A single point mutation in class III ribonucleotide reductase promoter renders Pseudomonas aeruginosa PAO1 inefficient for anaerobic growth and infection

Abstract

Pseudomonas aeruginosa strain PAO1 has become the reference strain in many laboratories. One enzyme that is essential for its cell division is the ribonucleotide reductase (RNR) enzyme that supplies the deoxynucleotides required for DNA synthesis and repair. P. aeruginosa is one of the few microorganisms that encodes three different RNR classes (Ia, II and III) in its genome, enabling it to grow and adapt to diverse environmental conditions, including during infection. In this work, we demonstrate that a lack of RNR activity induces cell elongation in P. aeruginosa PAO1. Moreover, RNR gene expression during anaerobiosis differs among P. aeruginosa strains, with class III highly expressed in P. aeruginosa clinical isolates relative to the laboratory P. aeruginosa PAO1 strain. A single point mutation was identified in the P. aeruginosa PAO1 strain class III RNR promoter region that disrupts its anaerobic transcription by the Dnr regulator. An engineered strain that induces the class III RNR expression allows P. aeruginosa PAO1 anaerobic growth and increases its virulence to resemble that of clinical strains. Our results demonstrate that P. aeruginosa PAO1 is adapted to laboratory conditions and is not the best reference strain for anaerobic or infection studies.

Figure 1

Elongated cell morphology of P. aeruginosa PAO1 under anaerobic conditions. (a) Fluorescence micrographs of P. aeruginosa PAO1, PAET1, PAET2, PA14, PA54, and PA166 cells grown anaerobically (to an OD₅₅₀ of approximately 0.5) and stained with the LIVE/DEAD assay. (b) RNR mutant strains (∆) of P. aeruginosa PAO1 (ETS102 (∆nrdJ), ETS103 (∆nrdD), and ETS125 (∆nrdJ∆nrdD) that contain complementation vectors (pETS159, pBBR1-NrdJab; pETS160, pBBR1-NrdDG; or pETS197, pUCP20T-DG)). Cells were stationary-cultured in the presence or absence of 1 μM vitamin B_12. Live cells stained with the LIVE/DEAD assay were visualized under a fluorescence microscope (1,000X magnification). The images are representative of three independent experiments with three replicates each. Cell length (mean ± standard deviation) was determined with ImageJ software. Scale bars, 10 μm.

Figure 2

Relative nrd gene expression in clinical isolates and laboratory P. aeruginosa strains. (a) Differences in the expression levels of nrd genes in PAO1 and in clinical isolate cells grown anaerobically or aerobically at mid-exponential phase (OD₅₅₀ = 0.5). The values in bold represent up-regulated nrdD gene. The log fold-change is shown as the mean ± standard deviation of three independent experiments. (b) Different RNR (nrd) gene expression levels in cells grown anaerobically versus cells grown aerobically. The induction expression factor of the nrdA (in white), nrdJ (in gray) and the nrdD (in black) in clinical isolates compared to PAO1 strain. The error bars represent the standard error of the mean. Significantly different from P. aeruginosa PAO1 in an unpaired t-test (*P < 0.05 and **P < 0.0001).

Figure 3

nrdD promoter variations in different P. aeruginosa strains. (a) Multiple alignment of the nrdD promoter region sequences from different P. aeruginosa PAO1 strains (PAO1-CECT, PAO1-UW, PAO1-JPN) and from strains isolated from patients with CF (PAET) and with acute infections (extensively drug resistant; XDR). P. aeruginosa PAO1-PAdb, PA7, PA14 and PA-LESB58 sequences, along with P. fluorescens and P. alcaligenes sequences, were obtained from the Pseudomonas database. The gray background indicates a mismatch in the sequence. Twenty nucleotides of PnrdD, corresponding to the Anr-Dnr binding box that predicted the different Pseudomonas strains, are magnified. The percentage conservation is indicated in the bars and grey background indicates a mismatch in the consensus sequence. (b) Relative fluorescence units of nrdD promoter activity (pETS136-C (PnrdD of PAO1) or pETS196-T (PnrdD C > T)) in P. aeruginosa PAO1, PAET1, PAET2, PA166, PA54 and PA14 strains. P. aeruginosa PAO1 isogenic ∆anr and ∆dnr mutants were used as controls for Anr/Dnr binding. A plasmid carrying an extra copy of the dnr gene (pETS195) was used to complement the ∆dnr mutation. Three independent experiments were performed, and the mean ± standard deviation is shown. *, values for pETS196-T (PnrdD C > T) significantly differ from those for pETS136 (PnrdD of PAO1) in an unpaired t-test (P < 0.05).

Figure 4

Role of NrdDG in infection. Mean fluorescence intensity values (sum of intensity/area of measurement) in individual embryos infected with (a) P. aeruginosa PAO1 containing the pETS130, pETS134 (PnrdA), pETS180 (PnrdJ), or pETS136 (PnrdD) vectors or (b) with different P. aeruginosa strains (PAO1, PA14 and PAET1) containing the pETS136-C and pETS196-T vectors over 24 h post-infection (hpi). The data represent three independent experiments, with 100 fish analyzed per strain. Statistics were performed to compare strains carrying pETS196-T with strains carrying pETS136-C in an unpaired t-test (*P < 0.05 and **P < 0.001). (c) Fluorescent and overlaid images of D. rerio embryos infected with PAO1, PA14 and PAET1 containing the pETS136-C or pETS196-T fluorescent reporter vectors at 16 hpi. Fluorescence was visualized with a fluorescence microscope (Leica MZ16F), quantified with Nikon Nis-element software and processed with ImageJ software. Bars represent 500 μm. (d) Kaplan-Meier plots of a survival experiment in D. rerio infected with different P. aeruginosa strains (P. aeruginosa PAO1, PA14, ETS103 (∆nrdD), ETS127 (∆nrdD PAO1 NrdDG⁺), ETS128 (∆nrdD PAO1 NrdDG (C > T)⁺), ETS129 (PAO1 NrdDG⁺) and ETS130 (PAO1 NrdDG (C > T)⁺)). The graph corresponds to a single representative experiment from a total of three independent experiments performed (each using 100 fish per condition). The number of hours’ post-infection (hpi) at which 50% of zebrafish survived are listed with standard deviation. Statistics were performed to compare different strains to P. aeruginosa PAO1 in a Mantel-Cox test. *P < 0.05, **P  > 0.001 and ****P < 0.0001; N.S., no significant difference).