Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains

The rumen is the fermentative forestomach of ruminant animals, and is host to a wide range of anaerobic bacteria whose primary function is to facilitate forage degradation. Butyrivibrio and Pseudobutyrivibrio are closely related proteolytic and fibrolytic genera within the family Lachnospiraceae, and are commonly isolated from the rumens of animals fed fibrous diets. The ribosomal RNA (rRNA) operon is an important phylogenetically informative locus that is present in multiple copies in bacterial genomes. Ribosomal RNA genes are typically arranged in the order 16S-23S-5S, with internal transcribed spacer (ITS) regions located between the genes. However, in the rumen bacterium, Butyrivibrio proteoclasticus B316, rRNA operons have a 16S-5S-23S rRNA gene arrangement, and analysis of bacterial genome projects revealed that this configuration was present in all publicly available complete genomes from members of the family Lachnospiraceae. The 16S-23S ITS region is commonly used to identify bacterial strains, thus we sought to determine the utility of this region from rumen Butyrivibrio and Pseudobutyrivibrio isolates for their rapid molecular identification. Polymerase chain reaction was used to amplify 16S-23S ITS regions, which were assessed for length polymorphism (ITS-LP), and restriction fragment length polymorphism (ITS-RFLP) using AluI, HaeIII and HhaI on a panel of 13 Butyrivibrio and Pseudobutyrivibrio reference strains. Cluster analysis of the resulting banding patterns revealed that while the ITS-LP method did not group the strains according to major Butyrivibrio and Pseudobutyrivibrio clades identified via 16S rRNA gene sequences, ITS-RFLP was more discriminative, and able to rapidly delineate the strains into these clades.