Analysis of the microbial gene landscape and transcriptome for aromatic pollutants and alkane degradation using a novel internally calibrated microarray system

Abstract

Despite various efforts to develop tools to detect and compare the catabolic potential and activity for pollutant degradation in environmental samples, there is still a need for an open-source, curated and reliable array method. We developed a custom array system including a novel normalization strategy that can be applied to any microarray design, allowing the calculation of the reliability of signals and make cross-experimental comparisons. Array probes, which are fully available to the scientific community, were designed from knowledge-based curated databases for key aromatic catabolic gene families and key alkane degradation genes. This design assigns signals to the respective protein subfamilies, thus directly inferring function and substrate specificity. Experimental procedures were optimized using DNA of four genome sequenced biodegradation strains and reliability of signals assessed through a novel normalization procedure, where a plasmid containing four artificial targets in increased copy numbers and co-amplified with the environmental DNA served as an internal calibration curve. The array system was applied to assess the catabolic gene landscape and transcriptome of aromatic contaminated environmental samples, confirming the abundance of catabolic gene subfamilies previously detected by functional metagenomics but also revealing the presence of previously undetected catabolic groups and specifically their expression under pollutant stress.

Selective Enrichment Establishes a Stable Performing Community for Microbial Electrosynthesis of Acetate from CO2

“…Headspace CH 4 and CO 2 were analysed during the enrichment phase on a GC (GC 14-B, Shimadzu) with a TCD using a Hayesep Q 80-100 column (2.74m…”

“…Genomic DNA was extracted from 2 mL samples using a fastprep method according to the protocol described by Vilchez-Vargas et al 4 The concentration of extracted DNA was measured using a nanodrop ND1000 spectrophotometer (Thermo scientific, USA) and QuantiT dsDNA BR Assay kits with a Qubit fluorometer (Life Technologies, VIC Australia). The V6 to V8 region of the 16S rRNA gene was amplified using the 926F (5'-AAACTYAAAKGAATTGRCGG-3') & 1392R (5'-ACGGGCGGTGWGTRC-3') primers.…”

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

“…Proteins from P. veronii 1YdBTEX2 and 1YB2 annotated by the RAST server (35) For RNA preparation, strain 1YdBTEX2 was grown with benzene or toluene, strain 1YB2 was grown with benzene as the carbon source (inducing conditions), and both strains were grown on glucose (2 g/liter) as a control (noninducing conditions). RNA extractions were performed as previously described (36), and cDNA was prepared from 800 ng of RNA by using 0.3 g of random primers and 400 U of Superscript III reverse transcriptase (RT) (Invitrogen, Life Technologies, Germany), according to the instructions of the providers.…”

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway

“…The data set of 106,966 sequences was then filtered to consider only phylotypes that ap-peared in at least one of the bioreactors at Ͼ0.02% abundance (a total of 90,661 sequences: PFR-1, 71,306; PFR-2, 19,355). Normalization to obtain the same number of reads per sample (19,355) and rarefaction analyses were performed using R (24).…”

“…To this purpose, the genetic potential for microbial toluene transformation was assessed by deciphering the microbial diversity via next generation sequencing and the catabolic gene landscape using a recently developed microarray (19), substantiated by quantitative PCR (qPCR) analysis of selected genes. These results on catabolic potentialities were combined with 13 C and 2 H stable isotope fractionation analysis to characterize the system regarding the predominant toluene degradation pathway (20).…”