Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

Abstract

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the β clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

RecA and Specialized Error-Prone DNA Polymerases Are Not Required for Mutagenesis and Antibiotic Resistance Induced by Fluoroquinolones in Pseudomonas aeruginosa

“…Deletion of recA in CIP R spontaneous mutants was obtained using the pDM4 ∆recA derivative (Table S1) as described [65]. Single and double deletion mutants in recA, polB, dinB and/or imuC of the reference strain PAO1 (Table S1) were previously generated [65,66].…”

“…Conjugations were performed as previously described [66]. The frequency of transconjugants for each donor/recipient pair was calculated as the ratio between the number of transconjugant colonies obtained on selective agar plates and the total number of colony forming units (CFU) for the P. aeruginosa recipient strain, determined by plating serial dilutions of the conjugation mixture onto LB agar plates supplemented with 10 µg/mL nalidixic acid to counter-select the E. coli S17.1 λpir donor strain [67].…”

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa

“…The introduction of a plasmid expressing an FtsZ-GFP fusion protein (pME ftsZ-GFP ; Table S2) ( 35 ) allowed demonstrating that the Z-ring is assembled and correctly localized at mid-cell in double and triple asmA -like gene mutants (Fig. S5), leading to hypothesizing that depletion of essential AsmA-like proteins does not directly affect the divisome assembly and, plausibly, cell constriction.…”

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters

“…Homologs of YhdP and TamB are found throughout Enterobacterales and more distantly related members of the AsmA family are widespread in gram-negative bacteria (97). In fact, TamB has recently been shown to play a role in anterograde intermembrane phospholipid transport in the diderm Firmicute Veillonella parvula (40,98) and four AsmA members play a role in phospholipid transport in Pseudomonas aeruginosa (41). Structural predictions of YhdP and TamB are strikingly similar between species (32,33).…”

“…This study also directly demonstrates that a phosphate containing substrate, putatively assigned to be phospholipids, can be crosslinked to the hydrophobic groove of YhdP (39). The role of YhdP, TamB, and YdbH in phospholipid transport may be widely conserved as recent evidence implicates TamB in anterograde phospholipid transport in Veillonella parvula, a diderm Fermicute (40) and YhdP, TamB, YdbH, and PA4735 in intermembrane phospholipid transport in Pseudomonas aeruginosa (41).…”