Phenazine-1-carboxylic Acid Research Articles

A Defect in the Twin-Arginine Translocation Pathway Decreases the Tolerance of Xanthomonas campestris pv. campestris to Phenazines.

Phenazine-1-carboxylic acid (PCA), a member of phenazines secreted by microorganisms, inhibits the growth of many bacteria and fungi. Xanthomonas campestris pv. campestris is the causal agent of black rot, the most important disease of cruciferous crops worldwide, and is more tolerant to PCA than other Xanthomonas species. Previous studies reported that reactive oxygen species (ROS) scavenging ability is involved in regulating the PCA tolerance of Xanthomonas species. Additionally, the cytochrome c maturation (CCM) system has been found to play a more important role in tolerance to phenazines than the ROS scavenging system. In this study, a highly PCA-sensitive insertion mutant of X. campestris pv. campestris, X-5, was identified and studied. The insertion site of X-5 was found to be in tatB gene (XC_4183), which encodes a subunit of the twin-arginine translocation (TAT) complex. Disruption of the three genes of TAT pathway resulted in decreased biological fitness and reduced tolerance to phenazines in comparison with the wild-type strain 8004. These results imply that the tolerance mechanism of the TAT pathway to phenazines is related to the CCM system, but not due to the ROS scavenging system. Furthermore, respiration-related characteristic tests and peptide analysis suggested that disruption of the TAT complex causes a defect in the cytochrome bc1 complex, which may be involved in the tolerance to phenazines. In summary, this study sheds new light on the critical role of the TAT pathway in influencing the fitness and phenazines tolerance of Xanthomonas species.

Construction of an Electron Transfer Mediator Pathway for Bioelectrosynthesis by Escherichia coli.

Microbial electrosynthesis (MES) or electro-fermentation (EF) is a promising microbial electrochemical technology for the synthesis of valuable chemicals or high-value fuels with aid of microbial cells as catalysts. By introducing electrical energy (current), fermentation environments can be altered or controlled in which the microbial cells are affected. The key role for electrical energy is to supply electrons to microbial metabolism. To realize electricity utility, a process termed inward extracellular electron transfer (EET) is necessary, and its efficiency is crucial to bioelectrochemical systems. The use of electron mediators was one of the main ways to realize electron transfer and improve EET efficiency. To break through some limitation of exogenous electron mediators, we introduced the phenazine-1-carboxylic acid (PCA) pathway from Pseudomonas aeruginosa PAO1 into Escherichia coli. The engineered E. coli facilitated reduction of fumarate by using PCA as endogenous electron mediator driven by electricity. Furthermore, the heterologously expressed PCA pathway in E. coli led to better EET efficiency and a strong metabolic shift to greater production of reduced metabolites, but lower biomass in the system. Then, we found that synthesis of adenosine triphosphate (ATP), as the “energy currency” in metabolism, was also affected. The reduction of menaquinon was demonstrated as one of the key reactions in self-excreted PCA-mediated succinate electrosynthesis. This study demonstrates the feasibility of electron transfer between the electrode and E. coli cells using heterologous self-excreted PCA as an electron transfer mediator in a bioelectrochemical system and lays a foundation for subsequent optimization.

Phenazine-1-carboxylic acid (PCA) from Cold-Adapted Yeast Glaciozyma antarctica PI12

A phenazine alkaloid, phenazine-1-carboxylic acid (PCA) was isolated from the ethyl acetate extract of cold-adapted yeast Glaciozyma antarctica PI12 (Kriegeriales). The compounds were isolated by several chromatography techniques i.e. radial chromatography (RC) and thin-layer chromatography (TLC). The chemical structure was elucidated by ultraviolet (UV), infrared (IR), nuclear magnetic resonance (1D and 2D NMR) spectroscopy and mass spectrometry.

Identification of a Strong Quorum Sensing- and Thermo-Regulated Promoter for the Biosynthesis of a New Metabolite Pesticide Phenazine-1-carboxamide in Pseudomonas strain PA1201.

