Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity
of [FeFe] hydrogenases in a representative set of strains from Clostridium
sp. and to reveal the existence of neither yet detected nor characterized [FeFe]
hydrogenases in hydrogen-producing strains.
Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic
species), representing six phylogenetic clusters based on their 16S rRNA
sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different
[FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe]
hydrogenase cluster-specific degenerate primers were newly designed. Ten Clostridium
strains were screened by PCRs to assess the specificity of the primers
designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this
approach, a diversity of hydrogenase genes was discovered in several species previously
shown to produce hydrogen in bioreactors: Clostridium sartagoforme,
Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum.
Conclusions: The newly designed [FeFe] hydrogenase cluster-specific primers,
targeting the cluster-conserved regions, allow for a direct amplification of a
specific hydrogenase gene from the species of interest.
Significance and Impact of the Study: Using this strategy for a screening of different
Clostridium ssp. will provide new insights into the diversity of hydrogenase
genes and should be a first step to study a complex hydrogen metabolism
of this genus.