Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz-) and Pyrrolnitrin (prn-) Defective Mutants of Pseudomonas chlororaphis PA23

Abstract

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

Forest Tree Associated Bacterial Diffusible and Volatile Organic Compounds against Various Phytopathogenic Fungi

“…The PCR results suggested that ST-TJ4 contains genes for the biosynthesis of PCA and PRN. Phenazine type of antibiotics, such as PCA, are particularly active against oomycetes [43,44]. Park et al [45] demonstrated that PRN produced by P. chlororaphis O6 plays a pivotal role in inhibiting Phytophthora infestans, which causes tomato late blight disease.…”

Colonization and degradation of polyhydroxyalkanoates by lipase-producing bacteria

“…P. chlororaphis PA23-63-1 is able to grow in RMM containing different vegetable oils as a sole carbon source and produce PHAs (Sharma et al 2018). The identity of this strain was confirmed on the basis of genome sequencing (Loewen et al 2014).…”

“…Bacterial cultures, media and growth conditions P. chlororaphis PA23 is a plant growth promoting bacterium isolated from the rhizosphere of soybeans, capable of suppressing growth of the fungal pathogen Sclerotinia sclerotiorum due to its ability to synthesize and secrete two anti-fungal compounds, phenazine and pyrrolnitrin (Fernando et al 2007;Savchuk and Fernando, 2004). P. chlororaphis PA23-63-1 is a mutant strain of P. chlororaphis PA23 that is defective in phenazine and pyrrolnitrin production, and is also able to synthesize mcl-PHAs (Sharma et al 2018). P. chlororaphis PA23-63-1 was kindly provided by All bacteria used in this study were grown and purified on LB plates incubated at 30 o C for 24 hours (h).…”

“…Out of seven esterases/lipases, three were secretory proteins with signal peptides (EY04_17885, EY04_21540, and EY04_32435). In addition to this, an ATP-binding cassette for export of lipase (EY04_16115) was also present (Sharma et al 2018). Six esterases/lipases encoding genes with signal peptides were identified in the A. lwoffii (Mok et al 2016).…”

Tung Oil-Based Production of High 3-Hydroxyhexanoate-Containing Terpolymer Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) Using Engineered Ralstonia eutropha

“…Ralstonia eutropha, one of the most well-known PHA-producing bacteria, can accumulate PHA at up to 80% of its dry cell weight [ 24 , 25 , 26 ]. However, the wild-type strain can synthesize only the P(3HB) monomer.…”