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We constructed a metagenomic fosmid bank from <jats:styled-content style=\"fixed-case\">DNA<\/jats:styled-content> 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 <jats:styled-content style=\"fixed-case\">DNA<\/jats:styled-content> 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 \u03b2\u2010lactams 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 <jats:styled-content style=\"fixed-case\">PCR<\/jats:styled-content> amplification. Several clones had an insert in a gene encoding a new type of \u03b2\u2010lactamase. The amplified fosmid <jats:styled-content style=\"fixed-case\">DNA<\/jats:styled-content> was fully sequenced revealing an insert of 41\u00a0kb containing 39 open reading frames (<jats:styled-content style=\"fixed-case\">ORF<\/jats:styled-content>s). Transposon insertions inactivating the resistance to \u03b2\u2010lactams were also found in the <jats:styled-content style=\"fixed-case\">ORF<\/jats:styled-content> upstream of the <jats:italic>blaA<\/jats:italic> 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 <jats:italic>relA<\/jats:italic>\/<jats:italic>spoT<\/jats:italic> gene, and genes belonging to type <jats:styled-content style=\"fixed-case\">II<\/jats:styled-content> toxin\u2013antitoxin system. This predicted system was experimentally validated in <jats:italic>Escherichia coli<\/jats:italic> using an inducible expression system.<\/jats:p>","DOI":"10.1002\/mbo3.104","type":"journal-article","created":{"date-parts":[[2013,7,22]],"date-time":"2013-07-22T07:26:47Z","timestamp":1374478007000},"page":"674-683","source":"Crossref","is-referenced-by-count":10,"title":["Identification of a metagenomic gene cluster containing a new class A beta\u2010lactamase and toxin\u2010antitoxin systems"],"prefix":"10.1002","volume":"2","author":[{"given":"Ken","family":"Vercammen","sequence":"first","affiliation":[{"name":"Department of Bioengineering Sciences Research group Microbiology and VIB Department of Structural Biology Vrije Universiteit Brussel Pleinlaan 2 1050 Brussels Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tamara","family":"Garcia\u2010Armisen","sequence":"additional","affiliation":[{"name":"Ecologie des Syst\u00e8mes Aquatiques Universit\u00e9 Libre 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