Phenazine-1-carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clusters, phz1 and phz2, for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional phzH-encoding glutamine aminotransferase. However, PCN levels in PA1201 constitute approximately one-fifth of PCA levels and the optimal temperature for PCN synthesis is 28 °C. In this study, the phzH open reading frame (ORF) and promoter region were investigated and reannotated. phzH promoter PphzH was found to be a weak promoter, and PhzH levels were not sufficient to convert all of the native PCA into PCN. Following RNA Seq and promoter-lacZ fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter PrhlI was identified and characterized. The activity of PphzH is approximately 1% of PrhlI in PA1201. After three rounds of promoter editing and swapping by PrhlI, a new PCN-overproducing strain UP46 was generated. The optimal fermentation temperature for PCN biosynthesis in UP46 was increased from 28 to 37 °C and the PCN fermentation titer increased 179.5-fold, reaching 14.1 g/L, the highest ever reported.

Phenazine 1- carboxylic acid (PCA) produced by Pseudomonas aeroginosa MUT.3: a study on its stability and antibacterial activity under various environmental conditions

Phenazine 1- carboxylic acid (PCA) produced by Pseudomonas aeroginosa MUT.3: a study on its stability and antibacterial activity under various environmental conditions

Phenazine-1-carboxylic acid-producing Pseudomonas synxantha LBUM223 alters the transcriptome of Streptomyces scabies, the causal agent of potato common scab

Common scab of potato is an important disease caused by Streptomyces scabies. Biological control of this disease was previously demonstrated under controlled and field conditions using Pseudomonas synxantha LBUM223, a phenazine-1-carboxylic acid (PCA)-producer. To better understand the impact of PCA on the pathogen, RNA-seq analyses of S. scabies confronted with wild-type LBUM223, its isogenic PCA-deficient mutant and purified PCA were performed. Following exposure to wild-type LBUM223 or purified PCA (but not to the isogenic PCR-deficient mutant), 12%–14% of all S. scabies genes were differentially expressed, including key genes involved in pathogenicity/virulence, mycelium differentiation, and increased oxidative stress in S. scabies.

Engineering of glycerol utilization in Pseudomonas chlororaphis GP72 for enhancing phenazine-1-carboxylic acid production

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4mg/L to 993.4mg/L at 36h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.

Co-culture of the bacterium Pseudomonas aeruginosa with the fungus Fusarium tricinctum induces bacterial antifungal and quorum sensing signaling molecules

Co-cultivation of viable cells of the human pathogenic bacterium Pseudomonas aeruginosa with the endophytic fungus Fusarium tricinctum induced silent bacterial biosynthetic gene clusters. Visual and chemical inspection of the co-cultures by comparison with the axenic cultures showed a clear reaction of P. aeruginosa towards its fungal competitor which was reflected by the deep greenish coloration of the co-cultures. Inspection of the HPLC chromatograms of the co-cultures revealed the induction of the pseudomonal quorum sensing molecule 2-heptyl-4-hydroxy-quinolone (HHQ) along with the antifungal phenazine alkaloids, phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN), which are well-known metabolites of P. aeruginosa. The latter compounds were not detected in axenic bacterial cultures. In contrast to P. aeruginosa, no metabolic responses were detected for F. tricinctum which is in sharp contrast to previous co-culture experiments with this fungus using other bacteria.

Overexpression of oxyR Increases Phenazine-1-Carboxylic Acid Biosynthesis via Small RNA phrS in the Rhizobacterium Strain Pseudomonas PA1201.

Phenazine-1-carboxylic acid (PCA) is the primary active component in the newly registered, commercial biopesticide Shenqinmycin and is produced during fermentation by the engineered rhizobacterium strain Pseudomonas PA1201. Both phz1 and phz2 gene clusters contribute to PCA biosynthesis. In this study, we evaluated the role of OxyR in the regulation of PCA biosynthesis in PA1201. We first showed a functional link between oxyR expression and PCA biosynthesis. Deletion of oxyR and overexpression of oxyR both increase PCA biosynthesis. The molecular mechanisms underlying OxyR regulation of PCA production were investigated using several approaches. OxyR acts divergently in phz1 and phz2. Overexpression of oxyR activated the expression of phz1 and phz1-dependent PCA production. However, overexpression of oxyR had little effect on phz2-dependent PCA biosynthesis, while deletion of oxyR promoted phz2-dependent PCA production and exerted a negative effect on phz2 expression. Further, OxyR directly bound to the phz2 promoter region. In addition, the regulation of PCA biosynthesis by OxyR was associated with quorum sensing (QS) systems. Overexpression of OxyR positively regulated pqs QS system. Finally, transcriptomic analysis and subsequent genetic analysis revealed the small RNA phrS plays a key role in OxyR-dependent PCA accumulation. Specifically, OxyR directly binds to the phrS promoter region to positively regulate phrS expression wherein PhrS regulates the PCA positive regulator MvfR in order to control PCA biosynthesis.

