Development of an mlrA Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms

Abstract

We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency.It has been predicted that the changing climatic conditions around the world are likely to increase both the occurrence and the intensity of blue-green algal (cyanobacterial) blooms (16). Of particular concern to the water industry are the blooms of Microcystis, Anabaena, Nostoc, and Planktothrix species, which are capable of producing microcystin toxins within surface water storages used for potable water supply (4,5,12). In dissolved (extracellular) form, microcystins are not efficiently removed by conventional water treatment processes (6), and more advanced treatment options, such as activated carbon application or ozonation, are usually employed. However, these are expensive alternatives, and removal efficiencies are often compromised by the presence of natural organic matter (15). Biological filtration of microcystins is now recognized as an alternative treatment barrier (1,7,8,14) and is favored by water utilities, as the process is generally low technology, chemical free, and requires little maintenance, where retrofitting of the process into existing water treatment plant (WTP) infrastructure is often feasible.To date, 10 different microcystin-degrading bacteria have been isolated from rivers, lakes, and biofilters (7), and the mlr gene cluster has been demonstrated to encode proteins involved in the initial steps of microcystin biodegradation by such organisms (2, 3). The MlrA protein is responsible for the initial hydrolytic cleavage of the cyclic microcystin structure, and conventional mlrA gene-directed PCR has been employed for qualitative detection of microcystin-degrading bacteria from lakes (18) and within the biofilm of biofilters (1,7,8). However, these conventional PCR assays do not allow for accurate quantitation of mlrA gene abundance and have not been designed with degenerate primer sequences to allow for variations that exist between different mlrA homologues. In this study, we report for the first time a quantitative mlrA genedirected TaqMan PCR assay, including degenerate oligonucleotides targeting conserved DNA regions, for the rapid detection of microcystin-degrading bacteria.Using all available mlrA nucleotide sequences to date (GenBank accession numbers DQ112243, AF411068, AB114203, AB161685, and AB114202), primers qmlrAf (5Ј-AGCCCKGG CCCRCTGC-3Ј) and qmlrAr (5Ј-ATGCCARGCCCACCAC AT-3Ј) and the TaqMan probe qmlrA-tm, which was labeled with 6-carboxyfluorescein (FAM) at the 5Ј end and labeled with black hole quencher 1 (BHQ1) at the 3Ј end (5Ј-FAM-TGCCSCAGCTSCTCAAGAAGTTTG-BHQ1-3Ј), were designed to target highly conserved regions of the mlrA gene for quantitative TaqMan PCR. Reactions resulted in t...

Assessment of the factors contributing to the variations in microcystins biodegradability of the biofilms on a practical biological treatment facility

“…3b, the increase of mlrA gene copy number to beyond 10 6.00 copies g À1 biofilm triggered much more rapid biodegradation, and the maximal rate cooccurred with the peak of mlrA gene abundance in October. This observation suggested that the seasonal variation in MCLR-biodegradability of biofilms on BTF was largely dependent on the dynamics in indigenous MC-degraders population, agreeing with the findings proposed previously (Ho et al, 2010;Hoefel et al, 2009). In addition, the enhanced biodegradation might be involved in MC-degrading enzyme activity, aside from MC-degrader population.…”

Ecological Dynamics of Toxic Microcystis spp. and Microcystin-Degrading Bacteria in Dianchi Lake, China

“…Previous studies proposed that MCs are used as secondary substrates by MC-degrading bacteria that come into contact with them (17,22). Microorganisms accumulate in polysaccharide matrices and form structural and functional microbial assemblies on surfaces submerged in water commonly known as biofilms.…”

“…To quantify the abundance of total Microcystis spp., toxic Microcystis spp., and MC-degrading bacteria in Dianchi Lake, a qPCR assay was performed using primers based on 16S rRNA genes, mcyD genes, and mlrA genes, according to previously described methods (6,9,22). Microcystis aeruginosa PCC7806 was used as the standard strain for the quantification of both total Microcystis spp.…”

“…In particular, the mlrA gene has been shown to encode an enzyme responsible for the hydrolytic cleavage of the cyclic structure of MCs (20). Conventional PCR techniques are able to identify the mlrA gene and detect MC-degrading bacteria from field water and from the biofilms of biofilters (21,22). However, these conventional PCR assays cannot reveal the abundance of the mlrA gene that is present in a sample.…”

“…However, these conventional PCR assays cannot reveal the abundance of the mlrA gene that is present in a sample. Recently, a quantitative mlrA gene-directed TaqMan PCR assay that can rapidly detect MC-degrading bacteria via the use of degenerate oligonucleotides that target conserved DNA regions has been developed (22). In this study, the TaqMan PCR assay was used to quantitatively detect total Microcystis spp., toxic Microcystis spp., and MC-degrading bacteria using primers that target the 16S rRNA gene, mcyD genes, and the microcystin degradation protein-encoding gene (mlrA).…”