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.
Highlights
Pseudomonas chlororaphis strain PA23 is a soybean rhizosphere isolate that has been developed as a biocontrol agent to protect canola from stem rot caused by Sclerotinia sclerotiorum [1,2]
Strain PA23 produces a number of compounds, including phenazines (PHZ), pyrrolnitrin (PRN), hydrogen cyanide (HCN), proteases, lipases and siderophores that contribute to its biological control potential [3,4]
In an earlier study examining the relationship between PHZ and PHA production, we discovered that the PHZ-minus mutant P. chlororaphis PA23-63 accumulated greater concentrations of mcl-PHAs compared to the wild type [23]
Summary
Pseudomonas chlororaphis strain PA23 is a soybean rhizosphere isolate that has been developed as a biocontrol agent to protect canola from stem rot caused by Sclerotinia sclerotiorum [1,2]. Strain PA23 produces a number of compounds, including phenazines (PHZ), pyrrolnitrin (PRN), hydrogen cyanide (HCN), proteases, lipases and siderophores that contribute to its biological control potential [3,4]. Phenazine-1-carboxylic acid (PCA), one of the major PHZs produced by the fluorescent pseudomonads, was commercially named as Shenqinmycin. Glucose and glycerol favor the production of PHZ in P. chlororaphis PCL1391 [9], while mannitol and fructose favor the production of PRN in P. protegens strain CHA0 [10]. A number of mutants defective in PHZ or PRN production, or both, were isolated from P. chlororaphis PA23 [12]. These mutations affected P. chlororaphis antibiotic production as well as biocontrol activity [13,14]. PHZ production imparts deep orange pigmentation to P. chlororaphis PA23 in glucose medium, while growth on octanoate results in colonies with a pale-yellow color, indicating reduced PHZ production
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