Abstract
MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.
Highlights
Quorum sensing (QS) is a communication system that many bacteria aggregates used to regulate their aggregate size via small molecules called autoinducers [1,2]
The PCR products were cloned into pET-24a(+) vectors via seamless cloning, and the recombinant plasmids were transformed into E. coli BL21(DE3)
Carotovorum (Pcc) other acyl homoserine lactones (AHLs) lactonases belonging to the metallo-β-lactonase superfamily, we found that Ile144, Val149, Asn179, were effects variableofamino acids in the conserved
Summary
Quorum sensing (QS) is a communication system that many bacteria aggregates used to regulate their aggregate size via small molecules called autoinducers [1,2]. The process of interfering with QS through degradation of signals is termed as quorum quenching (QQ). N-acyl homoserine lactones (AHLs) are QS signals used by a wide range of Gram-negative bacteria. AHL lactonase is one major type of AHL-degrading enzymes, which hydrolyses the lactone ring of AHL molecule to produce corresponding N-acyl-homoserine. Since some bacteria use QS to mediate virulence factors and antimicrobial resistance, QQ is considered to be a promising alternative for bacterial disease control, which can attenuate QS-regulated virulence factors production in many bacterial pathogens without any lethal effect and impart less-selective pressures for resistant mutants than conventional antibiotics [3,4,5].
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