Gloria Soberón-Chávez

Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications.

Pseudomonas aeruginosa produces and secretes rhamnose-containing glycolipid biosurfactants called rhamnolipids. This review describes rhamnolipid biosynthesis and potential industrial and environmental applications of rhamnolipids. Rhamnolipid production is dependent on central metabolic pathways, such as fatty acid synthesis and dTDP-activated sugars, as well as on enzymes participating in the production of the exopolysaccharide alginate. Synthesis of these surfactants is regulated by a very complex genetic regulatory system that also controls different P. aeruginosa virulence-associated traits. Rhamnolipids have several potential industrial and environmental applications including the production of fine chemicals, the characterization of surfaces and surface coatings, as additives for environmental remediation, and as a biological control agent. Realization of this wide variety of applications requires economical commercial-scale production of rhamnolipids.

Production of rhamnolipids by Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces glycolipidic surface-active molecules (rhamnolipids) which have potential biotechnological applications. Rhamnolipids are produced by P. aeruginosa in a concerted manner with different virulence-associated traits. Here, we review the rhamnolipids biosynthetic pathway, showing that it has metabolic links with numerous bacterial products such as alginate, lipopolysaccharide, polyhydroxyalkanoates, and 4-hydroxy-2-alkylquinolines (HAQs). We also discuss the factors controlling the production of rhamnolipids and the proposed roles this biosurfactant plays in P. aeruginosa lifestyle.

Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di‐rhamnolipid biosynthesis

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso‐active glycolipids containing one (mono‐rhamnolipid) or two (di‐rhamnolipid) l‐rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono‐rhamnolipid from dTDP‐l‐rhamnose and β‐hydroxydecanoyl‐β‐hydroxydecanoate, whereas di‐rhamnolipid is produced from mono‐rhamnolipid and dTDP‐l‐rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di‐rhamnolipid (l‐rhamnose‐l‐rhamnose‐β‐hydroxydecanoyl‐β‐hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of l‐rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII‐2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di‐rhamnolipid, whereas mono‐rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1‐rhlA) was incapable of producing both mono‐ and di‐rhamnolipid. Complementation of mutant RTII‐2 with plasmid pRTII‐26 containing rhlC restored the level of di‐rhamnolipid production in the recombinant to a level similar to that of the wild‐type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A σ54‐type promoter for the PA1131–rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131–rhlC operon was dependent on the P. aeruginosa rhl quorum‐sensing system, and a well‐conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di‐rhamnolipid.

Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter

Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P. aeruginosa virulence-associated traits.

Biosurfactants: A General Overview

This Microbiology Monographs volume covers the current and most recent advances in the field of microbial surfactants. There is increasing interest in microbial biosurfactants for several reasons. First, biosurfactants are considered environmentally “friendly” since they are relatively nontoxic and biodegradable. Second, biosurfactants have unique structures that are just starting to be appreciated for their potential application to many different facets of the industry, ranging from biotechnology to environmental cleanup. The aim of this introductory chapter is to give a general overview of biosurfactants, their properties, their relationship to the synthetic surfactant industry, and their distribution in the environment.

Monorhamnolipids and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) production using Escherichia coli as a heterologous host

Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-l-rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.

The Pseudomonas aeruginosa rhlAB Operon Is Not Expressed during the Logarithmic Phase of Growth Even in the Presence of Its Activator RhlR and the Autoinducer N-Butyryl-Homoserine Lactone

The Pseudomonas aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid; rhlAB induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer N-butyryl-homoserine lactone (C(4)-HSL). In this work we studied rhlAB induction in a P. aeruginosa and Escherichia coli background. We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C(4)-HSL. Additionally, we found that rhlAB expression is partially sigma(s) dependent.

The Pseudomonas aeruginosa RhlA enzyme is involved in rhamnolipid and polyhydroxyalkanoate production

Pseudomonas aeruginosa produces the biosurfactant rhamnolipid, which has several potential biotechnological applications. The synthesis of this surfactant is catalyzed by rhamnosyltransferase 1, composed of the proteins RhlA and RhlB. Here we report that RhlA plays a role not only in surfactant synthesis, but also in the production of polyhydroxyalkanoates, polymers that can be used for the synthesis of biodegradable plastics.

Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters

The study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio‐technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase‐guano‐sine diphospho‐D‐mannose pyrophosphorylase (PMI‐GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP‐mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD‐independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.