rhlAB Genes Research Articles

Получение гетерологичных продуцентов рамнолипидов для промышленного производства высокоэффективного биосурфактанта

Rhamnolipids are promising microbial glycolipid biosurfactants that can be used as preparations for soil bioremediation in oil pollution. The expression of the rhamnolipid biosynthesis gene cluster in the main producer, Pseudomonas aeruginosa, is regulated by quorum sensing signals (QS). Complex biosynthesis regulation does not allow achieving high product yields. The production of heterologous rhamnolipid producers is a promising way to overcome the complex QS-dependent regulation of rhamnolipid biosynthesis, and also avoids the use of opportunistic Pseudomonas aeruginosa for the industrial production of rhamnolipids. Heterologous Escherichia coli BLWT-based producers with a transformant E14 productivity of 3.40±0.96 g/L were obtained on the basis of the plasmid vector pAl2–T with a construct from the native promoter and rhlAB genes, and Pseudomonas viridiflava AlC1223-based ones with the productivity of transformants P5, P6, P8 332.00±0.10, 200.00±0.10 and 180.00±0.10 g/L, respectively. The presence of rhlAB genes in the expression vector allows the obtaining monorhamnolipids for further use as an agent for the solubilization of soil hydrophobic pollutant (e. g. petroleum products), which will intensify bioremediation processes.

Interspecies Interaction between Pseudomonas aeruginosa, Staphylococcus aureus and E. coli in vitro

Microbial interactions are frequently categorized according to how they affect each population in a binary system. We aimed to determine the interaction between P . aeruginosa, S . aureus, and E . coli in-vitro. In this experimental hospitalized patients’ sputum, urine, and blood samples were used to collect a total of 90 clinical isolates for the study in Damanhour Medical National Institute, Behira, Egypt, followed by accurate identification and testing for antibiotic sensitivity. To examine the effect of the supernatant of P. aeruginosa on S. aureus and E. coli determined MIC using broth microdilution method. We also measured the activity of lasA protease by assessing the S. aureus cell lysis potential of P. aeruginosa culture supernatants. Extraction of pyocyanin was made to determine the change in the cell nature of S. aureus upon exposure to pyocyanin by using a scanning electron microscope and the shape of colonies on the culture media was determined. Finally, we detect lasA, operon phz, phzM, phzS and rhlAB genes for P. aeruginosa. P. aeruginosa showed a great impact on S. aureus isolates resistant to different antibiotics as it facilitates their killing and may drive the normal colonies of S. aureus into SCVs. The ability to form biofilm by S. aureus and E. coli decreased in the presence of Pseudomonas supernatant.

ALIPHATIC HYDROCARBONS BIODEGRADATION BY A Pseudomonas STRAIN

The present study aimed to evaluate the potential of a Pseudomonas aeruginosa strain to tolerate and degrade aliphatic petroleum hydrocarbons. P. aeruginosa IBB<sub<Ct8</sub< was isolated from a seawater sample using crude oil as the sole carbon source in a minimal medium. The bacterium was identified as P. aeruginosa based on its morphological, biochemical, and molecular characteristics. P. aeruginosa IBB<sub<Ct8</sub<, which possesses alkB (870 bp) and rhlAB (216 bp) genes in its genome, showed a higher tolerance and degradation efficiency of the n-hexadecane, n-decane, cyclohexane, or paraffin oil, compared with the tolerance to n-hexane and its degradation. P. aeruginosa IBB<sub<Ct8</sub< cells produced rhamnolipid biosurfactants which showed a very good emulsification activity against the tested aliphatic hydrocarbons. Due to its characteristics, the isolated bacterium could be used in the bioremediation of petroleum-polluted environments.

Characterization of Enterobacter cloacae BAGM01 Producing a Thermostable and Alkaline-Tolerant Rhamnolipid Biosurfactant from the Gulf of Mexico.

