Metagenomic Screening for Bleomycin Resistance Genes

Abstract

A metagenomic library of activated sludge was screened for bleomycin resistance genes. Two genes were identified that differed greatly from each other, from the genes of bleomycin-producing actinomycetes, and from those of clinical isolates. Therefore, the nonclinical environment is a rich reservoir of new resistance elements, and metagenomics can be used to sample the resistome rapidly.The antibiotic bleomycin (Bm) was first discovered in the culture broth of "Streptomyces verticillus" (14,17), an antitumor agent used in clinical settings. Bm-producing actinomycetes have resistance genes (ble) that are often clustered in biosynthetic operons (3,12,15). However, resistance has spread to bacteria that do not produce Bm, and the genes that confer this resistance have been identified in clinical isolates, Tn5 of gram-negative bacteria (4), and pUB110 of gram-positive bacteria (11). Nonetheless, the resistance mechanism is conserved; the acidic Bm resistance protein (BRP) sequesters the basic Bm to prevent DNA cleavage by Bm (17).The natural, or nonclinical, environment is also a reservoir for various antibiotic resistance genes (1, 2, 8, 10). Because ble genes have been reported only from clinical isolates and actinomycetes, we explored nonclinical environments for these genes to better understand the origin and distribution of antibiotic resistance. Our aims were to determine the following: (i) whether ble could be retrieved from a nonclinical environment, (ii) the evolutionary position of environmental BRPs, and (iii) the resistance mechanism of environmental BRPs. We produced a metagenomic library (5) using activated sludge as a DNA source (13). Activated sludge, which is used to treat industrial wastewater polluted with phenolic compounds, contains a dense microbial community. To survive in this niche, each bacterium must develop resistance to myriad natural antibiotics, mostly those produced by actinomycetes (1). The library contained 3.2 Gb of DNA comprising 96,000 clones of Escherichia coli EPI300-T1 R , each carrying a fosmid with an ϳ33-kb insert (13). One hundred clones were mixed in each well of a 96-well plate, and a total of 10 plates were screened in 100 l of LB medium containing 12.5 g/ml chloramphenicol and 50 g/ml phleomycin, in which the host E. coli alone could not grow. After 14 h at 37°C, three resistant clones were identified, 4H7, 7A10, and 8D4. For each fosmid, a shotgun library was produced; each library comprised 388 clones, each with an ϳ2-to 3-kb insert in pUC118. The libraries were used for DNA sequencing and functional screening.The 4H7 fosmid contained a 37.4-kb insert (see Table S1 in the supplemental material). Putative ble (open reading frame 17 [ORF17]) was identified from among 30 ORFs. For the 7A10 and 8D4 fosmids, sequences were not assembled into a long contig. However, the read sequences from these two fosmids were identical. Therefore, we assumed that they shared the same region of environmental DNA and combined them to generate a single contig. The 31.07-kb fragment contain...

Association of the Emerging Carbapenemase NDM-1 with a Bleomycin Resistance Protein in Enterobacteriaceae and Acinetobacter baumannii

“…We showed that this BRP was functional and caused decreased susceptibility to bleomycin and bleomycin-like molecules, such as zeocin. Since Walsh et al demonstrated the large spread of the bla NDM-1 gene in bacteria recovered from environmental samples in New Delhi (32), it can be hypothesized that bleomycin or bleomycin-like molecules exert a selective pressure in the environment, in accordance with a metagenomic study that showed that BRP-encoding genes could be retrieved from activated sludge (14). In the United Kingdom and Germany, studies showed that bleomycin was present in sewage effluents, rivers, and potable water (1,29).…”

“…In the case of Tn5, the bleomycin resistance gene is associated with a neomycin-kanamycin resistance gene (12). A recent metagenomic study revealed that the nonclinical environment might constitute a large reservoir of Bm resistance genes (14). Despite a high divergence between each other at the sequence level, those genes encode proteins conferring a similar resistance mechanism (14).…”

“…A recent metagenomic study revealed that the nonclinical environment might constitute a large reservoir of Bm resistance genes (14). Despite a high divergence between each other at the sequence level, those genes encode proteins conferring a similar resistance mechanism (14). It is based on the production of an acidic protein (named BRP, for Bm resistance protein) that is able to complex Bm and thus prevent the Bm-induced DNA cleavage.…”

Crystallization and preliminary X-ray diffraction analysis of a putative two-domain-type laccase from a metagenome

“…During the course of metagenomic library screening for various genes (Suenaga et al, 2007;Mori, Mizuta et al, 2008;, we constructed a metagenomic sequence database. In the search for various potentially useful enzymes, we identified a gene containing a copper-binding motif.…”

Functional metagenomic characterization of antibiotic resistance genes in agricultural soils from China

“…However, activity-based functional screening of metagenomic libraries, which provide direct evidence for antibiotic resistant phenotype, still remains a critical means to discover novel ARGs and identify their function that could be missed when using sequence based analysis only. Using functional screening of metagenomic libraries, novel functional ARGs have been identified from human microbiome (Sommer et al, 2009), organic pig gut (Kazimierczak et al, 2009), activated sludge (Mori et al, 2008) and soils (Allen et al, 2009;Lang et al, 2010;McGarvey et al, 2012). Most of these ARGs have not been identified previously and are evolutionarily distant from known resistance genes.…”