Refinement of metabolite detection in cystic fibrosis sputum reveals heme correlates with lung function decline.

The bacterial growth environment within cystic fibrosis (CF) sputum is complex, dynamic, and shaped by both host and microbial processes. Characterization of the chemical parameters within sputum that stimulate the in vivo growth of airway pathogens (e.g. Pseudomonas aeruginosa) and their associated virulence factors may lead to improved CF treatment strategies. Motivated by conflicting reports of the prevalence and abundance of P. aeruginosa-derived metabolites known as phenazines within CF airway secretions, we sought to quantify these metabolites in sputum using quadrupole time-of-flight mass spectrometry. In contrast to our previous work, all phenazines tested (pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine) were below detection limits of the instrument (0.1 μM). Instead, we identified a late-eluting compound that shared retention time and absorbance characteristics with PCA, yet generated mass spectra and a fragmentation pattern consistent with ferriprotoporphyrin IX, otherwise known as heme B. These data suggested that UV-vis chromatographic peaks previously attributed to PCA and PYO in sputum were mis-assigned. Indeed, retrospective analysis of raw data from our prior study found that the heme B peak closely matched the peaks assigned to PCA, indicating that the previous study likely uncovered a positive correlation between pulmonary function (percent predicted forced expiratory volume in 1 second, or ppFEV1) and heme B, not PCA or any other phenazine. To independently test this observation, we performed a new tandem mass-spectrometry analysis of 71 additional samples provided by the Mountain West CF Consortium Sputum Biomarker study and revealed a positive correlation (ρ = −0.47, p<0.001) between sputum heme concentrations and ppFEV1. Given that hemoptysis is strongly associated with airway inflammation, pulmonary exacerbations and impaired lung function, these new data suggest that heme B may be a useful biomarker of CF pathophysiology.

An angular dioxygenase gene cluster responsible for the initial phenazine-1-carboxylic acid degradation step in Rhodococcus sp. WH99 can protect sensitive organisms from toxicity

A bacterial strain, Rhodococcus sp. WH99, capable of degrading phenazine-1-carboxylic acid (PCA) was isolated and characterized. Genome comparison revealed that a 21499-bp DNA fragment containing a putative angular dioxygenase gene cluster consisting of the dioxygenase-, ferredoxin reductase- and ferredoxin-encoding genes (pzcA1A2, pzcC and pzcD) is missed in the PCA degradation-deficient mutant WH99M. The pzcA1A2CD genes were expressed in Escherichia coli respectively and hydroxylation of PCA to 1,2-dihydroxyphenazine occurred in vitro only when all components were present. However, in vivo analyses showed that pzcA1A2 and pzcD were indispensable for PCA degradation, while PzcC can be partially replaced by other ferredoxin reductases. Hydroxylation of PCA not only initiates degradation of PCA in strain WH99 but also provides protection to sensitive organisms that would otherwise be inhibited by PCA toxicity. This study illustrates a new initial PCA degradation step in Gram-positive bacteria and enhances our understanding of the genes responsible for PCA hydroxylation, thus enabling targeted studies on protection by PCA degradation in diverse environments.

Phenazine-1-Carboxylic Acid-Producing Bacteria Enhance the Reactivity of Iron Minerals in Dryland and Irrigated Wheat Rhizospheres.

