Construction of an experimental study and addition of adapter sequences using HiDi DNA polymerase for improving DNA normalization methods relevant to novel gene discovery.

Microorganisms in the environment can be distinguished into dominant and rare microbial species based on their genes. It is difficult to obtain genetic information derived from rare microbial species (rare genes) because of the differences in relative abundance. DNA normalization is an approach that is used to obtain genetic information derived from rare microbial species from an environmental sample. This method involves the addition of adapter sequences for the amplification, denaturation, and reassociation of the DNA fragments and single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) separation. In this method, the amount of a high-copy-number of DNA fragments and a low-copy-number of DNA fragments can be equalized. Improvements in this technique are expected to provide novel genetic information or genes in rare microbial species. However, few model experimental systems have been reported to validate the DNA normalization techniques. This study is aimed to improve the DNA normalization technique used to obtain genetic information of rare genes from rare microbial species. An experimental study was constructed with two antibiotic resistance genes, whose copy numbers differed up to a million-fold. Both genes were mixed and the mixture of DNA fragments, of high- and low-copy-number, containing these genes was normalized by separating ssDNA/dsDNA fragments using hydroxyapatite. Normalized DNA fragments were introduced into Escherichia coli and DNA normalization was evaluated by counting colonies. Moreover, we improved the method to amplify a low-copy-number of DNA fragments by the addition of adapter sequences to DNA fragments using HiDi DNA polymerase to increase the efficiency of DNA normalization. This normalization method was achieved with a 100,000-fold difference. These methods allowed for quantitative evaluation of the DNA normalization efficiency. The experimental data and methods obtained in this study are expected to improve the DNA normalization efficiency to obtain novel genetic information or genes.

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-ß-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution.

Gram-negative bacteria producing metallo-ß-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the ß-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h-1, kslow = 0.108 h-1 for IMP-1, and kfast = 14.0 h-1 and kslow = 1.66 h-1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against ß-lactam antibiotics, and the inhibitory effect of thiol compounds.