Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing.

Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa. We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa. Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions.

Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110: A modular metabolic engineering approach.

Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in Escherichia coli W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W-H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W-H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.

An atypical 3-ketoacyl ACP synthase III required for acyl homoserine lactone synthesis in Pseudomonas syringae pv. syringae B728a.

The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from ß-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

Antibacterial mechanism of Pseudomonas aeruginosa UKMP14T rhamnolipids against multidrug resistant Acinetobacter baumannii.

Rhamnolipids, a major category of glycolipid biosurfactant, have recently gained enormous attention in medical field because of their relevance as effective antibacterial agents against a wide variety of pathogenic bacteria. Our previous studies have shown that rhamnolipids from an environmental isolate of Pseudomonas aeruginosa UKMP14T possess antibacterial, anti-adhesive and anti-biofilm activity against multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.) pathogens. However, the mechanism of their antibacterial action remains unclear. Thus, this study aimed to elucidate the mechanism of the antibacterial action of P. aeruginosa UKMP14T rhamnolipids by studying the changes in cells of one of the ESKAPE pathogens, Acinetobacter baumannii, which is the most difficult strain to kill. Results revealed that rhamnolipid treatment rendered A. baumannii cells more hydrophobic as evaluated through contact angle measurements. It also induced the release of cellular proteins measuring 510 µg/mL at a rhamnolipid concentration of 1000 µg/mL. In addition, rhamnolipids were found to be bactericidal in their action as they could permeate the inner membranes, leading to a leak-out of nucleotides. More than 50 % of the cells were found to be killed upon 1000 µg/mL rhamnolipid treatment as observed through fluorescence microscopy. Other cellular changes such as irregular shape and size, membrane perturbations, clumping, shrinkage and physical damage were clearly visible in SEM, FESEM and laser micrographs. Furthermore, rhamnolipid treatment inhibited the levels of acyl-homoserine lactones (AHLs) in A. baumannii, which are vital for their biofilm formation and virulence. The obtained results indicate that P. aeruginosa UKMP14T rhamnolipids target outer and inner bacterial membranes through permeation, including physical damage to the cells, leading to cell leakage. Furthermore, AHL inhibition appears to be the mechanism behind their anti-biofilm action. All these observations can be correlated to rhamnolipids' antibacterial effect against A. baumannii.

Quorum Quenching in Membrane Bioreactors for Fouling Retardation: Complexity Provides Opportunities.

The occurrence of biofouling restricts the widespread application of membrane bioreactors (MBRs) in wastewater treatment. Regulation of quorum sensing (QS) is a promising approach to control biofouling in MBRs, yet the underlying mechanisms are complex and remain to be illustrated. A fundamental understanding of the relationship between QS and membrane biofouling in MBRs is lacking, which hampers the development and application of quorum quenching (QQ) techniques in MBRs (QQMBRs). While many QQ microorganisms have been isolated thus far, critical criteria for selecting desirable QQ microorganisms are still missing. Furthermore, there are inconsistent results regarding the QQ lifecycle and the effects of QQ on the physicochemical characteristics and microbial communities of the mixed liquor and biofouling assemblages in QQMBRs, which might result in unreliable and inefficient QQ applications. This review aims to comprehensively summarize timely QQ research and highlight the important yet often ignored perspectives of QQ for biofouling control in MBRs. We consider what this "information" can and cannot tell us and explore its values in addressing specific and important questions in QQMBRs. Herein, we first examine current analytical methods of QS signals and discuss the critical roles of QS in fouling-forming microorganisms in MBRs, which are the cornerstones for the development of QQ technologies. To achieve targeting QQ strategies in MBRs, we propose the substrate specificity and degradation capability of isolated QQ microorganisms and the surface area and pore structures of QQ media as the critical criteria to select desirable functional microbes and media, respectively. To validate the biofouling retardation efficiency, we further specify the QQ effects on the physicochemical properties, microbial community composition, and succession of mixed liquor and biofouling assemblages in MBRs. Finally, we provide scale-up considerations of QQMBRs in terms of the debated QQ lifecycle, practical synergistic strategies, and the potential cost savings of MBRs. This review presents the limitations of classic QS/QQ hypotheses in MBRs, advances the understanding of the role of QS/QQ in biofouling development/retardation in MBRs, and builds a bridge between the fundamental understandings and practical applications of QQ technology.

Development of an analytical method to quantify N-acyl-homoserine lactones in bacterial cultures, river water, and treated wastewater.

