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.
Highlights
The genome of the wild-type P. aeruginosa PAO1 strain is relatively large (6.3 Mbp) and contains paralogues of various genes that perform different metabolic activities, thereby allowing for adaption to and exploration of different ecological niches
PAO1 cells were elongated under anaerobic conditions but were restored to their normal rod shape when the class II ribonucleotide reductase (RNR) cofactor vitamin B12 was added to the culture medium (Fig. 1b), indicating that the enzymatic activation of this RNR class restores proper DNA synthesis and replication
Our results clearly demonstrated deficient P. aeruginosa PAO1 DNA replication under anaerobic conditions that was reversed by increasing class III RNR expression levels or gene copy number
Summary
The genome of the wild-type P. aeruginosa PAO1 strain is relatively large (6.3 Mbp) and contains paralogues of various genes that perform different metabolic activities, thereby allowing for adaption to and exploration of different ecological niches. Members of the class III RNR, encoded by the nrdDG genes, carry a stable but oxygen-sensitive glycyl radical plus an iron-sulfur center that catalyzes the reduction of S-adenosylmethionine to generate its radical. This class can only be active under anaerobic conditions[3,4]. The P. aeruginosa strain PAO1 cannot properly grow anaerobically[2,9] This common reference laboratory strain is a spontaneous chloramphenicol-resistant mutant strain that was isolated in 1954 from a patient wound in Melbourne, Australia (American Type Culture Collection ATCC 15692)[10] and was found to be equipped with endogenous virulence-suppression mechanisms and to be highly adapted to growth under laboratory conditions. These pathogenic islands occur in several cystic fibrosis (CF) P. aeruginosa isolates but are absent from P. aeruginosa PAO1
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