Preferential isolation of Megasphaera elsdenii from pig feces.

Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60-100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9-99.2% identity). These three strains were approximately 1.5 µm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.

γ-Resorcylate catabolic-pathway genes in the soil actinomycete Rhodococcus jostii RHA1.

The Rhodococcus jostii RHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.

Valerate production by Megasphaera elsdenii isolated from pig feces.

Megasphaera elsdenii is able to produce several short-chain fatty acids (SCFAs), such as acetate, propionate, butyrate, and valerate. These SCFAs serve as an energy source for host animals and play an important role in gut health. In this study, M. elsdenii was isolated from pig feces that had been collected from two farms located in distinct areas of Japan. These M. elsdenii isolates were genotyped, and 7 representative strains were selected. When these 7 strains and M. elsdenii JCM 1772T were cultured with lactate for 24 h, all 7 strains produced valerate as a predominant SCFA. Therefore, the valerate-producing M. elsdenii inhabits a wide area of Japan. In contrast, M. elsdenii JCM 1772T produced acetate, propionate, butyrate, and valerate at similar levels. When the Y2 strain, one of the 7 representative strains, was cultured without lactate, low levels of valerate accumulated. In contrast, in a time course of lactate fermentation by the Y2 strain, lactate was rapidly consumed, and acetate and propionate were produced after 6 h of incubation. Thereafter, acetate and propionate were consumed from 6 to 12 h after the start of the incubation, and valerate and butyrate were produced. In most of the previously described M. elsdenii strains, valerate was not a predominant SCFA. Therefore, the M. elsdenii Y2 strain showed an unique metabolism in which valerate was produced as a primary end product of lactate fermentation.