N-Acyl-homoserine lactones (AHL) play a major role in the communication of Gram-negative bacteria. They influence processes such as biofilm formation, swarming motility, and bioluminescence in the aquatic environment. A comprehensive analytical method was developed to elucidate the "chemical communication" in pure bacterial cultures as well as in the aquatic environment and engineered environments with biofilms. Due to the high diversity of AHLs and their low concentrations in water, a sensitive and selective LC-ESI-MS/MS method combined with solid-phase extraction was developed for 34 AHLs, optimized and validated to quantify AHLs in bacterial conditioned medium, river water, and treated wastewater. Furthermore, the developed method was optimized in terms of enrichment volume, internal standards, limits of detection, and limits of quantification in several matrices. An unanticipated variety of AHLs was detected in the culture media of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs), which to our knowledge have not been described for these bacterial cultures so far. Furthermore, AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs). Several detected AHLs were quantified (in total 24) using a standard addition method up to 7.3±1.0 µg/L 3-Oxo-C12-AHL (culture media of P. aeruginosa).

Structural characterization of N-acyl-homoserine lactones from bacterial quorum sensing using LC-MS/MS analyses after Paternò-Büchi derivatization in solution.

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs.

The effect of quorum sensing on cadmium- and lead-containing wastewater treatment using activated sludge: Removal efficiency, enzyme activity, and microbial community.

Quorum sensing (QS) is prevalent in activated sludge processes; however, its essential role in the treatment of heavy metal wastewater has rarely been studied. Therefore, in this study, acyl homoserine lactone (AHL)-mediated QS was used to regulate the removal performance, enzyme activity, and microbial community of Cd- and Pb-containing wastewater in a sequencing batch reactor (SBR) over 30 cycles. The results showed that exogenous AHL strengthened the removal of Cd(II) and Pb(II) in their coexistence wastewater during the entire period. The removal of NH4+-N, total phosphorus, and chemical oxygen demand (COD) was also enhanced by the addition of AHL despite the coexistence of Cd(II) and Pb(II). Meanwhile, the protein content of extracellular polymeric substances was elevated and the microbial metabolism and antioxidative response were stimulated by the addition of AHL, which was beneficial for resistance to heavy metal stress and promoted pollutant removal by activated sludge. Microbial sequencing indicated that AHL optimized the microbial community structure, with the abundance of dominant taxa Proteobacteria and Unclassified_f_Enterobacteriaceae increasing by 73.9% and 59.2% maximally, respectively. This study offers valuable insights into the mechanisms underlying Cd(II) and Pb(II) removal as well as microbial community succession under AHL availability in industrial wastewater.

The potential effects of N-Acyl homoserine lactones on aerobic sludge granulation during phenolic wastewater treatment.

The formation of aerobic granular sludge (AGS) is relatively difficult during the treatment of refractory wastewater, which generally shows small granular sizes and poor stability. The formation of AGS is regulated by N-Acyl homoserine lactones (AHLs)-mediated quorum sensing (QS). However, the potential role of AHLs in AGS formation under the toxic stress of refractory pollutants and the heterogeneity in the distribution and function of AHLs across different aggregates are not well understood. This study investigated the potential effects of AHLs on the formation of AGS during phenolic wastewater treatment. The distribution and succession of AHLs across varying granular sizes and development stages of AGS were investigated. Results showed that AGS was successfully formed in 13 days with an average granular size of 335 ± 39 µm and phenol removal efficiency of >99%. The levels of AHLs initially increased and then decreased. C4-HSL and 3-oxo-C10-HSL were enriched in large granules, suggesting they may play a pivotal role in regulating the concentration and composition of extracellular polymeric substances (EPS). The content of EPS constantly increased to 149.4 mg/gVSS, and protein (PN) was enriched in small and large granules. Luteococcus was the dominant genus constituting up to 62% after the granulation process, and exhibited a strong association with C4-HSL. AHLs might also regulate the bacterial community responsible for EPS production, and pollutant removal, and facilitate the proliferation of slow-growing microorganisms, thereby enhancing the formation of AGS. The synthesis and dynamics of AHLs were mainly governed by AHLs-producing bacterial strains of Rhodobacter and Pseudomonas, and AHLs-quenching strains of Flavobacterium and Comamonas. C4-HSL and 3-oxo-C10-HSL might be the major contributors to promoting sludge granulation under phenol stress and play critical roles in large granules. These findings enhance our understanding of the roles that AHLs play in sludge granulation under toxic conditions.

