Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

Abstract

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H 2 -producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

Thermophilic anaerobic digestion of thermal pretreated sludge: Role of microbial community structure and correlation with process performances

“…The sequence of the PCR primer SYN961R from the study of Horz et al (2006) was used to generate a FISH probe targeting the phylum Synergistetes and members of the genus Anaerobaculum; coverage and efficiency of the probe were evaluated using the software TestProbe (Yilmaz et al, 2013, http://www.arb-silva.de/search/ testprobe/, SSU database version 117) and mathFISH (Yilmaz et al, 2011, http://mathfish.cee.wisc.edu/index.html). In order to identify thermophilic Methanobacteriales with ARC915 and MB311 probes and improve the probe penetration, the protocol was modified by using an enzymatic pretreatment of fixed samples with pseudomurein endopeptidase (Pei), as described in Nakamura et al (2006). A recombinant form of Pei (rPeiW) originated from cloning and expression of pseudomurein endoisopeptidase gene from Methanothermobacter wolfeii DSM2970 (peiW) was used.…”

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method

“…Membrane hybridization technique, together with the use of a comprehensive set of rRNA-targeted oligonucleotide probes, can detect most methanogens at different levels of specificity (that is, order and genus) in various anaerobic processes (Raskin et al, 1994a, b;Zheng and Raskin, 2000;McMahon et al, 2004). These rRNA-targeted oligonucleotide probes are further used in fluorescence in situ hybridization (FISH) analysis (Crocetti et al, 2006;Nakamura et al, 2006;Zheng et al, 2006) and PCR-based methods (Banning et al, 2005;Hori et al, 2006;Yu et al, 2006) to quantify the abundance of different methanogens in environments. Though providing quantitative measurement of different methanogens in environments, the detection specificity of these molecular methods is rather limited to the order or family level, or to functionally important methanogens in environments, and their operations tend to be laborious and time consuming for on-site detection in the biological systems.…”

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field

“…1b). Ultrathin sections of cells were also prepared and observed by TEM (Nakamura et al, 2006). Cells of strain RMAS T possessed a thicker cell wall, which presumably consists of pseudomurein, with a thickness of about 21 nm (Fig.…”