Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification

• Published in: Journal of microbiological methods Vol. 49; no. 3; pp. 235 - 245
• Main Authors: Bach, H.-J, Tomanova, J, Schloter, M, Munch, J.C
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.05.2002; Elsevier Science
• Subjects: 16S rDNA copy number; Bacillus cereus - genetics; Bacillus cereus - isolation & purification; Bacillus subtilis - genetics; Bacillus subtilis - isolation & purification; Bacteria - enzymology; Bacteria - genetics; Bacteria - isolation & purification; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Biotechnology; Colony Count, Microbial; DNA Primers; DNA, Bacterial - analysis; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; In vitro gene amplification. Pcr technique; Methods. Procedures. Technologies; Microbiology; Organic Chemicals; Peptidase genes; Peptide Hydrolases - genetics; Polymerase Chain Reaction - methods; Pseudomonas fluorescens - genetics; Pseudomonas fluorescens - isolation & purification; Reproducibility of Results; RNA, Ribosomal, 16S - genetics; rRNA Operon; Soil bacteria; Soil Microbiology; SybrGreen-PCR; Taq Polymerase; TaqMan-PCR; Total counts of bacteria; SybrGreen-PCR; Total counts of bacteria; Peptidase genes; Soil bacteria; TaqMan-PCR; 16S rDNA copy number; Pseudomonadales; Enumeration; Methodology; Enzyme; Bacillus subtilis; Escherichia coli; Ribosomal DNA; Pseudomonas fluorescens; Bacillaceae; Polymerase chain reaction; Peptidases; Bacillus cereus; Bacillales; Soil test; Detection limit; 16S-RNA; Bacteria; Hydrolases; Pseudomonadaceae; Enterobacteriaceae; Quantitative analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(01)00370-0

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed. Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.