Distribution and diversity of autotrophic bacteria in groundwater systems based on the analysis of RubisCO genotypes

A molecular approach, based on the detection of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes, was applied to investigate the distribution and diversity of autotrophic bacteria in groundwater systems. DNA extracts from 48 sampling stations, including a variety of pristine and polluted, shallow and deep-subsurface groundwater samples obtained from Germany and Austria, served as a template for the PCR amplification of form I (cbbL) and form II (cbbM) large subunit RubisCO genes. The majority of the samples (>80%) contained two different forms of RubisCO. In 17 samples, all three forms of RubisCO were identified. PCR products from four selected groundwater habitats containing all three forms of RubisCO were used to construct clone libraries. Based on restriction fragment length polymorphism (RFLP) analysis, 109 RubisCO-clone-inserts were subjected to sequencing and phylogenetic analysis. With the exception of a form IA RubisCO sequence cluster obtained from deep subsurface samples, which was identical to the RubisCO genes described for Ralstonia metallidurans CH34, most sequences were distantly related to a variety of RubisCO species in chemolithoautotrophic Proteobacteria. Several sequences occurred in isolated lineages. These findings suggest that autotrophic bacteria with the capability to assimilate CO₂ via the Calvin Cycle pathway are widespread inhabitants of groundwater systems.