The search for novel biosurfactants (Bs) requires the isolation of microorganisms from different environments. The Gulf of Mexico (GoM) is a geographical area active in the exploration and exploitation of hydrocarbons. Recent metagenomic and microbiologic studies in this area suggested a potential richness for novel Bs microbial producers. In this work, nineteen bacterial consortia from the GoM were isolated at different depths of the water column and marine sediments. Bs production from four bacterial consortia was detected by the CTAB test and their capacity to reduce surface tension (ST), emulsion index (EI24), and hemolytic activity. These bacterial consortia produced Bs in media supplemented with kerosene, diesel, or sucrose. Cultivable bacteria from these consortia were isolated and identified by bacterial polyphasic characterization. In some consortia, Enterobacter cloacae was the predominant specie. E. cloacae BAGM01 presented Bs activity in minimal medium and was selected to improve its Bs production using a Taguchi and Box-Behnken experimental design; this strain was able to grow and presented Bs activity at 35gL-1 of NaCl. This Bs decreased ST to around 34.5 ± 0.56mNm-1 and presented an EI24 of 71 ± 1.27%. Other properties of this Bs were thermal stability, stability in alkaline conditions, and stability at high salinity, conferring important and desirable characteristics in multiple industries. The analysis of the genome of E. cloacae BAGM01 showed the presence of rhlAB genes that have been reported in the synthesis of rhamnolipids, and alkAB genes that are related to the degradation of alkanes. The bioactive molecule was identified as a rhamnolipid after HPLC derivatization, 1H NMR, and UPLC-QTOF-MS analysis.

Evaluating temperature-induced regulation of a ROSE-like RNA-thermometer for heterologous rhamnolipid production in Pseudomonas putida KT2440

The microbial production of rhamnolipids has been in the focus of research for the last decades. Today, mainly heterologous production systems are targeted due to the advantage of non-pathogenic hosts as well as uncoupling from complex quorum sensing regulatory networks compared to their natural producer Pseudomonas aeruginosa. In the recent past, the presence and function of a ROSE-like RNA-thermometer located in the 5′UTR of the rhamnosyltransferase genes rhlAB has been reported in wild type P. aeruginosa. In this study, the temperature-induced regulation of this native RNA-thermometer for heterologous rhamnolipid production was evaluated and its potential application for process control is discussed. For this purpose, the non-pathogenic production host P. putida KT2440 containing the rhlAB genes with the native P. aeruginosa 5′-UTR region was used. The system was evaluated and characterized regarding the effect of temperature on growth and product formation, as represented by efficiency parameters and yields. Experimental data suggests a major effect of temperature on specific rhamnolipid production rates. With maximum values of 0.23 g/(g h) at 37 °C, this constitutes a more than 60% increase compared to the production rate of 0.14 g/(g h) at the growth optimum of 30 °C. Interestingly however, control experiments unveiled that besides the regulatory effect of the RNA-thermometer, multiple metabolic effects may contribute equally to the observed increase in production rate. As such, this work constitutes an important step towards the utilization of temperature-based process designs and enables the possibility for novel approaches for process control.

Oxygen effects on rhamnolipids production by Pseudomonas aeruginosa

BackgroundRhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa. This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl-genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements.ResultsQuantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC–MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C8-C10, Rha–Rha-C10-C12:1 and Rha–Rha-C8-C10; anaerobically produced rhamnolipids mainly contained Rha-C10-C12 and Rha-C10-C10. Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air–water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI24 = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI24 = 62.3%. Both two rhamnolipids products retained surface activity (ST < 35.0 mN/m) and emulsifying activity (EI24 > 60.0%) under temperatures (4–121 °C), pH values (4–10) and NaCl concentrations less than 90 g/L.ConclusionsOxygen affected the rhl-genes expression in P. aeruginosa, thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

Characterization of rhamnolipid biosurfactants produced by recombinant Pseudomonas aeruginosa strain DAB with removal of crude oil.

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids. Strain DAB had a higher yield of 17.3g rhamnolipids l-1 in the removal process with crude oil as the sole carbon source than 10g rhamnolipids l-1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m-1 with CMC of 90mgl-1. The predominant rhamnolipid congeners were Rha-C10-C10 and Rha-Rha-C10-C10 detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated. Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

In silico Assessment of the Genotypic Distribution of Virulence and Antibiotic Resistance Genes in Pseudomonas aeruginosa