Phosphate recovery from urban sewage by the biofilm sequencing batch reactor process: Key factors in biofilm formation and related mechanisms.

The biofilm sequencing batch reactor (BSBR) technique has been deployed in the laboratory to enrich phosphorus from simulated wastewater, but it is still not clear what its performance will be when real world sewage is used. In this work, the effluent from the multi-stage anoxic-oxic (AO) activated sludge process at a sewage plant was used as the feed water for a BSBR pilot system, which had three reactors operating at different levels of dissolved oxygen (DO). The phosphorus adsorption and release, the biofilm growth, and the extracellular polymeric substances (EPS) components and contents were examined. The microbial communities and the signaling molecules N-acyl-l-homoserine lactones (AHLs) were also analyzed. Gratifyingly, the BSBR process successfully processed the treated sewage, and the biofilm developed phosphorus accumulation capability within 40 days. After entering stable operation, the system concentrated phosphate from 2.59 ± 0.77 mg/L in the influent to as much as 81.64 mg/L in the recovery liquid. Sludge discharge had profound impacts on all aspects of BSBR, and it was carried out successfully when the phosphorus absorption capacity of the biofilm alone was comparable to that of the reactor containing the activated sludge. Shortly after the sludge discharge, the phosphate concentration of the recovery liquid surged from 50 to 140 mg/L, the biofilm thickness grew from 20.56 to 67.32 µm, and the diversity of the microbial population plunged. Sludge discharge stimulated Candidatus competibacter to produce a large amount of AHLs, which was key in culturing the biofilm. Among the AHLs, both C10-HSL and 3OC12-HSL were significantly positively correlated with EPS and the abundance of Candidatus competibacter. The current results demonstrated BSBR as a viable option to enrich phosphorus from real world sewage with low phosphorus content and fluctuating chemistry. The mechanistic explorations also provided theoretical guidance for cultivating phosphorus-accumulating biofilms.

The quorum sensing molecule C12-HSL promotes biofilm formation and increases adrA expression in Salmonella Enteritidis under anaerobic conditions.

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.

Facilitating nitrification and biofilm formation of Vibrio sp. by N-acyl-homoserine lactones in high salinity environment.

The N-acyl-homoserine lactones (AHLs)-mediated quorum-sensing (QS) system played a crucial role in regulating biological nitrogen removal and biofilm formation. However, the regulatory role of AHLs on nitrogen removal bacteria in high salinity environment has remained unclear. This study evaluated the roles and release patterns of AHLs in Vibrio sp. LV-Q1 under high salinity condition. Results showed that Vibrio sp. primarily secretes five AHLs, and the AHLs activity is strongly correlated with the bacterial density. Exogenous C10-HSL and 3OC10-HSL were found to significantly enhance ammonium removal, while making a minor contribution to the growth rate. Both the C10-HSL and 3OC10-HSL promoted the biofilm formation of Vibrio sp. with an enhancement of 1.64 and 1.78 times, respectively. The scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) observations confirmed the biofilm-enhancing effect of AHLs. Further analysis revealed that AHLs significantly improved bacterial self-aggregation and motility, as well as the level of extracellular polymeric substances (EPS). These findings provide significant guidance on construction of nitrification system at high salinity.

N-acyl homoserine lactones (AHLs) enhanced removal of cadmium and other pollutants by algae-bacteria consortia.

Signal transduction is an important mode of algae-bacteria interaction, in which bacterial quorum sensing (QS) may affect microalgal growth and metabolism. Currently, little is known whether acyl homoserine lactones (AHLs) released by bacteria can affect the pollutant removal by algae-bacteria consortia (ABC). In this study, we constructed ABC using Chlorella vulgaris (Cv) with two AHLs-producing bacteria and investigated their performance in the removal of multiple pollutants, including chemical oxygen demand (COD), total nitrogen (TN), phosphorus (P), and cadmium (Cd). The AHLs-producing bacteria, namely Agrobacterium sp. (Ap) and Ensifer adherens (Ea), were capable of forming a symbiosis with C. vulgaris. Consortia of Cv and Ap with ratio of 2:1 (Cv2-Ap1) showed the optimal growth promotion and higher removal of Cd, COD, TN, and P compared to the C. vulgaris monoculture. Cv2-Ap1 ABC removed 36.1-47.5% of Cd, 94.5%-94.6% COD, 37.1%-56.0% TN, and 90.4%-93.5% P from the culture medium. In addition, increase of intracellular neutral lipids and extracellular protein, as well as the types of functional groups on cell surface contributed to Cd removal and tolerance in the Cv2-Ap1 ABC. Six AHLs were detected in the Cv2-Ap1 culture. Among these, 3OC8-HSL and 3OC12-HSL additions promoted the ABC growth and enhanced their Cd accumulation. These findings may contribute to further understanding of AHL-mediated communication between algae and bacteria and provide support bioremediation efforts of metal-containing wastewater.

