Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA

A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as sitespecific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease Sl is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in ØX174 DNA as the model system. Studies on the changes A → G and G → A at position 587 of ØX174 viral DNA (am 3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

In VitroProtein-primed Initiation of Pneumococcal Phage Cp-1 DNA Replication Occurs at the Third 3′ Nucleotide of the Linear Template: A Stepwise Sliding-back Mechanism

Phage Cp-1 fromStreptococcus pneumoniaemakes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5′ dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5′ end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infectedS. pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication. A mutational analysis of the 3′ terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and thatin vitroinitiation of Cp-1 DNA replication is directed by the third nucleotide of the template. However, the two terminal nucleotides are recovered during the first steps of elongation. A new variant of the sliding-back mechanism for protein-primed initiation, firstly described forBacillus subtilisphage φ29, is proposed to account for the maintenance of Cp-1 DNA ends. The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication.     

Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX² DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung^? (uracil N-glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al., 1980a).Using (5′-³²P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA.Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro. Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas &; Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA.Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.     

Cell-Size Homeostasis and the Incremental Rule in a Bacterial Pathogen

How populations of growing cells achieve cell-size homeostasis remains a major question in cell biology. Recent studies in rod-shaped bacteria support the “incremental rule” where each cell adds a constant length before dividing. Although this rule explains narrow cell-size distributions, its mechanism is still unknown. We show that the opportunistic pathogen Pseudomonas aeruginosa obeys the incremental rule to achieve cell-length homeostasis during exponential growth but shortens its cells when entering the stationary phase. We identify a mutant, called frik, which has increased antibiotic sensitivity, cells that are on average longer, and a fraction of filamentous cells longer than 10 μm. When growth slows due to entry in stationary phase, the distribution of frik cell sizes decreases and approaches wild-type length distribution. The rare filamentous cells have abnormally large nucleoids, suggesting that a deficiency in DNA segregation prevents cell division without slowing the exponential elongation rate.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

Participation of bacteriophage T4 gene 41 in replication repair

The location of the non-essential T4 mutant uvs79, with defective replication repair, is described. After crosses with double mutants dispersed over the early region of T4, a linkage was observed with the double mutant am41 : am42. For more accurate location, crosses were made with single mutants. Uvs79 proved to be located between mutants amC23 and amN81 in gene 41, as shown by 3-point crosses. No genetic complementation with respect to multiplicity reactivation was found between amN81 and uvs79 after a co-infection of an su^? host. Apparently, mutant amN81 is disturbed as to replication repair and, owing to its lack of DNA synthesis, also in replication-dependent recombination repair. Consequently, the product of gene 41 has a function additional to its RNA-primer induction during replication of undamaged DNA. Presumably, the product of gene 41 induces RNA primers opposite DNA regions containing lesions. This capability is believed to be specifically affected by the uvs79 mutation.     

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.     

Mutation to Overproduction of Bacteriophage T4 Gene Products

R9 was isolated as one of several mutations that enhanced the growth of a leaky amber (am) mutant of bacteriophage T4 gene 62 (product required for phage DNA synthesis) under conditions of partial suppression by ribosomal ambiguity. R9 also enhanced the growth of leaky am mutants of some, but not all, other T4 “early” gene functions. R9 mapped between mutations in genes 43 and 62. By using assays involving polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, we observed the following. (i) R9 resulted in an overproduction of many T4 “early” proteins in infected cells. The most pronounced effects of R9 were observed when phage DNA synthesis and/or the functions of maturation genes 55 and 33 were not expressed. (ii) In rifampintreated infected cells, the capacity to synthesize T4 “early” proteins decayed more slowly in the presence of the R9 mutation than in the presence of the wild-type counterpart of R9. R9 appeared to have no effect on the rates of RNA synthesis either during early or late times after infection. The results suggest that the R9 mutation leads to increased functional stability of T4 “early” messengers.     

