Studies on fatty acid and acylated homoserine lactone biosynthesis in Pseudomonas aeruginosa

Abstract

This study was initiated to define the pathways for fatty acid (FA) and acylated homoserine biosynthesis in Pseudomonas aeruginosa. To date, a complete FA biosynthetic pathway has only been defined in Escherichia coli. This pathway is essential and is central to the biosynthesis of membrane phospholipids. LPS, lipoproteins, lipoic acid, and biotin. and therefore offers attractive targets for various antimicrobial agents. At the onset of this study, there were conflicting results in the literature concerning the role of FA synthesis in acylated homoserine lactone (HSL or autoinducers) synthesis and thus provided additional incentives to define both pathways. Some investigators believed that the acyl groups of HSLs were derived from the FA biosynthetic pathway, while others argued that the FA degradative pathway may be the source of acyl groups. HSLs in P. aeruginosa are responsible for regulation of the majority of the virulence factors. Hence, the goals of the present study were to define the FA biosynthetic pathway in conjunction with HSL biosynthesis to not only reveal potential targets for inhibition of growth, but also inhibition of virulence gene expression in this bacterium. Some unique genetic technologies were developed to facilitate the process of achieving the above goals. First, the development of a phage CTX-based chromosomal integration system allows the integration of gene cassettes at a defined location, attB, in the P. aeruginosa chromosome without disrupting any cellular functions. The mini-CTX integration plasm ids are equipped with FRT sites that were configured to allow in vivo removal of unwanted plasmid DNA sequences with Saccharomyces cerevisiae Flp-recombinase. In two steps, the newly described system allows stable, single-copy, chromosomal integration of any DNA segment to be studied, e.g. autoinducer regulated transcriptional lacZ gene fusions. Deletion of plasmid-borne promoters by Flp-recombinase-mediated excision and flanking of the integrated gene cassette by strong phage T4 transcriptional terminators minimizes transcriptional interferences from external promoters. This system will facilitate studies involving various reporter gene fusion constructs in P. aeruginosa (and potentially other Pseudomonads) from a single chromosomal copy. It will also allow for the genetic engineering of many useful laboratory strains, as well as strains for use in industrial settings where gene expression from plasmids may not be desirable. Second, a broad-host-range system for isolation of marked and unmarked chromosomal mutations was developed utilizing an engineered Flp-FRT system. A series of new gene-replacement vectors was constructed which facilitate mutant construction in P. aeruginosa and other bacteria. Together, these two newly developed systems allow easy P. aeruginosa chromosome manipulation by integration and/or deletion of genes or sequences. Novel genetically engineered strains and vectors were created to allow the purification of problem proteins. This was in part necessitated since the present study involved the purification of 14 different proteins for complete assembly of the FA and HSL biosynthetic pathways in vitro. During this project, three proteins (RhII, LasI, and ACP) were especially problematic and could only be expressed and purified after the development of low-copy number expression vectors and appropriate host strains. RhII and LasI (the two autoinducer synthases) were extremely insoluble when overexpressed from high copy number plasmids, and ACP (acyl carrier protein) requires post-translational modification for function. So far, this system provides the best way to purify ACP, in terms of yield and percentage of holo-ACP, as well as ease and expense of the procedures involved. A connection of the FA biosynthetic (Fab) pathway to HSL biosynthesis was made possible by the combination of the technologies developed above, followed by in vitro assembly of the complete pathway. Purified RhII could efficiently synthesize biologically active N-butyryl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) only from the respective acyl-ACP substrates but was very inefficient when the respective acyl-CoA substrates were present. This corroborated the notion that the Fab pathway provided the acyl groups for HSL synthesis. Mutant analysis demonstrated that FabI (enoyl-ACP reductase) was required for efficient synthesis of both autoinducers (BHL and HHL) in P. aeruginosa. In a RhII/FabI coupled reaction, the synthesis of BHL was inhibited by triclosan, an inhibitor of FabI. The studies also demonstrated that P. aeruginosa contains another FabI homologue. Utilizing purified proteins, the complete Fab pathway was assembled in vitro and coupled to purified LasI for the synthesis of N-[3-oxo-dodecanoyl]-L-homoserine lactone (3-oxo-C12-HSL). Fab pathway inhibitors inhibited 3-oxo-C12-HSL synthesis, indicating that this in vitro system may be useful for drug screening. Finally, the E. coli acyl-ACP synthase membrane-associated protein was purified by affinity chromatography and used to label ACP with various fatty acids. This enzyme will be useful in future experiments because it allows enzymatic synthesis of substrates for various stages of FA biosynthesis, as well as substrates for the synthesis of HSL.