Pseudomonas aeruginosa harbours virulence and antibiotic resistance genes that contribute to life-threatening infections. In silico PCR amplification detected both virulence and antibiotic resistance genes of eighteen Pseudomonas aeruginosa isolates. L lipoprotein (oprL) gene was found in 72.22% of the isolates and is used usually to rapidly identify Pseudomonas aeruginosa species. Alkaline protease (apr) was detected in 66.67% of the isolates. Protein biosynthesis inhibited by exotoxin A (exoA), was found in 55.56% of the isolates. Fifty percent (n=9) of the isolates had the exoenzyme S (exoS) and elastase (lasB) genes. Thirteen isolates (72.22%) which harboured the phospholipid C (plcH) genes, were also found to be positive for rhamnolipid AB (rhlAB) genes. Thirteen isolates (72.22%) were found to possess alginate (algD) and neuraminidae (nan2) genes, respectively. Pseudomonas aeruginosa PA7 harboured both aph(3”) and aph(6)-1d genes. Only sulfonamide resistance gene, sul1 was present in 11.11% (n=2) of the isolates. Beta-lactamase gene, blaTEM was present in only one isolate and no tetracycline resistance gene was found. Fluoroquinolone resistance-determining region of the gyrA and parC genes were present in fourteen (77.78%) and thirteen (72.22%) isolates, respectively. Pseudomonas aeruginosa NCGM2.S1 is a multidrug resistant bacterium since it harboured class 1 integrase gene. In silico pulsed-field gel electrophoresis (PFGE) analysis was able to group eighteen isolates into three genotypes. Gene distribution pattern within genotypes was almost similar and not dependent on genotypes. The data generated here helps to predict virulence and antibiotic resistance profile of Pseudomonas aeruginosaspecies based on genotype.

Response Mechanisms in Serratia marcescens IBBPo15 During Organic Solvents Exposure.

Serratia marcescens strain IBBPo15 (KT315653) which possesses serratiopeptidase (ser) gene (KT894207) exhibited good solvent tolerance. During the exposure of S. marcescens IBBPo15 cells to 5% organic solvents, n-decane was less toxic for this bacterium, compared with n-hexane, cyclohexane, ethylbenzene, toluene, and styrene. The exposure of the S. marcescens IBBPo15 cells to n-hexane, cyclohexane, ethylbenzene, toluene, and styrene induced the formation of large clusters, while in control and n-decane-exposed cells, only organization into small clusters was observed. The data obtained suggested that S. marcescens IBBPo15 cells produced some secondary metabolites (i.e., surfactant serrawettin, red pigment prodigiosin) which are well known as valuable molecules due to their large applications. The exposure of the bacterial cells to organic solvents induced secondary metabolites profile modifications. However, S. marcescens IBBPo15 possesses only alkB1, todM, rhlAB, pswP, mpr, and ser genes, the unspecific amplification of other fragments being acquired also when the primers for alkM1, xylM, ndoM, and C23DO genes were used. Modifications of DNA patterns were not depicted in S. marcescens IBBPo15 cells exposed to organic solvents.

Enhanced rhamnolipid production of Pseudomonas aeruginosa SG by increasing copy number of rhlAB genes with modified promoter

Increasing the copy number ofrhlABgenes with a modified promoter efficiently enhanced the production of rhamnolipid byP. aeruginosa.

The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis.

BackgroundPseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent β-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here.ResultsUsing 5′-RACE PCR, a luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified σ70- and σ54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis.ConclusionThis first detailed study of rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis.

Statistical Screening of Medium Components for Recombinant Production of Pseudomonas aeruginosa ATCC 9027 Rhamnolipids by Nonpathogenic Cell Factory Pseudomonas putida KT2440

Rhamnolipids (RLs) produced by the opportunistic human pathogen Pseudomonas aeruginosa are considered as potential candidates for the next generation of surfactants. Large-scale production of RLs depends on progress in strain engineering, medium design, operating strategies, and purification procedures. In this work, the rhlAB genes extracted from a mono_RLs_producing strain of P. aeruginosa (ATCC 9027) were introduced to an appropriate safety host Pseudomonas putida KT2440. The capability of the recombinant strain was evaluated in various media. As a prerequisite for optimal medium design, a set of 32 experiments was performed in two steps for screening a number of macro-nutritional compounds. In the experiments, a two-level fractional factorial design resolution IV was followed by a two-level full factorial one. By means of this approach, it was observed that glycerol, yeast extract, and peptone have significant positive influence on recombinant RLs production while the yeast extract/peptone two-factor and glycerol/yeast extract/peptone three-factor interactions have considerable negative effects. A wide range of variation from 0 to 570mg/l was obtained for RLs production during the screening experiments indicating the importance of medium optimization. The results point out the opportunity for possible higher yields of RLs through further screening, mixture/combined mixture designs, and high-cell-density cultivations.

