Development of a signature probe targeting the 16S-23S rRNA internal transcribed spacer of a ruminal Ruminococcus flavefaciens isolate from reindeer.

The cellulolytic Ruminococcus flavefaciens has previously been introduced into the ruminant rumen to increase microbial degradation of plant cell wall carbohydrates. The functional effect of an introduced bacterium depends on its ability to establish in the digestive tract, and signature probes can be used as a tool to track and quantify introduced strains. The purpose of this current study was to develop an oligonucleotide signature probe targeting the 16S-23S rRNA internal transcribed spacer (ITS) of a putative probiotic cellulolytic isolate (R. flavefaciens strain 8/94-32) from the rumen of reindeer (Rangifer tarandus tarandus). The 16S-23S rRNA gene ITS of three Ruminococcus strains; R. flavefaciens strain 8/94-32, R. flavefaciens FD-1 and Ruminococcus albus Ra-8, was investigated. The ITS region has been reported to vary more between closely related bacteria compared to the widely used 16S rRNA gene, and a high degree of sequence polymorphism was indeed detected between the three Ruminococcus strains studied. Based on observed sequence differences, two oligonucloetide probes, ITSRumi1 and ITSRumi2, targeting the ITS region of the R. flavefaciens isolate 8/94-32 were developed. Probe specificity was evaluated in dot blot hybridisations with R. flavefaciens isolate 8/94-32 and four other Ruminococcus-strains tested. The probe ITSRumi1 gave positive signals for the R. flavefaciens isolate 8/94-32 only, while probe ITSRumi2 gave positive signals for R. flavefaciens isolate 8/94-32 as well as for R. albus Ra-8. The result of hybridisations with the probe ITSRumi1 indicates that the probe is specific for the R. flavefaciens strain 8/94-32 amongst the four Ruminococcus-strains tested, and is promising for further studies using it as a signature probe for tracking this strain when re-introduced to the reindeer rumen.

Effect of insertional mutations in the pueA and pueB genes encoding two polyurethanases in Pseudomonas chlororaphis contained within a gene cluster.

AIMS: To better understand the role of PueA and PueB from Pseudomonas chlororaphis in polyurethane degradation, the present study was conducted to create insertional mutants in their respective genes. METHODS AND RESULTS: Growth kinetic studies showed that the pueA knockout mutant had a greater effect than the pueB knockout mutant. The pueA mutant had an 80% decrease in cell density from that of the wild type, while the pueB mutant had an 18% decrease in cell density. Polyurethane utilization followed Michaelis-Menten kinetics. The pueA and pueB mutants exhibited a 17% and 10% decrease respectively in growth rate using polyurethane when compared with the wild type. CONCLUSIONS: In this present study, pueA and pueB, are shown to be part of an ABC transporter gene cluster that consists of seven open reading frames. Mutational analysis results suggest that PueA may play a more major role in polyurethane degradation than PueB based on cell density and growth rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from this study provide a starting point for the eventual enhancement and bioremediation of polyurethane waste. Understanding the role of polyurethane-degrading enzymes is useful for the creation of strains for this purpose.