Lake Pavin History, geology, biogeochemistry, and sedimentology of a deep meromictic maar lake

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Box 16.1: Which Nucleic-Acids Biomarkers May Be Used

to Investigate the Diversity of Methanogens and

Methanotrophs?

Investigation of prokaryotic composition in environ-

mental samples by culturing techniques is laborious

and prone to strong bias since the growth requirements

of many bacteria are still unknown. Therefore, nucleic-

acid biomarker sequencing has become the gold stan-

dard in environmental microbiology to explore the

diversity of microorganisms. One obvious marker fre-

quently used in molecular ecology particularly for the

taxonomic identification and the phylogenetic affilia-

tion of prokaryotes is the 16S rRNA gene. A comple-

mentary option is to use functional genes encoding

proteins (i.e., specific enzymes). This option allows (in

some cases) establishing a relationship between the

taxonomic identity and the functional role of microor-

ganisms in ecosystems.

The 16S rRNA gene as a phylogenetic gene

The 16S rRNA gene emerged, since early, as a suit-

able phylogenetic marker. This gene is universally

present in prokaryotes, is not submitted to lateral gene

transfert, and has a relatively short sequence (~1.5 kb)

making its sequencing fast and (relatively) cheap.

Moreover, the degree of conservation differs con-

siderably within 16S rRNA gene. Conserved regions

of the gions contain specific sites unique to a species.

The uniqueness enables designing primers targeting

different taxonomic ranks (i.e., Archaea → Euryarcha

eota → Methanosarcinales → Methanosaeta →

Methanosaeta concilii).

rpoB gene as an alternative to 16S rRNA

Various artifacts are reported with the used of 16S

rRNA (e.g., multiple copies within a genome, hetero-

geneity of rates of change between the sequences,

Brochier-Armanet et al. 2008 ). The increase in protein

sequence data, through the sequencing of complete

genomes enables to make phylogenies based on pro-

tein markers. For example, alternative core-house

keeping genes, such as the RNA polymerase β subunit

gene (rpoB), have emerged as an alternative choice

16S rRNA gene illustrating the conserved (grey) and variable

(green) regions

(Case et al. 2007 ). The rpoB inferred phylogenies are

congruent with those based on 16S rRNA, show a

higher resolution of some nodes and are less sensitive

to artifact reconstruction.

The mcrA gene as a diagnostic indicator of methano-

genesis (and of anaerobic methanotrophy)

The methyl-coenzyme M reductase is an enzymatic

complex encoding by a gene cluster. The genomes of

all known methanogens contain at least one copy of the

mcrA which is a highly conserved gene. This property

makes mcrA a suitable candidate to study the diversity

of methanogens and of anaerobic methanotrophs.

Furthermore, mcrA and 16S rRNA phylogenies are

congruent (Hallam et al. 2003 ; Luton et al. 2002 ).

The pmoA gene constitutes a functional marker of

aerobic methanotrophs

The methane monooxygenase (MMO) is an enzy-

matic complex unique to aerobic methanotrophs. Two

distinct forms of the MMO complex have been identi-

fied at different cellular locations, a cytoplasmic solu-

ble form (sMMO) and a particulate form generally

bound to the intracytoplasmic membrane (pMMO)

(Lipscomb 1994 ). Those enzymes are encoded by dif-

ferent gene clusters.

A large dataset of pmoA sequences is available in

public database and pmoA and 16S rRNA phylogenies

are congruent; therefore pmoA is a suitable candidate

to study the diversity of aerobic methanotrophs.

(continued)

Schematic representation of the mcr operon structure (Adapted

from Hallam et al. 2003 )

sMMO and pMMO gene clusters from Methylocystis sp.

(Adapted from K. Iwasaki (http://www.nies.go.jp/kenko/bio-

tech/topi_1-3/iwakiku.html))

Box 16.1 (continued)

16 Methanogens and Methanotrophs in Lake Pavin

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