In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the P. putida wild-type strain and its Pol IV-defective dinB knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type P. putida populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving P. putida cells was observed. The occurrence of 1-bp deletions in P. putida cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the P. putida dinB gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving P. putida cells.During the past several years our understanding of mutation mechanisms has been expanded by the discovery of a new superfamily of DNA polymerases, called the Y family (46). The Y-family polymerases have been identified in prokaryotes, archaea, and eukaryotes. Members of this superfamily are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA with low fidelity and low processivity; many of these enzymes can bypass DNA lesions that block chain elongation by replicative DNA polymerases (21-23). According to the concept of specialized polymerases some of these polymerases are able to copy cognate lesions with high genetic fidelity (22). On the other hand, the specialized DNA polymerases are involved in mutation processes when copying noncognate DNA lesions or normal DNA.In a growth-restricting environment (e.g., during starvation), mutants arise that are able to take over bacterial populations by a process known as stationary-phase mutation (15). One widely discussed idea is that genetic adaptation of microbial populations under environmental stress might be accelerated by stress-induced activation of error-prone DNA polymerases (see, for example, references 16, 50, and 63). In Escherichia coli, two error-prone DNA polymerases, Pol V (UmuDЈC) and Pol IV (DinB), and one high-fidelity DNA polymerase, Pol II, are upregulated during the SOS response (23). SOS induction has also been shown to occur spontaneously in static bacterial populations (62). It has been recently demonstrated that errorprone DNA polymerases Pol IV and Pol V are involved in stationary-phase mutagenesis in E. coli (4,7,42). The involvement of SOS-induced polymerase...
Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.
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...
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