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.

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 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. While 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 phz-boxes 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.

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