Prospects for using native and recombinant rhamnolipid producers for microbially enhanced oil recovery

EOR effects of cell-containing supernatants from cultures of native rhamnolipid producer Pseudomonas aeruginosa PA14, recombinant Escherichia coli carrying genes responsible for rhamnolipid production and P. aeruginosa PDO111 with repressed transcription of rhamnolipid production genes were studied and compared to EOR effects of chemical surfactants in experiments with sandpack columns. The P. aeruginosa PA14 rhlAB genes responsible for production of mono-rhamnolipids were PCR amplified and inserted into pNOT19 plasmid to produce a vector pF1bR4 that was used for transformation of E. coli TG2 to get recombinant strain E. coli pF1bR4. P. aeruginosa PA14, E. coli pF1bR4, and P. aeruginosa PDO111 were grown for 24h at 37°C and centrifuged at 3000rpm for 12min. MALDI-TOF analysis allowed finding previously described rhamnolipids in P. aeruginosa PA14 culture supernatant extract, but not in E. coli pF1bR4 or P. aeruginosa PDO111 extracts. Flooding and incubation tests with sandpack model columns containing two types of oil showed that culture supernatants did have EOR potential. Presence of rhamnolipids and some other biosurfactants including cell surface parts and/or bacterial cells led to enhancement of oil recovery when two different oil production mechanisms, water-flood drive and solution-gas drive, were applied.

Rhamnolipids: diversity of structures, microbial origins and roles.

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C10-C10), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.

Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts

The high-level production of rhamnolipid biosurfactants is a unique feature of Pseudomonas aeruginosa and is strictly regulated in response to environmental conditions. The final step in rhamnolipid biosynthesis is catalyzed by the rhlAB genes encoding a rhamnosyltransferase. The expression of the cloned rhlAB genes was studied in heterologous hosts, either under the control of the rhlR and rhlI rhamnolipid regulatory elements or under the control of the tac promoter. A recombinant P. fluorescens strain harboring multiple plasmid-encoded copies of the rhamnolipid gene cluster produced rhamnolipids (0.25 g liter(sup-1)) when grown under nitrogen-limiting conditions. The highest yields (0.6 g liter(sup-1)) and productivities (24 mg liter(sup-1) h(sup-1)) were obtained in a recombinant Pseudomonas putida strain, KT2442, harboring promoterless rhlAB genes fused to the tac promoter on a plasmid. Active rhamnosyltransferase was synthesized, but no rhamnolipids were produced, by recombinant Escherichia coli upon induction of rhlAB gene expression.

Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis.

Transposon Tn5-GM-induced mutant strains of Pseudomonas aeruginosa which are unable to produce rhamnolipid biosurfactants and lack rhamnosyltransferase activity have been isolated. The DNA regions flanking the transposon were cloned and used as specific probes for the isolation of the corresponding wild-type genes from a P. aeruginosa wild-type cosmid gene library. Single cosmid clones capable of restoring rhamnolipid synthesis in the mutant strains were isolated and further subcloned and sequenced, resulting in the identification of two genes (rhlAB) which are organized as an operon upstream of the previously identified rhlR regulatory gene. The RhlA protein (32.5 kDa) harbors a putative signal sequence, suggesting that this protein is located in the periplasm, while the RhlB protein (47 kDa) contains at least two putative membrane-spanning domains. The expression of the rhlAB genes was found to be enhanced 20-fold during the stationary phase of growth under conditions of nitrogen limitation, as measured by using rhlA::lacZ fusions. Moreover, the transcriptional activation of the rhlAB genes appears to depend on a functional RhlR regulatory protein. The sequence upstream of the rhlA promoter contains two inverted repeats which define putative binding sites for the RhlR regulator. The controlled expression of the rhlAB genes in Escherichia coli led to the formation of active rhamnosyltransferase. This provides direct evidence for the fact that the rhamnosyltransferase encoding genes have been identified.