The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30-84

Abstract

Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30-84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surface-attached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30-84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30-84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage-derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30-84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.

Fig 1. Production of non-attached biofilm and eDNA by 30-84ZN (no phenazine) 30-84PCA, 30–84 wild type, and 30-84O*.

(A) Bacteria were grown in AB minimal media + 2% casamino acid in static plates for up to 72 h. Biofilm matrix production by P. chlororaphis 30–84 and derivatives at 48 and 72 h was quantified by weight. (B) Visualization of biofilm formed after culturing in static plates for 72 h and being collected by centrifugation. (C) Production of eDNA by different derivatives was quantified using the double stranded DNA quantifying fluorescent dye assay from Invitrogen. The data are the average of three biological replicates and error bars indicate the standard deviation.

Fig 2. Production of biofilm matrix and eDNA in the presence or absence of DNase I.

(A) Biofilm production after bacterial strains were grown in static plates in AB minimal media + 2% casamino acid either with or without DNase I for 72 h and biofilms were collected by centrifugation. Data are the weight of the biofilm matrix. (B) Production of eDNA in the presence or absence of DNase I in the same experiment. The data represent the average of at least three biological replicates and error bars indicate the standard deviation.

Fig 3. The number of differentially expressed genes in 30-84PCA, wild type and 30-84O* compared with the 30-84ZN.

Differential expressed genes are those exhibiting over twofold change and a P value < 0.05. (A) Phenazine Induced and Suppressed genes are those that are expressed in at a higher or lower level, respectively, by the phenazine producing strains compared to 30-84ZN. (B) Venn diagram showing the number of genes differentially expressed in 30-84PCA, wild type and 30-84O* compared with 30-84ZN.

Fig 4. The effect of holin mutation on eDNA and biofilm matrix production.

(A) Production of eDNA by P. chlororaphis 30–84 wild type and the holin mutant after growing 48 and 72 hr in static culture. (B) Biofilm matrix production by 30–84 wild type and the holin mutant at 72 h. The data are the average of three biological replicates and error bars indicate the standard deviation.

Fig 5. Possible metabolic fates for chorismic acid in P. chlororaphis 30–84.

Chorismic acid is produced via the shikimic acid pathway and is a metabolic precursor for a number of different end products in pseudomonads, including phenazines (indicated by solid lines). The negative symbols (-) indicate competing pathways in which transcript levels for key genes are reduced in 2-OH-PCA producing strains compared to 30-84ZN (Table 4).