Abstract
We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.
Keywords: CTX-M-14, CTX-M-15, DNA shuffling, Hybrid enzyme, Evolution
Background
DNA recombination is a natural process by which genetic materials are exchanged among bacteria to enhance survival fitness under environmental stresses. Several hybrid CTX-M-lactamases (CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132), presumably resulting from recombination between the blaCTX-M-14 and blaCTX-M-15 genes, the most common variants worldwide, have been reported in recent years (Nagano et al., 2009; Tian et al., 2014; He et al., 2015; Liu et al., 2015). Among these hybrid enzymes, CTX-M-64, which contained the N- and C-terminal portions of CTX-M-15 and the middle fragment of CTX-M-14, exhibited even higher catalytic activity than their parental prototypes (He et al., 2015). DNA shuffling is a molecular approach designed to mimic and accelerate the evolution process through PCR-mediated random combinations of two target genes (Crameri et al., 1998). Our previous study demonstrated the use of DNA shuffling to investigate the molecular events driving the evolution of the CTX-M-type β-lactamases (Po et al., 2017). Mutants with or without cefotaximase activity were recovered. Important amino acid residues that played a role in conferring enzyme activity were identified by comparative analysis of the genotypes and phenotypes of the mutants. Such approach can be employed to characterize other functional proteins. Here we describe a detailed protocol of in vitro DNA shuffling.
Materials and Reagents
Equipment
Software
Procedure
The outline of the protocol is shown in Figure 1. Figure 1. Scheme of DNA shuffling
Data analysis
Sequence the target insert in two directions (forward and reverse). Align the sequences of mutants with that of the wildtype to identify mutations. Obtain protein structures from the Protein Data Bank (http://www.rcsb.org/pdb/). Analyze protein structures with the PyMOL software.
Recipes
Acknowledgments
This work has been previously published in Po et al., 2017. This work was supported by the Collaborative Research Fund of the Research Grant Council (C7038-15G and C5026-16G), and the Health and Medical Research Fund of the Food and Health Bureau, the Government of the Hong Kong SAR (HMRF: 14130422 to SC).
Competing interests
Potential conflicts of interest: All authors declare there are no conflicts to disclose. All authors declare there are no conflicts to disclose.
References
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