Comparison of Methods for Quantification of Cytochrome cd ₁ -Denitrifying Bacteria in Environmental Marine Samples

Abstract

Two PCR primer sets were developed for the detection and quantification of cytochrome cd 1 -denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd 1 denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd 1 denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.It is generally believed that only a small fraction of environmental bacteria are recovered by current cultivation techniques and that the quantification of microorganisms is therefore biased. The most prominent methods which have been suggested for studying this noncultivated fraction of indigenous community bacteria are based on using nucleic acids. Techniques such as most-probable-number (MPN) PCR and competitive PCR have been developed to quantify specific groups of bacteria by amplifying the 16S fragment in the ribosomal DNA (17,25,28,31) or in the functional gene (15,21,35). In using studies which target metabolic function, in some cases all the organisms of a species or genus possess the same metabolic function (nitrification or sulfate reduction, for example). Using a probe which targets a specific part of the ribosomal gene can therefore give an indication of the presence of the bacterial group which is capable of that function. On the contrary, if the function is spread among a variety of bacterial species, and only a small number of strains of each species possess that function, it is not possible to perform the classic approach of targeting the ribosomal gene. In this case the conserved region of a functional gene may serve as a suitable target. The use of a functional gene requires sufficient genetic homology of the structural genes and the availability of multiple sequences in order to reliably design primers. When the function is widely spread over the phylogenic groups, the primers used for molecular detection become more degenerated. This increases the risk of nonspecific annealing of the primer onto nontarget sequences, which in turn leads to low specificity and low sensitivity of the technique. Denitrification is a good example of a process which is performed by a great diversity of bacterial strains which come from all the major physiological groups, with the exception of Enterobacteriaceae. The nitrite reductase gene is a key enzyme for this metabolic process. Depending on the strain, denitrifying b...

Effect of pyrene on denitrification activity and abundance and composition of denitrifying community in an agricultural soil

“…nirK, nirS and nosZ gene fragments were amplified with the primer pairs 1F and 5R (Braker et al, 1998), R3cd and cd3aF (Michotey et al, 2000), Z-F and 1622R (Throbäck et al, 2004), respectively. The amplicons were gel-purified using the Gel Clean-up System (Promega, Madison, USA) and cloned using the pGEM-T Easy cloning kit according to the manufacturer's instructions.…”

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium

“…Although the primers (Michotey et al, 2000;Throbäck et al, 2004) used for quantification (quantitative PCR) may not cover all environmental nirS sequences and thereby underestimated denitrifiers at the gene level, these primers have been tested to exhibit high specificity (Throbäck et al, 2004) and cover the vast majority of the expressed complementary DNA sequences except for two OTUs (six sequences) retrieved in our study. In contrast, the primer nirS3R (Braker et al, 1998) used to quantify denitrifiers by Ward et al (2009) has sequence mismatches with all complementary DNA sequences obtained in our study, as well as with a fair number of cultured strains (Braker et al, 1998) and published environmental sequences (for example, 67 out of the 113 denitrifier sequences shown in Supplementary Figure S6), including some retrieved from the Arabian Sea.…”

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

“…We constructed the phylogenetic tree based on the sequences of the catalytic site (including cytochrome heme d1; 530-570 bp) in NirS. All widely used conventional primers, such as cd3aF/R3cd and nirS2F/nirS4R (Braker et al, 1998);Michotey et al, 2000;Throbäck et al, 2004, were also designed to amplify the sequence of this site. The tree was divided into three clusters (that is, Clusters I-III) with high bootstrap support (487%) (Figure 1b; see Supplementary Figure S2 for species names).…”

“…Therefore, nirK and nirS are used as marker genes to study the ecological behavior of denitrifying microorganisms in environments. Many attempts have been made to design and modify the polymerase chain reaction (PCR) primers required to amplify nirK and nirS (Braker et al, 1998;Hallin and Lindgren, 1999;Braker et al, 2000;Michotey et al, 2000;Throbäck et al, 2004). These approaches were conducted based on the available nirK and nirS sequences from cultivable denitrifying bacterial strains, most of which belong to the classes of alpha-, beta-and gamma-proteobacteria.…”