Identification of a metagenomic gene cluster containing a new class A beta-lactamase and toxin-antitoxin systems

Abstract

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.

Keywords: Antibiotic resistance; metagenomic DNA; toxin-antitoxin systems; β-lactamase.

Figure 1

Growth of the Amp^R clone in the presence of different concentrations of ampicillin (Oxoid strips). Although some growth inhibition could be observed, there is regrowth of colonies inside the inhibition zone.

Figure 2

Genomic organization of ORFs 1–6: ORF1 (algC) encodes a phosphomannomutase, ORF2 a peptidase, ORF3 a tryptophanyl-tRNA synthetase, ORF4 a methionine aminopeptidase, ORF5 an aminotransferase, and ORF6 a periplasmic β-lactamase. The black triangles represent the transposon insertions resulting in the inactivation of the resistance to ampicillin.

Figure 3

Alignment of the protein sequences corresponding to ORF6 encoded β-lactamase (1) and Herminiimonas arsenoxydans β-lactamase (2). The identical residues are highlighted in gray and the amino acids highlighted in yellow are those found in the class A β-lactamases as described in the results section.

Figure 4

Phylogenetic tree of different class A β-lactamases (see text for details).

Figure 5

ORFs 28 and 29 constitute a RelBE toxin–antitoxin system, whereas ORF26 is a lone toxin. Serial dilutions of the DJ624Δara containing the pBAD33lev control vector or its derivatives with ORFs 24, 26, or relE (ORF28) and the control vector pKK223-3lev or its derivatives with ORF25, ORF27, or relB (ORF29) were plated on M9 minimal media containing arabinose (1%) and IPTG (0,1 mmol/L) and the appropriate antibiotics. Plates were incubated overnight at 37°C.