Phenazine-1-carboxylic acid (PCA) is a broad-spectrum antibiotic produced by rhizobacteria in the dryland wheat fields of the Columbia Plateau. PCA and other phenazines reductively dissolve Fe and Mn oxyhydroxides in bacterial culture systems, but the impact of PCA upon Fe and Mn cycling in the rhizosphere is unknown. Here, concentrations of dithionite-extractable and poorly crystalline Fe were approximately 10% and 30-40% higher, respectively, in dryland and irrigated rhizospheres inoculated with the PCA-producing (PCA+) strain Pseudomonas synxantha 2-79 than in rhizospheres inoculated with a PCA-deficient mutant. However, rhizosphere concentrations of Fe(II) and Mn did not differ significantly, indicating that PCA-mediated redox transformations of Fe and Mn were transient or were masked by competing processes. Total Fe and Mn uptake into wheat biomass also did not differ significantly, but the PCA+ strain significantly altered Fe translocation into shoots. X-ray absorption near edge spectroscopy revealed an abundance of Fe-bearing oxyhydroxides and phyllosilicates in all rhizospheres. These results indicate that the PCA+ strain enhanced the reactivity and mobility of Fe derived from soil minerals without producing parallel changes in plant Fe uptake. This is the first report that directly links significant alterations of Fe-bearing minerals in the rhizosphere to a single bacterial trait.

Screening Effective Antifungal Substances from the Bark and Leaves of Zanthoxylum avicennae by the Bioactivity-Guided Isolation Method.

To find good antifungal substances by the bioactivity-guided isolation method, we tracked down the effective antifungal substances in the bark and leaves of Zanthoxylum avicennae, and isolated three antifungal compounds 1, 2, and 3. The structures were identified as xanthyletin, luvangetin, and avicennin by 1H-NMR, 13C-NMR, and HRMS spectra. Particularly, compound 2 had several isomers, and the 1H-NMR spectra of 2 in different solvents showed a significant difference. To determine the stereo structure of 2, a single crystal was prepared and identified by X-ray diffraction as Luvangetin. Moreover, the difference of 1H-NMR data of 2 between in solvent dimethyl sulfoxide-d6 (DMSO-d6) and deuterated chloroform (CDCl3), and other reported isomers were discussed for the first time. The bioassay results indicated that the three compounds 1, 2, and 3 displayed low to high antifungal activities against tested phytopathogenic fungi. In particular, all compounds 1, 2, and 3 showed excellent antifungal activities against Pyricularia oryzae and Z. avicennae, with the values of half maximal effective concentration (EC50) ranging from 31 to 61 mg/L, and compound 3 was also identified as a more potent inhibitor against Fusaium graminearum (EC50 = 43.26 ± 1.76 mg/L) compared with fungicide PCA (phenazine-1-carboxylic acid) (EC50 = 52.34 ± 1.53 mg/L). The results revealed that compounds 1, 2, and 3 were the main antifungal substances of Z. avicennae, and can be used as lead compounds of a fungicide, which has good development value and prospect.

Characterization of phenazine-1-carboxylic acid by Klebsiella sp. NP-C49 from the coral environment in Gulf of Kutch, India.

Coral-associated microbes from Marine National Park (MNP), Gulf of Kutch (GoK), Gujarat, India, were screened for siderophore production. Maximum siderophore-producing isolate NP-C49 and its compound were identified and characterized. The isolate was identified as Klebsiella sp. through 16S rRNA genes sequencing (GenBank accession nos. KY412519 and MTCC 25160). Antibiotic susceptibility profile against 20 commercial antibiotics showed its more sensitivity compared to human pathogenic strain, i.e., Klebsiella pneumonia. The compound was identified as phenazine-1-carboxylic acid (PCA) using the multinuclear ID (1H and 13C) and 2D (1H-1H COSY and 1H-13C HETCOR) NMR along with high-resolution mass spectrometry. No significant difference in the bacterial growth in the presence of PCA, FeCl3 and Fe(OH)3 indicated involvement of factors other than PCA in bacterial growth. The study first reports the identification and characterization of PCA from Klebsiella sp. both from terrestrial and marine sources.

Phyllosphere Fungal Communities of Plum and Antifungal Activity of Indigenous Phenazine-Producing Pseudomonas synxantha Against Monilinia laxa.

