Harnessing PGPR inoculation through exogenous foliar application of salicylic acid and microbial extracts for improving rice growth.

Plant-growth-promoting rhizobacteria (PGPR) should effectively colonize along the plant root to enhance the plant and soil health. The present investigation aims to improve the PGPR-mediated plant health benefits through above-ground foliar management. A green fluorescent protein-tagged PGPR strain, Pseudomonas chlororaphis (ZSB15-M2) was inoculated in a nonautoclaved agricultural soil before rice culturing. Salicylic acid and cell extracts of Corynebacterium glutamicum and Saccharomyces cerevisiae as a supply of hormonal and inducer compounds were applied on the foliage of the 10-days-old rice plants and subsequently observed the colonizing ability of ZSB15-M2. The cell extracts of Corynebacteria and yeast showed a 100-fold increase in the ZSB15-M2 population in the rhizosphere of rice, whereas salicylic acid had a 10-fold increase in relation to mock control. The rice root exudates collected after the spraying of salicylic acid and microbial extracts showed significantly enhanced release of total carbon, total protein, total sugar, total amino nitrogen, total nitrogen, and phenol content. In vitro assays revealed that these root exudates collected after exogenous spray of these chemicals enhanced the chemotactic motility and biofilm formation of ZSB15-M2 compared to the control plant's root exudate. Metabolomic analysis of root exudates collected from these rice plants by gas chromatography-mass spectrometry revealed that the Corynebacteria and yeast cell extracts enhanced the divergence of metabolites of rice root exudate. Further, due to these cumulative effects in the rice rhizosphere, the total chlorophyll, total protein, total nitrogen, and total phosphorus of rice were significantly improved. These observations provide insights into the rhizosphere functioning of rice plants as modulated by above-ground treatments with improved colonization of inoculant strains as well as the plant growth.

Quorum sensing and the anaerobic regulator (ANR) control polyhydroxyalkanoate (PHA) production in Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 is a biocontrol agent that, in addition to producing antifungal compounds, synthesizes polyhydroxyalkanoate (PHA) polymers as a carbon and energy sink. Quorum sensing (QS) and the anaerobic regulator (ANR) are required for PA23-mediated fungal suppression; however, the role of these regulators in PHA production is unknown. Strains lacking either QS or ANR accumulated less PHA polymers when propagated on Ramsay's minimal medium (RMM) with glucose or octanoate as the carbon source. In the acyl-homoserine lactone (AHL)-deficient background, all six of the genes in the pha locus (phaC1, phaC2, phaZ, phaD, phaF, phaI) showed reduced expression in RMM glucose, and all except phaC2 were repressed in RMM octanoate. Although changes in gene activity were observed in the anr mutant, they were less pronounced. Analysis of the promoter regions for QS- and ANR-binding consensus sequences revealed putative phzboxes upstream of phaZ and phaI, but no anr boxes were identified. Our findings indicate that altered pha gene expression likely contributes to the lower PHA accumulation in the QS- and ANR-deficient strains, which may be in part indirectly mediated. This study is the first to show that mcl-PHA production is under QS and ANR control.

Enhanced biosynthesis of arbutin by engineering shikimate pathway in Pseudomonas chlororaphis P3.

BACKGROUND: Arbutin is a plant-derived glycoside with potential antioxidant, antibacterial and anti-inflammatory activities. Currently, it is mainly produced by plant extraction or enzymatic processes, which suffers from expensive processing cost and low product yield. Metabolic engineering of microbes is an increasingly powerful method for the high-level production of valuable biologicals. Since Pseudomonas chlororaphis has been widely engineered as a phenazine-producing platform organism due to its well-characterized genetics and physiology, and faster growth rate using glycerol as a renewable carbon source, it can also be engineered as the cell factory using strong shikimate pathway on the basis of synthetic biology. RESULTS: In this work, a plasmid-free biosynthetic pathway was constructed in P. chlororaphis P3 for elevated biosynthesis of arbutin from sustainable carbon sources. The arbutin biosynthetic pathway was expressed under the native promoter Pphz using chromosomal integration. Instead of being plasmid and inducer dependent, the metabolic engineering approach used to fine-tune the biosynthetic pathway significantly enhanced the arbutin production with a 22.4-fold increase. On the basis of medium factor optimization and mixed fed-batch fermentation of glucose and 4-hydroxybenzoic acid, the engineered P. chlororaphis P3-Ar5 strain led to the highest arbutin production of 6.79 g/L with the productivity of 0.094 g/L/h, with a 54-fold improvement over the initial strain. CONCLUSIONS: The results suggested that the construction of plasmid-free synthetic pathway displays a high potential for improved biosynthesis of arbutin and other shikimate pathway derived biologicals in P. chlororaphis.

