Improved strategy for presumptive identification of methanogens using 16S riboprinting

Abstract

The predicted 16S riboprint patterns of 10 restriction endonucleases for 26 diverse methanogens were compared to actual patterns produced on agarose gels. The observed patterns corroborated the expected riboprints. Our analyses confirmed that the endonuclease HaeIII gave the best results generating 15 different riboprint sets. Six of these 15 riboprints represented more than one strain. Of these, three riboprint sets were further differentiated: Methanomicrobium mobile, Methanolacinia paynteri, and Methanoplanus petrolearius were differentiated from each other by the endonuclease AluI; Methanofollis liminatans, Methanospirillum hungatei, and Methanoculleus bourgensis were differentiated from each other by HpaII; and the combination of FokI and MluNI was used to differentiate Methanobrevibacter sp. ZA-10, and Methanobrevibacter arboriphilicus strains DH-1, AZ, and DC from each other. We could not differentiate the following pairs of strains from each other: Methanosarcina mazeii S-6 and C16, Methanobacterium bryantii MoH and MoH-G, Methanobacterium thermoautotrophicum GC-1 and ΔH, and Methanobrevibacter arborophillicus DC and A2. This riboprint strategy provided a simple and rapid method to presumptively identify 22 of the 26 diverse strains of methanogens belonging to 13 genera from a range of environments.

Introduction

The methanogenic archaea are a morphologically diverse group of strict anaerobes that can be extremely thermophilic, moderately thermophilic, or mesophilic. Although they resemble bacteria, existing as cocci, spirillum, and rods, methanogenic archaea are phylogenetically and physiologically distinct from bacteria. Methanogens use hydrogen to reduce carbon dioxide to methane gas, hence their common name—methanogens. However, some methanogens use methyl compounds or acetic acid instead of or as alternatives to hydrogen and carbon dioxide for methanogenesis.

Several species of methanogens have been isolated from the gastrointestinal tract of ruminants and other vertebrates, as well as from invertebrates, marine and bog sediments, lakes rich in decaying vegetation, sewage sludge, and hydrothermal vents. These microbes play a significant part in the biological breakdown of organic matter in these anaerobic environments. For example, methane is produced by domesticated ruminants, particularly sheep, cattle, and goats as part of the normal process of fermentation of feed in the rumen. However, methane is a very potent greenhouse gas that is estimated to be 23 times more potent than carbon dioxide. Hence, the growing interest in isolating and identifying methanogens from various environments, especially those from ruminants.

Methanogens have few morphological traits making them difficult to identify. They also have limited physiological diversity and some are either difficult to grow, or grow very slowly. With the advent of molecular technology, the methanogens were one of the first groups to have their taxonomy based upon 16S rRNA gene comparisons (Balch et al., 1979). Thus, many species can be easily identified by their 16S gene sequence. Consequently, a number of methanogen-specific fingerprinting assays have been developed with the aim of being more simple and rapid than conventional phenotypic characterizations Amann et al., 1995, Hiraishi et al., 1995, Jeanthon et al., 1999, Dollhopf et al., 2001, Lueders et al., 2001, Ramakrishnan et al., 2001, Weber et al., 2001, Huang et al., 2002, Pesaro and Widmer, 2002. Recently, as an alternative to the 16S gene, the methyl-coenzyme M reductase (mcr) gene has been analysed by phylogenetic analysis, denaturing gradient gel electrophoresis (DGGE), and restriction fragment length polymorphism (RFLP) to study and identify the methanogen populations in peat bogs in the United Kingdom and Finland Nercessian et al., 1999, Galand et al., 2002.

Methanogens are significant contributors to greenhouse gases. Here, we propose an improved strategy to use HaeIII and a suite of other endonucleases to presumptively identify nearly one-third of the known methanogens belonging to 13 genera from a range of environments.