A DNA Polymerase V Homologue Encoded by TOL Plasmid pWW0 Confers Evolutionary Fitness on Pseudomonas putida under Conditions of Environmental Stress

Abstract

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P. putida. When rulAB-carrying cells from an 8-day-old culture were mixed with Pol V-negative cells from a 1-day-old culture, cells derived from the aged culture out-competed cells from the nonaged culture and overtook the whole culture. At the same time, bacteria from an aged culture lacking the rulAB genes were only partially able to out-compete cells from a fresh overnight culture of the parental P. putida strain. Thus, in addition to conferring resistance to DNA damage, the plasmid-encoded Pol V genes significantly increase the evolutionary fitness of bacteria during prolonged nutritional starvation of a P. putida population. The results of our study indicate that RecA is involved in the control of expression of the pWW0-encoded Pol V.In natural environments, bacteria are faced with many different types of stresses. Among them, nutritional stress is the most common for bacteria occupying various water and soil habitats (71). Additionally, environmental bacteria are frequently exposed to cycles of drying and rehydration. Microorganisms living in geographic areas where the temperature sometimes drops below zero are faced with series of freezing and melting. Many bacteria, especially those living in a phyllosphere, are exposed to UV irradiation. The UV wavelengths that reach the earth's surface can cause direct DNA damage by inducing the formation of DNA photoproducts whose accumulation can be lethal to cells through the blockage of DNA replication and RNA transcription (45). Some data published already more than 20 years ago indicate that freeze-thaw stress and drying cycles can also cause DNA damage which is mutagenic to a bacterium (3,10,65,73). It has also been shown that oxidative DNA damage generated from endogenous metabolism in growth-arrested cells accumulates during stasis (7,8).In Escherichia coli, DNA polymerases Pol II, Pol IV, and Pol V are induced as part of the SOS regulon in response to DNA damage (23). The LexA repressor binds to a 20-bp consensus sequence in the operator region of the SOS regulon genes, suppressing their expression. RecA/single-stranded DNA nucleoprotein filament functions as a coprotease that stimulates LexA autoproteolysis (37). The timing, duration, and level of expression can vary for each LexA-regulated gene, depending on the location and binding affinity of the SOS boxes relative to the strength of the promoter. Therefore, some genes may be part...

Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida

“…The plasmid pUCNotrulAB (74) carrying the rulAB operon, originated from the catabolic TOL plasmid pWW0 This study pUCNotrulAB pUC18Not containing rulAB genes 74 pBK-miniTn7-⍀Sm1rulAB rulAB genes from pUC18NotrulAB in NotI site of pBK-miniTn7-⍀SmI This study pKTpheA56ϩA…”

“…The P. putida strain carrying the rulAB genes in its chromosome was designated as PaWRulAB. The expression of the rulAB genes in the P. putida chromosome was verified by using a UV irradiation survival assay described previously (74). In order to obtain P. putida GO repair-deficient strains carrying the rulAB genes in their chromosomes (strains PaWMutYRulAB, PaWMutMRulAB, and PaWMutTRulAB), PaWRulAB was used as a recipient for the replacement of original mut sequences with the interrupted ones by homologous recombination, as described above.…”

“…Instead, DNA polymerase PolV homologues are frequently encoded by naturally occurring conjugative plasmids in these bacteria (e.g., see reference 74 and references therein). Our previous studies have revealed that the PolV homologue RulAB from TOL plasmid pWW0 increases the evolutionary fitness of starving P. putida bacteria (74). This raises the question of whether this polymerase could be involved in stationary-phase mutagenesis via a mechanism which is dependent on oxidative damage of DNA.…”

Culture History and Population Heterogeneity as Determinants of Bacterial Adaptation: the Adaptomics of a Single Environmental Transition

“…The genes carried in extragenomic elements have the potential to alter not only the physiology and metabolism of a cell but also its capacity to generate mutations. The error-prone DNA polymerases can be encoded in plasmids and contribute to mutational adaptation (122,324). Together with the variation in plasmid content itself as a result of environmental stresses (358), a bacterial population may be heterogeneous because of these plasmid-encoded factors.…”

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes

“…Expression of these genes in E. coli and Ps. putida responds to DNA damage (Kitagawa et al, 1985;Tark et al, 2005), suggesting that umuDpR-umuC * genes from pUM505 may also respond likewise. Supporting this hypothesis, when total RNA from PS.…”