European plum (Prunus domestica L.) is a significant commercial crop in Serbia in terms of total fruit production, and is traditionally processed into slivovitz brandy. The brown rot disease caused by Monilinia laxa drastically reduces plum yield almost every year. Fungal communities associated with leaves and fruits of four local Serbian plum cultivars (Požegača, Ranka, Čačanska Lepotica and Čačanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Alpha diversity indices showed that fungal communities were heterogeneous and Beta diversity indicated that autochthonous fungal communities depended upon seasonal changes and the cultivars themselves. The phylum Ascomycota was the most abundant in all samples, with relative abundance (RA) between 46% in the Požegača cultivar (May) and 89% in the Lepotica cultivar (July). The most abundant genus for all plum cultivars in May was Aureobasidium, with RA from 19.27 to 33.69%, followed by Cryptococcus, with 4.8 to 48.80%. In July, besides Cryptococcus, different genera (Metschnikowia, Fusarium, and Hanseniaspora) were dominant on particular cultivars. Among all cultivable fungi, molecular identification of eleven M. laxa isolates from four plum cultivars was performed simultaneously. Bacterial isolates from the plum phyllosphere were tested for their potential antifungal activity against indigenous M. laxa isolates. The most potent antagonist P4/16_1, which significantly reduced mycelial growth of M. laxa, was identified as Pseudomonas synxantha. Further characterization of P4/16_1 revealed the production of volatile organic compounds and phenazine-1-carboxylic acid (PCA). Crude benzene extract of PCA exhibited 57–63% mycelial growth inhibition of M. laxa. LC/MS analysis of the crude extract confirmed the presence of phenazine derivatives amongst other compounds. Scanning electron microscopy revealed morpho-physiological changes in the hyphae of M. laxa isolates caused by the cell culture and the P. synxantha P4/16_1 crude benzene extract. This is the first report of antagonistic activity of P. synxantha against M. laxa induced by diffusible and volatile antifungal compounds, and it appears to be a promising candidate for further investigation for potential use as a biocontrol agent against brown rot-causing fungi.

Boosting Heterologous Phenazine Production in Pseudomonas putida KT2440 Through the Exploration of the Natural Sequence Space.

Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pseudomonas putida KT2440, we compared PCA production from both operons originating from P. aeruginosa strain PAO1 (O1.phz1 and O1.phz2) as well as from P. aeruginosa strain PA14 (14.phz1 and 14.phz2). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes phzM and phzS required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, P. putida harboring 14.phz2 produced 4-times higher PCA concentrations (80 μg/mL), which resulted in 3-times higher current densities (12 μA/cm2) compared to P. putida 14.phz1. The respective PCA/PYO producer containing the 14.phz2 operon was the best strain with 80 μg/mL PCA, 11 μg/mL PYO, and 22 μA/cm2 current density. Tailoring phenazine production also resulted in improved oxygen-limited metabolic activity of the bacterium through enhanced anodic electron discharge. To elucidate the reason for this superior performance, a detailed structure comparison of the PCA-synthesizing proteins has been performed. The here presented characterization and optimization of these new strains will be useful to improve electroactivity in P. putida for oxygen-limited biocatalysis.

Isolation and characterization of antiprotozoal compound-producing Streptomyces species from Mongolian soils

Natural resources are recognized as important sources of potential drugs for treating various infections, and microorganisms are a rich natural source of diverse compounds. Among the world's microorganisms, actinomycetes, which are abundant in soil and marine, are the well-known producers of a wide range of bioactive secondary metabolites and antibiotics. In the present study, four actinomycetes (samples N25, N6, N18, and N12) were isolated from soil samples in Mongolia. Phylogenetic analysis of these isolates revealed that they share the highest similarity with Streptomyces canus (N25), S. cirratus (N6), S. bacillaris (N18) and S. peucetius (N12), based on 16S rRNA gene sequencing. Crude extracts were obtained from them using ethyl acetate, and the crude fractions were separated by thin layer chromatography. The fractions were then evaluated for their cytotoxicities and their anti-Toxoplasma and antimalarial activities in vitro. The S. canus (N25) crude extract was selected for further chemical characterization based on its antiprotozoal activities. Using liquid chromatography-high resolution mass spectrometry, phenazine-1-carboxylic acid (PCA) was detected and identified in the active fractions of the metabolites from strain N25. We next confirmed that commercially available PCA possesses antiprotozoal activity against T. gondii (IC50: 55.5 μg/ml) and Plasmodium falciparum (IC50: 6.4 μg/ml) in vitro. The results of this study reveal that soil actinomycetes are potential sources of antiprotozoal compounds, and that PCA merits further investigation as an anti-protozoal agent.

The influence of steric configuration of phenazine-1-carboxylic acid-amino acid conjugates on fungicidal activity and systemicity.