Zinc and lead detoxifying abilities of humic substances relevant to environmental bacterial species.

The effect of humic substances (HS) and their different fractions (humic acids (HA) and hymatomelanic acids (HMA)) on the toxicity of zinc and lead to different strains of bacteria was studied. All tested bacteria demonstrated a lower resistance to zinc than lead showing minimum inhibitory concentrations of 0.1 - 0.3mM and 0.3-0.5mM, respectively. The highest resistance to lead was characteristic of Pseudomonas chlororaphis PCL1391 and Rhodococcus RS67, while Pseudomonas chlororaphis PCL1391 showed the greatest resistance to zinc. The combined fractions of HS and HA alone reduced zinc toxicity at all added concentrations of the organic substances (50 - 200mgL-1) to all microorganisms, while hymatomelanic acids reduced zinc toxicity to Pseudomonas chlororaphis PCL1391 at 200mgL-1 organic concentration only. The HS fractions imparted similar effects on lead toxicity also. This study demonstrated that heavy metal toxicity to bacteria could be reduced through complexation with HS and their fractions. This was particularly true when the metal-organic complexes held a high stability, and low solubility and bioavailability.

Production of trans-2,3-dihydro-3-hydroxyanthranilic acid by engineered Pseudomonas chlororaphis GP72.

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic ß-amino acid that can be used for the synthesis of chiral materials and nonnatural peptides. The aim of this study was to accumulate DHHA by engineering Pseudomonas chlororaphis GP72, a nonpathogenic strain that produces phenazine-1-carboxylic acid and 2-hydroxyphenazine. First, the phzF deletion mutant DA1 was constructed, which produced 1.91 g/L DHHA. Moreover, rpeA and pykF were disrupted and then ppsA and tktA were co-expressed in strain DA1. The resulting strain DA4 increased DHHA concentration to 4.98 g/L, which is 2.6-fold than that of DA1. The effects of the addition of glucose, glycerol, L-tryptophan, and Fe3+on DHHA production were also investigated. Strain DA4 produced 7.48 g/L of DHHA in the culture medium in the presence of 12 g/L glucose and 3 mM Fe3+, which was 1.5-fold higher than the strain in the original fermentation conditions. These results indicate the potential of P. chlororaphis GP72 as a DHHA producer.

Ag nanoparticles generated using bio-reduction and -coating cause microbial killing without cell lysis.

Cost-effective "green" methods of producing Ag nanoparticles (NPs) are being examined because of the potential of these NPs as antimicrobials. Ag NPs were generated from Ag ions using extracellular metabolites from a soil-borne Pythium species. The NPs were variable in size, but had one dimension less than 50 nm and were biocoated; aggregation and coating changed with acetone precipitation. They had dose-dependent lethal effects on a soil pseudomonad, Pseudomonas chlororaphis O6, and were about 30-fold more effective than Ag(+) ions. A role of reactive oxygen species in cell death was demonstrated by use of fluorescent dyes responsive to superoxide anion and peroxide accumulation. Also mutants of the pseudomonad, defective in enzymes that protect against oxidative stress, were more sensitive than the wild type strain; mutant sensitivity differed between exposure to Ag NPs and Ag(+) ions demonstrating a nano-effect. Imaging of bacterial cells treated with the biocoated Ag NPs revealed no cell lysis, but there were changes in surface properties and cell height. These findings support that biocoating the NPs results in limited Ag release and yet they retained potent antimicrobial activity.