Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1

Abstract

Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria.

Fig 1

Pyocyanin production from P. aeruginosa PAO1 and PW5126 grown in PB. Cultures were assayed for OD₆₀₀ and pyocyanin via spectrophotometry. Individual points represent mean values of triplicate samples, and standard error bars are shown.

Fig 2

LC-MS analysis of phenazines produced from P. aeruginosa PAO1 and PW5126. Strains were grown in PB for 24 h at 37°C. Total phenazines were extracted and subsequently resuspended in water-methanol for LC-MS. Ion extraction of m/z 224.75 to 225.5 and m/z 210.75 to 211.5 was used to detect the [M+H]⁺ for PCA (A) and pyocyanin (B), respectively. In contrast to P. aeruginosa PAO1, PW5126 did not produce pyocyanin but generated trace quantities of PCA. Expression of the PA2449 ORF from the lac promoter of pBBR1MCS-5 (pBRL435) restored PCA and pyocyanin biosynthesis in PW5126. Expression of the phzA2B2C2D2E2F2G2 cluster from the lac promoter of pBBR1MCS-5 (pBRL413) allowed PW5126 to produce PCA but not pyocyanin. These data indicate that production of pyocyanin in P. aeruginosa PAO1 requires the PA2449 gene.

Fig 3

Relative expression levels of the phzB1, phzB2, phzM, and phzH genes in P. aeruginosa PAO1 and PW5126. The 5′ regulatory DNA regions consisting of either ∼1,000 bp (phzB1 [A], phzB2 [B], and phzM [C]) or 500 bp (phzH [D]) of the designated phz ORFs were cloned upstream of an E. coli lacZ ORF in a promoter-less pBBR1MCS-5 plasmid. P. aeruginosa strains harboring the phz-lacZ constructs were grown in PB at 37°C and periodically assayed for LacZ activity. Individual points represent mean values of triplicate samples, and standard error bars are shown.

Fig 4

LC-MS analysis of HSLs produced from P. aeruginosa PAO1 and PW5126. Strains were grown in PB at 37°C to an OD₆₀₀ of 0.7. Total HSLs were extracted and subsequently resuspended in acetonitrile for LC-MS analysis. HSLs were detected by ion extraction of their respective [M+H]⁺ m/z's. Chromatograms are shown for authentic 3-oxo-C₁₂-HSL (m/z of 298 to 299) (A) and C₄-HSL (m/z of 172 to 173) (B). Wild-type P. aeruginosa PAO1 did not produce detectable levels of 3-oxo-C₁₂-HSL (C), but C₄-HSL (D) was observed. Conversely, PW5126 produced 3-oxo-C₁₂-HSL (E) and insignificant quantities of C₄-HSL (F).

Fig 5

HPLC analysis of free amino acids present in spent PB media of P. aeruginosa PAO1 and PW5126. Strains were grown in PB at 37°C. At 6 and 24 h postinoculation, free amino acids present in spent media were derivatized and subsequently separated on a Zorbax Eclipse AAA column. Represented chromatograms are shown for an amino acid mixture (A), uninoculated PB (B), PAO1 at 6 h (C), PW5126 at 6 h (D), PAO1 at 24 h (E), and PW5126 at 24 h (F). Glycine concentrations represent mean values of triplicate samples, and standard deviations are shown.

Fig 6

Key pathways of glycine assimilation that might be regulated by PA2449 in P. aeruginosa PAO1. Several genes whose products are predicted to be involved in glycine/serine metabolism were observed to be transcriptionally downregulated in the absence of a functional PA2449 gene. The approximate fold changes in the transcription of individual genes of interest (boldface) are given in parentheses.

Fig 7

Relative expression levels of the gcvP2, hcnA, and metE genes in P. aeruginosa PAO1 and PW5126. The 5′ regulatory DNA regions consisting of ∼1,000 bp (gcvP2 [A]) or 500 bp (hcnA [B]and metE [C]) upstream of the designated gene ORF were fused with the E. coli lacZ ORF. The resulting lacZ constructs were cloned into a promoter-less pBBR1MCS-5 plasmid. P. aeruginosa strains harboring the gcvP2- (A), hcnA- (B), and metE–lacZ (C) constructs were grown in PB at 37°C and periodically assayed for LacZ activity. Individual points represent mean values of triplicate samples, and standard error bars are shown.

Fig 8

Serine dehydratase (encoded by sdaA) is necessary for the optimal growth of P. aeruginosa PAO1 in PB. (A) Pyocyanin production from P. aeruginosa mutants possessing transposon insertions within genes that are potential candidates of regulation by PA2449. Pyocyanin titers represent mean values from triplicate samples, and standard error bars are shown. wt, wild type. (B) The transposon insertion sdaA mutant has a characteristic cessation in growth (arrow) following entry into stationary phase when cultured in PB. Individual points represent mean values derived from triplicate samples, and standard error bars are shown.

Fig 9

Proposed models for PA2449 in regulating the biosynthesis of C₄-HSL and pyocyanin in P. aeruginosa PAO1. (A) Transcription of mexEF-oprN, encoding an efflux pump, is repressed by PA2449. This prevents the efflux of intracellular PQS, thus enhancing the expression of rhl-related genes and phenotypes, including phenazine production. (B) The PA2449 protein directly activates the transcription of the C₄-HSL synthase gene, rhlI, thereby facilitating the expression of the rhl network. (C) PA2449 operates independently of the rhl network to activate the expression of phz-related genes for the biosynthesis of pyocyanin.