Design and Validation ofureC-based Primers for Groundwater Detection of Urea-Hydrolyzing Bacteria

Abstract

Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater ureahydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the α, β, and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.

Bioremediation of lead by ureolytic bacteria isolated from soil at abandoned metal mines in South Korea

“…For amplification of the 323 bp amplicons from the ureC gene, 2 universal primers, ureC-F (5 0 -AAG STS CAC GAG GAC TGG GG-3 0 ) and ureC-R (5 0 -AGG TGG TGG CAS ACC ATS AGC AT-3 0 ) were used (Gresham et al, 2007). PCR amplification was performed under the following conditions: initial denaturation for 5 min at 94 C; 30 cycles of denaturation for 1 min at 94 C, annealing for 1.5 min at 53 C, extension for 2 min at 72 C; and a final extension for 7 min at 72 C. The PCR products were analyzed by 1% agarose gel electrophoresis.…”

“…A primer pair (ureC-F and ureC-R) was designed to specifically recognize the ureC sequence (Gresham et al, 2007). When this PCR primer pair was tested against all six isolated strains (Fig.…”

Final report for DOE Grant No. DE-FG02-07ER64404 - Field Investigations of Microbially Facilitated Calcite Precipitation for Immobilization of Strontium-90 and Other Trace Metals in the Subsurface

“…The coordinates are in meters and referenced to the midpoint of the inter-wellbore zone with positive coordinates being north and east of the midpoint. precipitation occurs under laboratory (Fujita et al 2000;Ferris et al 2004) and field conditions (Colwell et al 2005;Fujita et al 2008) and can incorporate substantially more strontium than abiotic precipitation Mitchell and Ferris 2005), (ii) developed and tested in the field the tools needed to determine in situ urease activity (expressed as ureolysis rates; Colwell et al 2005) and (iii) developed biomolecular tools to track changes in subsurface microbial communities (Colwell et al 2005;Petzke et al 2006;Gresham et al 2007). However, the coupling between ureolysis-driven calcite precipitation and advective groundwater fluxes remains poorly understood.…”

Phylogenetic Relationships and Functional Genes: Distribution of a Gene ( mnxG ) Encoding a Putative Manganese-Oxidizing Enzyme in Bacillus Species

“…Similarities in tree topologies are interpreted as representing the vertical inheritance of genes (23,27,28), while incongruities can signify the occurrence of lateral gene transfer events (14), simultaneous evolution of genes with similar functions in different organisms (28), divergence of genes from a common origin (5), or gene loss (20). If positive correlations can be made between 16S rRNA gene sequences and the presence or absence of functional genes, then 16S rRNA surveys of environmental samples can be used to understand the functional potential of the community.…”