Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.

Kitagawa, Yuichi; Akaboshi, Eiko; Shinagawa, Hideo; Horii, Toshihiro; Ogawa, Hideyuki; Kato, Takao

doi:10.1073/pnas.82.13.4336

Cited by 128 publications

(78 citation statements)

References 38 publications

(30 reference statements)

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“…However, quantitative mutagenesis assays with MMS gave essentially similar results (Fig. 8) (30). Likewise, posttranslational cleavage of UmuD to mutagenically active UmuD' is also inefficient (8,56).…”

Section: Resultssupporting

confidence: 49%

Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremel useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

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Section: Resultssupporting

confidence: 49%

Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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“…A putative promoter region of the rulAB genes is similar to the promoter region of the umuDC genes characterized in E. coli (31) (Fig. 4A).…”

Section: Resultsmentioning

confidence: 95%

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

et al. 2005

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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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“…The sequence of the SOS box in S. typhimurium was identical to that of the SOS box upstream of umuD in E. coli but was located 4 bp closer to the initiation codon. In E. coli there is a second but apparently nonfunctional SOS box beginning 4 bp upstream from the first box (21) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon

Smith¹,

Koch²,

Franklin³

et al. 1990

J Bacteriol

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In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDCrelated sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDt restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs' (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24-Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence' of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25-to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore,' that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago.' These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S.typhimurium and E. coli.

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Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.

Cited by 128 publications

References 38 publications

Isolation and characterization of novel plasmid-encoded umuC mutants

Isolation and characterization of novel plasmid-encoded umuC mutants

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

Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon

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