Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Groundwater and Aquifer Microorganisms

Abstract

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.

Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats

“…Despite the good results previously obtained with these primers for the haloalkaliphilic SOB (Tourova et al, 2006(Tourova et al, , 2007, they gave positive amplification among the halophilic SOB only for representatives of the genera Halothiobacillus, Thiohalomonas, Thiohalophilus and Thiohalobacter. Of the other primer pairs tested (Table 1), the one specific for the 'green-like' form IA (Alfreider et al, 2003) gave the best results, but the amplified cbbL fragment only had a small overlap with the fragments obtained with the RubIgF/RubIgR primers and with most of the cbbL sequences deposited in GenBank. Therefore, we designed an additional set of primers to amplify the cbbL gene in representatives of the genera Thiohalospira, Thiohalorhabdus and Thioalkalibacter.…”

A new cell morphotype among methane oxidizers: a spiral-shaped obligately microaerophilic methanotroph from northern low-oxygen environments

“…Partial fragments of the pmoA genes were amplified using the primers A189f and A682r and the reaction conditions described by Holmes et al (1995). PCR-based retrieval of the cbbL gene coding for the large subunit of form I RubisCO was performed using the primers and the reaction conditions described by Alfreider et al (2003). PCR mixtures (100 μl) contained 1 μl of template DNA, 50 μl of 2 × MasterMix (Promega, Madison, WI, USA) and 0.3 μM of each primer (Syntol, Moscow, Russia).…”

“…This PmoA lineage is also addressed as OSC (Organic Soil Cluster) cluster of uncultivated methanotrophs, which occur predominantly in peatlands and in some upland soils (Knief, 2015). Given that the presence of RubisCO is one of the characteristic features of type X (Methylococcus-like) methanotrophs (Whittenbury and Dalton, 1981;Whittenbury, 1981;Baxter et al, 2002), we made an attempt to detect genes coding for this enzyme in spiral-shaped methanotrophs using the primer set developed by Alfreider et al (2003). PCR product of the expected size (approximately 600 bp) was successfully obtained from DNA extract of the studied culture, cloned and analyzed.…”

Autotrophic fixation of geogenic CO<sub>2</sub> by microorganisms contributes to soil organic matter formation and alters isotope signatures in a wetland mofette

“…Form II RubisCO consists only of large subunits. Because of its low CO 2 affinity and high O 2 sensitivity, it represents an early form, evolved under anaerobic conditions and high CO 2 concentrations (Alfreider et al, 2003). Form II RubisCO might be favorable under conditions prevailing in mofettes.…”