Rhamnlopids are biosurfactants with a wide range of industrial applications. The understanding of their biosynthesis and the genetic regulation of their production have impacted the development of ...strains that are suitable for industrial production of these biosurfactants.
Summary
Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B‐30761, which have acquired RL‐producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence‐related traits by the quorum‐sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.
All these goals are interconnected, and there are many ways that microbial technologies have impacted each of them. ...the examples of microbial technologies that may impact SDG that I include in ...this ‘Burning Questions’ editorial do not wish to be exhaustive, but to highlight the enormous potential of microbial biotechnology. Human population is increasing at a very high rate, and it increased 2.5 times from 1960 to 2020 reaching 8 billion people on November 15, 2022, so land for agriculture and farming is increasingly limited. ...new strategies should be developed for food production that optimizes land productivity, without harming the environment, and new sources of nutrients should be made available. ...making symbiotic nitrogen fixation available to non-legume crops would represent a major breaking point in agriculture and in the possibility of ending hunger in the world. The advantage of biosurfactants over chemically synthetized surfactants that are widely used in practically all industrial fields is that they are non-toxic and biodegradable and show unexpected activities that are useful in many biotechnology areas (Kossmann et al., 2023, for example). ...the use of these bioactive compounds instead of their chemically synthetized counterparts will considerably reduce pollution and will benefit different industrial areas.
Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum‐sensing response regulates the expression of ...several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame‐shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE‐dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR‐dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE‐dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains.
Highlights
Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR.
This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE.
Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE.
The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction.
The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation.
Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.
We described that the production of Pseudomonas aeruginosa virulence factors, pyocyanin, and rhamnolipids, which are regulated by the transcriptional factor RhlR is differentially affected by the PqsE protein that has been reported to form a complex with RhlR, being pyocyanin production dependent on PqsE overexpression while rhamnolipids synthesis is only slightly affected. In addition, we show that some P. aeruginosa strains, such as ATCC 9027, produce pyocyanin even when having a defective PqsR protein.
In several Gram‐negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, ...regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB‐like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti‐sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three‐hybrid system. We propose that both RssB and RssC are necessary for the ClpXP‐dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.
In bacteria belonging to the Pseudomonadaceae family, such as Azotobacter vinelandii and Pseudomonas aeruginosa, the regulation of proteolysis of the alternative sigma factor RpoS remains poorly understood. Here, we demonstrate that during the exponential phase of growth, two proteins (RssB and RssC) are necessary for the degradation of RpoS by the proteolytic complex ClpXP.
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.
Pseudomonas aeruginosa is an important opportunistic pathogen. Several of its virulence‐related processes, including the synthesis of pyocyanin (PYO) and biofilm formation, are controlled by quorum ...sensing (QS). It has been shown that the alternative sigma factor RpoS regulates QS through the reduction of lasR and rhlR transcription (encoding QS regulators). However, paradoxically, the absence of RpoS increases PYO production and biofilm development (that are RhlR dependent) by unknown mechanisms. Here, we show that RpoS represses pqsE transcription, which impacts the stability and activity of RhlR. In the absence of RpoS, rhlR transcript levels are reduced but not the RhlR protein concentration, presumably by its stabilization by PqsE, whose expression is increased. We also report that PYO synthesis and the expression of pqsE and phzA1B1C1D1E1F1G1 operon exhibit the same pattern at different RpoS concentrations, suggesting that the RpoS‐dependent PYO production is due to its ability to modify PqsE concentration, which in turn modulates the activation of the phzA1 promoter by RhlR. Finally, we demonstrate that RpoS favors the expression of Vfr, which activates the transcription of lasR and rhlR. Our study contributes to the understanding of how RpoS modulates the QS response in P. aeruginosa, exerting both negative and positive regulation.
In Pseudomonas aeruginosa, RpoS favors the expression of Quorum‐Sensing (QS) regulators while it reduces the synthesis of pyocyanin and biofilm formation, both processes induced by QS. We disentangle this apparent contradiction demonstrating that RpoS indirectly induces the transcription of QS regulators LasR and RhlR by activating the expression of Vfr while it represses the expression of PqsE, a protein that stabilizes and modulates RhlR activity, resulting in the reduction of pyocyanin and biofilm production.
Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas ...putida due to the health risks associated with their large‐scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono‐RL or di‐RL, respectively) can be produced by the innocuous soil‐bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl‐homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3‐hydroxy‐hexanoyl homoserine lactone, 3OH‐C6‐HSL, or 3‐oxo‐hexanoyl homoserine lactone, 3O‐C6‐HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH‐C6‐HSL is the primary activator of gene expression for phenazine‐1‐carboxylic acid (PCA) and phenazine‐1‐carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH‐C6‐HSL or 3O‐C6‐HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH‐C6‐HSL or CsaR/3O‐C6‐HSL cannot activate the expression of the rhlAB operon to produce mono‐RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum‐sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB‐R or rhlAB‐R‐C for the industrial production of RL.
The heterologous production of the biosurfactant rhamnolipids in Pseudomonas chlororaphis ATCC 9446 was attained by the expression of the P. aeruginosa genes coding for the RhlA, RhlB, and RhlC enzymes involved in their synthesis. The P. aeruginosa quorum sensing transcriptional regulator RhlR coupled with acylhomoserine lactones endogenous produced by P. chlororaphis activate the transcription of the rhamnolipids structural genes forming a positive autoregulated circuit. The levels of rhamnolipids produced in the heterologous system are similar to those produced by P. aeruginosa PAO1.
In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5′ untranslated region (UTR) that block ...translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5′ UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.
Significance Several bacteria that are pathogens of humans regulate the production of virulence factors in response to temperature changes, expressing them only at 37 °C. This thermoregulation is commonly due to the presence of RNA structures (RNA thermometers) in the 5′ regions of transcripts specifying regulatory proteins responsible for the expression of virulence-associated traits. At environmental conditions, RNA thermometers possess structures that block translation initiation of mRNAs, whereas at body temperature these structures are no longer stable, allowing the synthesis of their corresponding proteins. We report for the first time, to our knowledge, the molecular basis of thermoregulation of virulence-factor production in the opportunistic pathogen Pseudomonas aeruginosa , and have determined that this regulation is achieved by two RNA thermometers with previously unidentified characteristics not previously reported in bacteria.
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that presents a complex regulatory network called ‘quorum-sensing’, which is responsible for the transcription of genes coding for several ...traits implicated in its pathogenicity. Strain 148 is a dolphin isolate that has been shown to produce quorum-sensing-regulated virulence traits and to be virulent in a mouse model, despite the fact that it contains a 20-kbp deletion that eliminates from the chromosome the lasR gene and the lasI promoter. LasR is a key quorum-sensing transcriptional regulator that, when coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) produced by LasI, activates transcription of genes coding for some virulence-associated traits such as elastase, lasI, rhlI and rhlR. RhlR is also a key quorum-sensing transcriptional regulator that, when interacting with the autoinducer butanoyl homoserine lactone (C4-HSL) that is produced by the synthase RhlI, activates the genes involved in the synthesis of some virulence-associated traits, as rhamnolipids and pyocyanin. We describe that in P. aeruginosa 148, the LasR/3O-C12-HSL-independent rhlR transcriptional activation is due to the release of the negative effect of Vfr (a CRP-ortholog) caused by the insertion of an IS element in vfr, and that rhlI transcription is driven from the rhlR promoter, forming the rhlR-I operon.
Pseudomonas aeruginosa 148 is able to express virulence factors that in typical strains depend on LasR transcriptional regulator, in the absence of this protein.
PDF
