Uncovering the Role of PhzC as DAHP Synthase in Shikimate Pathway of Pseudomonas chlororaphis HT66

DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic acid, and ubiquinone in Pseudomonas chlororaphis. In this study, we identified and characterized one DAHP synthase encoding gene phzC, which differs from the reported DAHP synthase encoding genes aroF, aroG and aroH in E. coli. PhzC accounts for approximately 90% of the total DAHP synthase activities in P. chlororaphis HT66 and plays the most critical role in four DAHP synthases in the shikimate pathway. Inactivation of phzC resulted in the reduction of PCN production by more than 90%, while the absence of genes aroF, aroG and aroH reduced PCN yield by less than 15%, and the production of PCN was restored after the complementation of gene phzC. Moreover, the results showed that phzC in P. chlororaphis HT66 is not sensitive to feedback inhibition. This study demonstrated that gene phzC is essential for PCN biosynthesis. The expression level of both phzC and phzE genes are not inhibited in feedback by PCN production due to the absence of a loop region required for allosteric control reaction. This study highlighted the importance of PhzC and applying P. chlororaphis for shikimate pathway-derived high-value biological production.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot

Background Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. Methods We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. Results P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. Conclusions P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Phytoremediation of Formaldehyde in Indoor Environment With Common House Plants and Pseudomonas Chlororaphis

Low light survivor house plants were assessed for their formaldehyde removal capacity from indoor environment. Low ventilation leading to poor air circulation in indoor environment has become a matter of grave concern as it leads to health issues. Phytoremediation technology is being studied in such situations. The capacity of plants in absorbing indoor pollutants can be enhanced through use of bacteria helping phytoremediation process. The gaseous formaldehyde of about 5 ppm was released into the static chamber of volume 1 m3 . Selected test plants were Aglaonema commutatum, Chlorophytum comosum, Sansevieria trifasciata and Epipremnum aureum. Medium in which plants were growing was inoculated with Pseudomonas chlororaphis, which helps the process of phytoremediation. Activated charcoal was also added in the medium, to increase the absorptive surface. The exposure given was for 24 hours. Experiment was replicated for three times. Air quality in the chamber was monitored on advanced Formaldehyde meter, at the start of the experiment and after 24 hours. Leaves of the plants were analysed by DNPH on LCMS method for quantification of Formaldehyde. Quantification of Formaldehyde from leaves ranged between 0.03–4.7 ppm. Formaldehyde meter showed reduction in formaldehyde quantity ranges from 1.999 to 0 ppm in 24 hours. This clearly indicates that selected plants have enhanced limited capacity of formaldehyde absorption in synergy with Pseudomonas chlororaphis.

Microencapsulation of a Pseudomonas Strain (VUPF506) in Alginate–Whey Protein–Carbon Nanotubes and Next-Generation Sequencing Identification of This Strain

Alginate is a common agent used for microencapsulation; however, the formed capsule is easily damaged. Therefore, alginate requires blending with other biopolymers to reduce capsule vulnerability. Whey protein is one polymer that can be incorporated with alginate to improve microcapsule structure. In this study, three different encapsulation methods (extrusion, emulsification, and spray drying) were tested for their ability to stabilize microencapsulated Pseudomonas strain VUPF506. Extrusion and emulsification methods enhanced encapsulation efficiency by up to 80% and gave the best release patterns over two months. A greenhouse experiment using potato plants treated with alginate–whey protein microcapsules showed a decrease in Rhizoctonia disease intensity of up to 70%. This is because whey protein is rich in amino acids and can serve as a resistance induction agent for the plant. In this study, the use of CNT in the ALG–WP system increased the rooting and proliferation and reduced physiological complication. The results of this study showed that the technique used in encapsulation could have a significant effect on the efficiency and persistence of probiotic bacteria. Whole genome sequence analysis of strain VUPF506 identified it as Pseudomonas chlororaphis and revealed some genes that control pathogens.

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18

Background 1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines. Results In this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h. Conclusions The 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production. Graphical Abstract

Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

Genetic engineering of Pseudomonas chlororaphis Lzh-T5 to enhance production of trans-2,3-dihydro-3-hydroxyanthranilic acid

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.