Mutsumi Nakamura

Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR.

ABSTRACT A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola,Prevotella albensis, Prevotella bryantii,Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens,Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii,Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenesand R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. TheR. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that ofP. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958T, P. bryantii B14T, P. albensis M384T, and P. brevis ATCC19188T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species.

The Replicon of the Cryptic Plasmid pSBO1 Isolated from Streptococcus bovis JB1

The cryptic plasmid pSBO1 (3904 bp) was isolated from Streptococcus bovis JB1. pSBO1 contained an open reading frame (ORF) that is homologous to sequences encoding the replication protein (Rep) in pEFC1 (isolated from Enterococcus faecalis), pSK639 (Staphylococcus epidermidis), pLA103 (Lactobacillus acidophilus), and pUCL287 (Tetragenococcus halophila). In addition, four 22-bp direct repeats (DRs) were located upstream of the putative replication gene (rep) of pSBO1. Recombinant plasmids (pSBE10 and pSBE11) containing the DRs and putative rep of pSBO1 replicated in S. bovis 12-U-1 and no8 strains. This result indicates that the putative rep encoded Rep and that the replicon of pSBO1 contained the DRs and the rep. Gel shift assays showed that the Rep of pSBO1 bound the 22-bp DRs.

Sequence Analysis of Small Cryptic Plasmids Isolated from Selenomonas ruminantium S20

Abstract. Two small cryptic plasmids designated pONE429 and pONE430 were isolated from a rumen bacterium, Selenomonas ruminantium S20. The complete sequence of pONE429 was 2100 bp and contained one open reading frame (ORF) of 201 amino acids. The sequence of pONE430 had 1527 bp and one ORF of 171 amino acids with the similarity of replication protein (Rep protein) of pOM1, pSN2, and pIM13 isolated from Butyrivibrio fibrisolvens, Staphylococcus aureus, and Bacillus subtilis, respectively. In these plasmids, the upstream nucleotide sequence of Rep protein had the conserved nucleotides which could be double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The plasmids of pONE429, pONE430, pJJMI, pJDB21, and pS23 were isolated from S. ruminantium strains and had similar regions that were located within a <450-bp nucleotide. These similar regions may be the location that was recognized by the host strain, S. ruminantium.

Characterization of the Cryptic Plasmid pSBO2 Isolated from Streptococcus bovis JB1 and Construction of a New Shuttle Vector

A cryptic plasmid designated pSBO2 (3582 bp) was isolated from Streptococcus bovis JB1. The pSBO2 contained putative sites for a double-strand origin (dso), a small transcriptional repressor protein (Cop), countertranscribed RNAs (ctRNAs), and a replication protein (Rep), which were similar to those from pMV158 and pLS1, which were isolated from S. agalactiae, and pWVO1, isolated from Lactococcus lactis. The putative single-strand origin (sso) of pSBO2 was similar to pER341 and pST1, which were isolated from S. thermophilus. Recombinant plasmid designated pSBE2 was constructed to bind pECM184 vector and the DNA fragment containing sso, dso, Cop, ctRNAs, and Rep of pSBO2. When pSBE2 was introduced into S. bovis 12-U-1 and no8, the plasmids in the transformants had deleted the 160-bp fragment between sso and dso. This plasmid, designated pSBE2A, was capable of transforming Escherichia coli and S. bovis strains 12-U-1 and no8 on high frequency; therefore, pSBE2A is an effective shuttle vector.

Expression of Ruminococcus albus xylanase gene ( xynA) in Streptococcus bovis 12-U-1.

The objective of this study was to ligate the xylanase gene A (xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [β-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37°C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood.

Construction of Prevotella ruminicola-Escherichia coli shuttle vector pRAM45 and transformation of P. ruminicola strains by electroporation

A new plasmid shuttle vector, pRAM45, was constructed from the Escherichia coli plasmid pACYC184 and the cryptic plasmid pRAM4, which was originally isolated by us from Prevotella ruminicola T31 (Ogata et al., Plasmid, 35, 91-97, 1996). The vector was electrotransformed into different P. ruminicola strains. Polymerase chain reaction and Southern hybridization experiments confirmed its integrity in P. ruminicola transformants.

Comparison of gut microbiota composition between laboratory-bred marmosets (Callithrix jacchus) with chronic diarrhea and healthy animals using T-RFLP analysis: Chronic diarrhea microbiota in marmoset

Chronic diarrhea in laboratory‐bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory‐bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory‐bred marmosets, the gut microbiota composition (GMC) of 58 laboratory‐bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α‐diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory‐bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory‐bred marmosets.