The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

Relationship of the transcription factor MexT to quorum sensing and virulence in Pseudomonas aeruginosa.

IMPORTANCE: Pseudomonas aeruginosa is an opportunistic bacterial pathogen. Many of its virulence genes are regulated by quorum sensing (QS), a form of cell-to-cell communication. P. aeruginosa QS consists of three interlinked circuits, LasI-R, Rhl-R, and Pseudomonas quinolone signal (PQS). Additionally, its QS system is interconnected with other regulatory networks, which help optimize gene expression under variable conditions. The numbers of genes regulated by QS differ substantially among P. aeruginosa strains. We show that a regulatory factor MexT, which is activated in response to certain antibiotics, downregulates the RhlI-R circuit and in turn measurably lowers virulence in a nematode worm infection model. Our findings help understand how existing and future therapeutic interventions for P. aeruginosa infections may impact this bacterium's gene regulation and physiology.

Growth rate and nutrient limitation as key drivers of extracellular quorum sensing signal molecule accumulation in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa.

A FadR-Type Regulator Activates the Biodegradation of Polycyclic Aromatic Hydrocarbons by Mediating Quorum Sensing in Croceicoccus naphthovorans Strain PQ-2.

Bacteria employ multiple transcriptional regulators to orchestrate cellular responses to adapt to constantly varying environments. The bacterial biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been extensively described, and yet, the PAH-related transcriptional regulators remain elusive. In this report, we identified an FadR-type transcriptional regulator that controls phenanthrene biodegradation in Croceicoccus naphthovorans strain PQ-2. The expression of fadR in C. naphthovorans PQ-2 was induced by phenanthrene, and its deletion significantly impaired both the biodegradation of phenanthrene and the synthesis of acyl-homoserine lactones (AHLs). In the fadR deletion strain, the biodegradation of phenanthrene could be recovered by supplying either AHLs or fatty acids. Notably, FadR simultaneously activated the fatty acid biosynthesis pathway and repressed the fatty acid degradation pathway. As intracellular AHLs are synthesized with fatty acids as substrates, boosting the fatty acid supply could enhance AHL synthesis. Collectively, these findings demonstrate that FadR in C. naphthovorans PQ-2 positively regulates PAH biodegradation by controlling the formation of AHLs, which is mediated by the metabolism of fatty acids. IMPORTANCE Master transcriptional regulation of carbon catabolites is extremely important for the survival of bacteria that face changes in carbon sources. Polycyclic aromatic hydrocarbons (PAHs) can be utilized as carbon sources by some bacteria. FadR is a well-known transcriptional regulator involved in fatty acid metabolism; however, the connection between FadR regulation and PAH utilization in bacteria remains unknown. This study revealed that a FadR-type regulator in Croceicoccus naphthovorans PQ-2 stimulated PAH biodegradation by controlling the biosynthesis of the acyl-homoserine lactone quorum-sensing signals that belong to fatty acid-derived compounds. These results provide a unique perspective for understanding bacterial adaptation to PAH-containing environments.

A Stenotrophomonas maltophilia TetR-Like Transcriptional Regulator Involved in Fatty Acid Metabolism Is Controlled by Quorum Sensing Signals.

