clearly distinguishable from other Paenibacillus species and thus represents a novel species of the genus Paenibacillus, for which the name Paenibacillus taichungensis sp. nov. is proposed. The type strain is V10537(T) (=BCRC 17757(T) =DSM 19942(T)).

Psychrotolerant endospore-forming bacteria Bacillus and Paenibacillus spp. are important spoilage organisms in fluid milk. A recently developed rpoB subtyping method was applied to characterize the diversity and phylogenetic relationships among Bacillus and related sporeformers associated with milk processing systems. Milk samples representing the processing continuum from raw milk to pasteurized products were collected from two fluid milk processing plants, held at 6 degrees C up to the code date that had been established by each processing plant (i.e., either 18 or 21 days), and plated for bacterial enumeration throughout storage. Bacterial colonies selected to represent the visible diversity in colony morphology on enumeration plates were examined further. Among 385 bacterial isolates characterized, 35% were Bacillus spp., and 65% were Paenibacillus spp. A total of 92 rpoB allelic types were identified among these isolates, indicating considerable diversity among endospore-forming spoilage organisms present in fluid milk systems. Of the 92 allelic types identified, 19 were isolated from samples collected from both processing plants. The same rpoB allelic types were frequently identified in paired raw milk and packaged product samples, indicating that Bacillus and Paenibacillus spp. can enter dairy processing systems through raw milk. Certain subtypes were found exclusively in pasteurized samples, including those that were temporally independent, suggesting the possibility of in-plant sources for these spoilage organisms, including through the persistence of selected subtypes in processing plants. Development of effective control strategies for the diverse array of psychrotolerant endospore-forming organisms that currently limit the shelf lives of high-temperature short-time fluid milk products will require comprehensive, integrated efforts along the entire milk processing continuum.