Multiplex PCR screening of soil isolates for novel Bacillus-related lineages

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze–heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.