Stenotrophomonas maltophilia is an environmental bacterium as well as an emerging opportunistic multidrug-resistant pathogen. They use the endogenous diffusible signal factor (DSF) quorum sensing (QS) system to coordinate population behavior and regulate virulence processes but can also respond to exogenous N-acyl-homoserine lactone (AHL) signals produced by neighboring bacteria. The effect of these QS signals on the global gene expression of this species remains, however, unknown. Whole-transcriptome sequencing analyses were performed for exponential cultures of S. maltophilia K279a treated with exogenous DSF or AHLs. Addition of DSF and AHLs signals resulted in changes in expression of at least 2-fold for 28 and 82 genes, respectively. Interestingly, 22 of these genes were found upregulated by both QS signals, 14 of which were shown to also be induced during the stationary phase. Gene functions regulated by all conditions included lipid and amino acid metabolism, stress response and signal transduction, nitrogen and iron metabolism, and adaptation to microoxic conditions. Among the common top upregulated QS core genes, a putative TetR-like regulator (locus tag SMLT2053) was selected for functional characterization. This regulator controls its own ß-oxidation operon (Smlt2053-Smlt2051), and it is found to sense long-chain fatty acids (FAs), including the QS signal DSF. Gene knockout experiments reveal that operon Smlt2053-Smlt2051 is involved in biofilm formation. Overall, our findings provide clues on the effect that QS signals have in S. maltophilia QS-related phenotypes and the transition from the exponential to the stationary phase and bacterial fitness under high-density growth. IMPORTANCE The quorum sensing system in Stenotrophomonas maltophilia, in addition to coordinating the bacterial population, controls virulence-associated phenotypes, such as biofilm formation, motility, protease production, and antibiotic resistance mechanisms. Biofilm formation is frequently associated with the persistence and chronic nature of nosocomial infections. In addition, biofilms exhibit high resistance to antibiotics, making treatment of these infections extremely difficult. The importance of studying the metabolic and regulatory systems controlled by quorum sensing autoinducers will make it possible to discover new targets to control pathogenicity mechanisms in S. maltophilia.

Identification and engineering efflux transporters for improved L-homoserine production in Escherichia coli.

AIMS: This study aimed to functionally identify the potential L-homoserine transporters in Escherichia coli, and to generate the promising beneficial mutants by targeted directed evolution for improving the robustness and efficiency of microbial cell factories. METHODS AND RESULTS: By constructing a series of gene deletion and overexpression strains, L-homoserine tolerance assays revealed that RhtA was an efficient and major L-homoserine exporter in E. coli, whereas RhtB and RhtC exhibited relatively weak transport activities for L-homoserine. Real-time RT-PCR analysis suggested that the expression levels of these three target mRNAs were generally variably enhanced when cells were subjected to L-homoserine stress. Based on in vivo continuous directed evolution and growth-couple selections, three beneficial mutations of RhtA exporter (A22V, P119L, and T235I) with clearly increased tolerance against L-homoserine stress were quickly obtained after two rounds of mutagenesis-selection cycles. L-homoserine export assay revealed that the RhtA mutants exhibited different degrees of improvement in L-homoserine export capacity. Further studies suggested that a combination of these beneficial sites led to synergistic effects on conferring L-homoserine-resistance phenotypes. Moreover, the introduction of RhtA beneficial mutants into the L-homoserine-producing strains could facilitate increased amounts of L-homoserine in the shake-flask fermentation. CONCLUSIONS: In this study, we provided further evidence that RhtA serves as a major L-homoserine exporter in E. coli, and obtained several RhtA beneficial mutants, including A22V, P119L, and T235I that contributed to improving the L-homoserine resistance phenotypes and the production efficiency in microbial chassis.

Development of a biofilm-forming bacterial consortium and quorum sensing molecules for the degradation of lignin-containing organic pollutants.

The presence of lignin along with other pollutants makes effluent more complex when it is discharged from Pulp and paper mills. The present study investigates the use of biofilm-forming bacteria isolated from pulp paper mill effluent contaminated sites (PPMECSs) for lignin degradation. Isolated biofilm-forming and lignin-degrading bacteria were identified as Bacillus subtilis, Enterobacter cancerogenus, and Bacillus licheniformis by 16S rRNA gene sequencing. Thin liquid chromatography (TLC) analysis showed that the consortium of bacteria produced acyl-homoserine lactone (AHL) as quorum sensing molecules and extracellular polymeric substances (EPS) that protect the bacterial consortium under unfavorable conditions. The potential consortium was able to reduce lignin (900 ppm) by 73% after 8 days of incubation in a minimal salt medium containing kraft lignin and glucose at pH 7.0 and 37 °C as compared to individual strains. The degradation by-products were identified as amides, alcohols, and acids. The major organic pollutants in the effluent were reduced after treatment of the constructed consortium, thus confirming active biotransformation and biodegradation of the lignin. Microscopic examination also indicated the presence of lignin induced biofilm formation. Hence, the constructed biofilm-forming bacterial consortia based on quorum sensing offered a sustainable and effective solution to treat lignin-containing complex pollutants.