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Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system., (© 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.)