Termination of Bacillus subtilis chromosome replication as visualized by autoradiography

A mutant of Bacillus subtilis W23 thy his, temperature sensitive for the initiation of rounds of chromosome replication, has been used to investigate the manner in which the two growing points on the replicating circular chromosome approach one another during termination (completion) of the round in progress.The mutant, growing exponentially at 34 °C, was transferred to 45 °C and [³H]thymidine added shortly afterwards. After a further short interval, the specific radioactivity was lowered by a factor of three and rounds of replication were allowed to complete under these conditions. The pattern of heavy and light grain density regions in chromosomal structures made visible by autoradiography was examined, and many with both ends more heavily labelled than their internal region were found. From an analysis of the relative frequency and size of these structures it is concluded that the two growing points in a single chromosome can continue to move at similar rates (within a factor of 2) as they approach one another to within < 10% of the length of the whole circular chromosome in the vicinity of the terminus. The data obtained do not prove that all chromosomes behave in this manner, but are consistent with such an interpretation.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota

Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn²⁺ ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson–Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg²⁺ reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson–Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Fate of Donor Deoxyribonucleic Acid in a Highly Transformation-Deficient Strain of Haemophilus influenzae

A transformation-deficient strain of Haemophilus influenzae (efficiency of transformation 10⁴-fold less than that of the wild type), designated TD24, was isolated by selection for sensitivity to mitomycin C. In its properties the mutant was equivalent to recA type mutants of Escherichia coli. The TD24 mutation was linked with the str-r marker (about 30%) and only weakly linked with the nov-r2.5 marker. The uptake of donor deoxyribonucleic acid (DNA) was normal in the TD24 strain, but no molecules with recombinant-type activity (molecules carrying both the donor and the resident marker) were formed. In the mutant the intracellular presynaptic fate of the donor DNA was the same as that in the transformation-proficient (wild-type) strain, and the radioactive label of the donor DNA associated covalently with the recipient chromosome in about the same quantity as in the wild type. However, many fewer donor atoms were associated with segments of the mutant's recipient chromosome as compared with segments of the wild-type chromosome. In the mutant the association was accompanied by complete loss of donor marker activity. The lack of donor marker activity of the donor-recipient complex of DNA isolated from the mutant was not due to lack of uptake of the complex by the second recipient and its inability to associate with the second recipient's chromosome. Because the number of donor-atom-carrying resident molecules was higher than could be accounted for by the lengths of presynaptic donor molecules, we favor the idea that the association of donor DNA atoms with the mutant chromosome results from local DNA synthesis rather than from dispersive integration of donor DNA by recombination.     

Processing of filamentous phage pre-coat protein: Effect of sequence variations near the signal peptidase cleavage site

The amber lesion of am8H1, the only conditional lethal mutant in a filamentous phage coat protein gene, lies two codons after the signal peptidase cleavage site (Boeke & Model, 1979). We sequenced the DNA of 15 independently isolated pseudorevertants of this mutant. We studied the production of unprocessed and processed coat protein in pseudorevertant-infected cells and in amber mutant-infected suppressor strains. These studies show that serine, glutamine, tyrosine or leucine residues can replace the glutamic acid residue found in the wild-type coat protein at position 2. Reversion to tyrosine or leucine was always accompanied by a second mutation, which leads to the replacement of asparagine by aspartic acid at position 12. Leucine and, to a lesser extent, tyrosine seem to inhibit processing since pre-coat protein accumulates in the infected cells. Filamentous phage particles were shown to migrate on agarose gels with a mobility that reflects the charge of the “external”, N-terminal domain of their major coat protein.     

T1 Genes Which Affect Transduction

Amber mutants of T1 were grown on each of three donor strains which were identical except that they carried different suppressors: respectively, supD, supE, and supB. The efficiency with which the mutants were able to transduce was tested after growth on each donor. In general, it was found that functions which control the synthesis of phage DNA usually caused significant increases in the efficiency of transduction (EOT). A few mutants located in genes essential for head production caused significant decreases in EOT. The presence of a particular suppressor in a donor can cause noteworthy changes in the EOT by certain of the mutant phages. Amber mutations in gene 3 of T1 were extremely sensitive to the particular suppressor present in the donor, showing a 17-fold decrease in EOT compared with other mutants after growth in donors with the supD suppressor and a 75-fold increase after growth in supE donors. Increases in EOT by early genes of T1 do not seem to be caused by a lack of competition of bacterial DNA with phage DNA during packaging since, in most instances, infective phage were produced in relatively normal amounts compared with wild-type T1. Phage DNA synthesis and degradations of the host chromosome are closely coupled in T1 infections; we believe that increases in EOT by mutants of early functions are due to inefficient degradation of the host chromosome.     

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and uv-induced mutagenesis.

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol^?) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol^? mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol⁺ parent. The presence of FP18 or FP111 in the Pol^? mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant.The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla.The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.