Conjugating an amino acid onto existing fungicidal parent structures has been demonstrated to be an effective way to endow non-phloem mobile fungicides with phloem mobility. To alter the systemicity of the fungicide PCA (phenazine-1-carboxylic acid), 10 amino acids derivatives of this fungicide were designed and synthesized, and their synthesis, characterization, phloem and xylem mobility in Ricinus communis L, and their fungicidal activity in vitro are described. The systemicity experiments in Ricinus communis system demonstrated that all conjugates exhibited obvious phloem mobility compared with non-phloem-mobile PCA, and the introduction of an L-amino acid to PCA more greatly enhanced the phloem mobility. The five D-amino acid conjugates exhibited higher xylem mobility than that of PCA and of each corresponding L-amino acid conjugate. Most conjugates were found to exhibit moderate in vitro fungicidal activities against six pathogenic fungi, which were lower than that of PCA. The results of the bioassay showed fungicidal activities of PCA-amino acid conjugates associated not only with different amino acids, but also with their conformation. Conjugation with D-amino acid contributed to the in vitro fungicidal activities of PCA-amino acid conjugates. The current research offers a new strategy for enhancing the systemicity of non-phloem-mobile fungicides and presents some useful information on the effects of introducing amino acids of different steric configurations on the fungicidal activity, phloem and xylem mobility of the parent fungicide. © 2019 Society of Chemical Industry.

The critical role of cytochrome c maturation (CCM) system in the tolerance of Xanthomonas campestris pv. campestris to phenazines

Phenazine-1-carboxylic acid (PCA), a secondary metabolite produced by Pseudomonas spp., exhibits a high inhibitory effect in Xanthomonas oryzae pv. oryzae (Xoo), but less inhibitory effect in Xanthomonas oryzae pv. oryzicola (Xoc), and almost no inhibitory effect in Xanthomonas campestris pv. campestris (Xcc). In our previous study, reactive oxygen species (ROS) scavenging system was reported to be involved in PCA tolerance in Xanthomonas spp. However, the PCA tolerance mechanism of Xanthomonas spp. is unclear. In the current study, we constructed a Tn5-based transposon mutant library in Xcc and four highly PCA-sensitive insertion mutants were obtained. TAIL-PCR further confirmed that the Tn5 transposon was inserted in the cytochrome c maturation (CCM) system (XC_1893, XC_1897) of these mutants. Disruption of the CCM system significantly decreased the growth, motility and tolerance of Xcc to PCA and other phenazines, such as phenazine and 1-OH-phenazine. The CCM system is responsible for the covalent attachment of the apocytochrome and heme. Disruption of the transmembrane thioredox protein (Dsb) pathway (XC_0531), an essential process for the formation of mature apocytochrome, also exhibited a decreased tolerance to PCA, suggesting that the defect of cytochrome c caused decreased tolerance of Xcc to PCA. Meanwhile, disruption of the CCM system or Dsb pathway interfered with the functions of cytochrome c proteins, causing an increased sensitivity to H2O2. Collectively, we concluded that the CCM system and Dsb pathway, regulate the tolerance of Xcc to phenazines by influencing the functions of cytochrome c. Therefore, these results provide important references for revealing the action mechanism of PCA in Xanthomonas spp.

Increasing in the Extraction Yield of Environmentally Friendly Antifouling Agent from Pseudomonas Aeruginosa MUT3 by Response Surface Methodology (RSM)

In the present study, the solvent-solvent extraction of phenazine 1-carboxylic acid (PCA) as an environmentally friendly antifouling agent from pseudomonas aeruginosa MUT3 culture was investigated. Accordingly, after screening the extraction ability of various solvents, the combined effects of operating parameters such as solvent type (ethyl acetate, dichloromethane, and n-hexane), solvent percent and mixing time on the PCA extraction process were analyzed using response surface methodology (RSM). As a consequence, ethyl acetate showed higher extraction yield (68%) and the optimum condition for PCA extraction were identified as 150% of solvent and 120 min mixing time. Meanwhile, the extraction yields for dichloromethane and n-hexane were measured by HPLC assay around 48.75 and 25.2%, respectively. The accuracy of the obtained model was proved by 99.90% R2 and 99.84% Adj R2. In addition, the disk diffusion test showed 9.2, 8 and 7.3 mm inhibition zone for ethyl acetate, dichloromethane and n-hexane, respectively. Consequently, the present study provided a great insight into the solvent-solvent extraction of antibiotics from the fermentation broth.