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...

Culture-dependent and culture-independent approaches were used for extensive retrieval of the extradiol dioxygenase (EDO) gene from the environment to investigate the relationship between the EDO genes from isolated bacteria and the metagenomic EDO genes from which they were isolated. In our previous study, we identified 91 fosmid clones showing EDO enzyme activity using a metagenomic approach. In the present study, we classified all these metagenome-derived EDOs and newly isolated 88 phenol-utilizing bacteria from the same sample and identified four EDO genes from them. Of these, two EDOs had amino acid sequences similar to those reported previously in aromatic-utilizing strains, and one EDO had a sequence almost identical to that of metagenomic EDOs identified in our previous study. Unexpectedly, one EDO showed no similarity to any class I EDOs and was categorized as class II, which has not been found in past metagenomic approaches. Quantitative polymerase chain reaction (PCR) assay indicated that the low-abundance class II EDO gene can be enriched by culturing approaches. We conclude that the combined use of the two approaches can explore the gene community more extensively than their individual use.

Extradiol dioxygenase (EDO) catalyzes metal-dependent ring cleavage of catecholic substrates. We previously screened a metagenomic library of activated sludge used to treat industrial wastewater contaminated with phenols and cyanide to identify 43 EDO genes. Here, we have characterized the enzymes belonging to novel I.2.G, I.3.M and I.3.N subfamilies. The I.3.M and I.3.N EDOs were Fe(II) dependent and preferred bicyclic substrates, whereas the I.2.G EDOs were Mn(II) dependent, preferred monocyclic substrates and had the highest affinity for catechol reported thus far. The I.2.G EDOs were more tolerant against heat (60 degrees C for 1 h) and chemical inhibitors (H(2)O(2) and NaCN) than I.3.M and I.3.N EDOs. Considering the dominance of the I.2.G EDOs over all retrieved EDOs (20 of 43 clones) and the presence of cyanide in the environment, this high affinity for substrate and structural robustness should provide survival advantages to host microorganisms. The 20 I.2.G EDOs were classified into six groups based on the amino acid sequence of the predicted ancestor, 1A1. Enzymes were chosen from each group and characterized. Two descendents, 1D2 and 5B2, each had a k(cat)/K(M) approximately twofold higher than that of 1A1 and reduced thermal stability, suggesting that descendents of 1A1 have adapted evolutionarily by a trade-off of inherent stability for increased activity.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate‐2‐sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK‐21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose‐6‐phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross‐correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.