ABSTRACT
REDUCING VIRULENCE: RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (BHL), with a minor product N-hexanoyl homoserine lactone (HHL). By using directed evolution and a genetic screen, RhlI has been engineered for enhanced production of both BHL and HHL at a similar level. Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhlI, an enzyme in the RhlI-RhlR quorum-sensing system of P. aeruginosa, to alter its substrate specificity and gain insight into the molecular mechanisms of quorum sensing. By using a genetic screen, we identified a mutant with improved production of RhlI's two signaling molecules, N-butanoyl- and N-hexanoyl-homoserine lactone (BHL and HHL). In particular, production of BHL has been enhanced by more than two-fold, and the synthesis of HHL has been improved from an undetectable level to a level similar to BHL; this change indicates a significant change in substrate specificity. No significant change in the gene expression level was observed. Sequence alignments suggest that the mutations are most likely to facilitate interactions between the enzyme and the two acylated ACP substrates. This work also demonstrates that the genetic screen/selection should be useful in engineering additional quorum-sensing components.
Subject(s)
Bacterial Proteins/metabolism , Directed Molecular Evolution , Pseudomonas aeruginosa/enzymology , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/chemistry , 4-Butyrolactone/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Humans , Molecular Structure , Pseudomonas aeruginosa/genetics , Quorum Sensing , Substrate SpecificityABSTRACT
Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.
Subject(s)
4-Butyrolactone/analogs & derivatives , Bacterial Proteins/genetics , Directed Molecular Evolution/methods , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Quorum Sensing , Transcription Factors/genetics , 4-Butyrolactone/biosynthesis , Chromatography, High Pressure Liquid , Escherichia coli/metabolism , Homoserine/analogs & derivatives , Homoserine/biosynthesis , Lactones , Plasmids , Selection, Genetic , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.
Subject(s)
Computer Simulation , Gene Expression Regulation/genetics , Genetic Engineering , HumansABSTRACT
We have previously reported the design and construction of positive feedback loops (PFLs) based on the LuxI-LuxR quorum-sensing system that can be used as modular transcriptional regulatory units for the construction of complex artificial genetic circuits. Here, we characterize these PFLs using single-cell and dynamic induction studies to fully understand their behavior and facilitate their incorporation into novel networks. The LuxR PFLs had graded responses to the OHHL signal molecule with inductions developing over time, causing a lag in response compared to a non-feedback control. The properties of the PFLs could be altered using LuxR mutants with altered sensitivities without changing the inherent properties of the systems. Because of their high sensitivity and ability to establish intercellular signaling, the LuxR PFLs described in this work could be used as well-defined modules for the construction of artificial genetic circuits.
