Vol. 171, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 975-9820021-9193/89/020975-08$02.00/0Copyright C) 1989, American Society for Microbiology

Effect of Degradative Plasmid CAM-OCT on Responses ofPseudomonas Bacteria to UV Light

DANI L. McBETH

Department of Microbiology, City University ofNew York Medical SchoollSophie Davis School of Biomedical Education,City College, 138th Street at Convent Avenue, New York, New York 10031

Received 20 July 1988/Accepted 8 November 1988

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa,in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes.

CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strainswithout CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesisof plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forwardmutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forwardmutation assay. These results suggest major differences in DNA repair between the two species and highlightthe presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carryingchromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and theeffect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P.putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesismutations, suppressing some and producing no suppression of others. On the basis of the results reported hereand results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may

differ either in regulation or in the number and functions of UV response genes encoded.

Several plasmids from a number of incompatibility groupsencode proteins capable of modulating the UV responses ofthe hosts (12, 25). The best studied of these is the R46derivative pKM101, which increases both survival and mu-tagenesis of UV-irradiated Escherichia coli, Salmonella ty-phimurium, and other enterobacteria by encoding two genes,mucA and mucB, which are analogs of the E. coli genesumuC and umuD (22, 26). Another plasmid, pMG2, an IncP2plasmid of Pseudomonas species, has been reported toencode a new polymerase activity responsible for increasingsurvival and mutagenesis in host cells (17, 18). Some otherIncP2 plasmids are also known to encode UV responsefunctions (13). Here I report on one such plasmid, CAM-OCT, a >300-kilobase megaplasmid which, besides modu-lating UV responses, also carries genes for alkane utiliza-tion, camphor utilization, and conjugative transfer (5, 10,12).The repair of UV-induced DNA damage has been studied

in only a few procaryotes, such as E. coli (26), Bacillussubtilis (19), other Bacillus species (8), Neisseria gonor-rhoeae (4), Streptococcus pneumoniae (9), and Pseudomo-nas aeruginosa (6, 15, 16). Detailed knowledge of theinduction, regulation, and mechanisms of UV-induced DNAdamage repair exists only for the E. coli system, in whichsuch repair is part of the inducible, error-prone SOS re-sponse (for a review, see reference 26). Evidence indicatesthat the E. coli model system is not wholly applicable to allprocaryotes. The apparent differences range from differ-ences in regulatory circuits (8, 19) to a fairly commonabsence of error-prone repair, both in other bacterial fami-lies (4, 9) as well as among different enterobacteria (22).Previous studies with P. aeruginosa have noted the UVsensitivity of this species (16), and recent studies on the recAgene of P. aeruginosa suggest that DNA repair functions inthese organisms may be regulated in a manner similar to thatseen in E. coli (11, 20).

In this study, the UV responses of P. aeruginosa and

Pseudomonas putida in the presence and absence of plasmidCAM-OCT were compared. In the absence of CAM-OCT,both the survival and the mutagenesis responses of the twospecies differed markedly, whereas in the presence of theplasmid, the responses were more similar. A number of UVsurvival and mutagenesis mutants of P. putida were isolated.CAM-OCT was found to suppress the mutations in all UVsurvival mutants tested and to at least partially suppress fiveof six low-mutagenesis mutations.

MATERIALS AND METHODS

Bacterial strains and plasmids. Most of the bacterial strainsused are listed in Table 1. Strains serving as donors fortransfer of plasmids into various strains included PpS145 forCAM-OCT and MRP119 for pMRP119. UV response mu-tants of P. putida are all derivatives of strain PpS102 and arenamed in Fig. 2 through 5 and Table 5.Media and culture conditions. Minimal medium (PA),

complete medium (TYE), and the conditions for growth oncamphor have been described elsewhere (2, 3, 10). Antibi-otics were added at the following concentrations (in micro-grams per milliliter): carbenicillin, 750 for P. putida and 500for P. aeruginosa; neomycin, 50; and rifampin, 75.

Genetic methods. (i) Bacterial matings. All conjugationswere performed by plate mating. Stationary-phase recipientsand exponential-phase donors were mixed and placed on thesurface of a TYE plate. After incubation overnight at 320C,cells were streaked on the appropriate selective medium.Selection included camphor utilization for CAM-OCT andneomycin resistance for pMRP119.

(ii) N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Pro-cedures for mutagenesis were basically as described byDrake and Baltz (7). A culture of PpS102 was grown tostationary phase. Cells were pelleted, washed with 0.1 Msodium citrate (pH 5.5), pelleted, and then resuspended in0.1 M sodium citrate (pH 5.5) containing N-methyl-N'-nitro-N-nitrosoguanidine at 100 [ug/ml. After 25 min at room

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TABLE 1. Bacterial strains and plasmids

Strain or Genotype or Source orplasmid characteristics reference

StrainP. aeruginosaPAC5 his-5 P. ClarkePAS106 his-106 J. Shapiro

P. putidaPpS102 trp-102 1PpS145 met-145 (CAM-OCT) 10PpS445 trp445 alcA437 2

E. coli MRP119 lacZ(Am) trp(Am) his J. ShapirorpsL (pMRP119)

PlasmidCAM-OCT IncP2; camphor' 5, 10

alkane' UV'pMRP119 IncP1; Tcr Kmr Tra' J. Shapiro (unpub-

CbS Am mutant of lished data)RP1

temperature, cells were again pelleted, washed twice withmedium, and suspended in TYE. Viable counts were mea-

sured by plating appropriate dilutions on TYE plates andcomparing counts with those obtained for unmutagenizedcontrols. Survival under these conditions was generallyabout 10%. Cultures were then grown for 3 h before platingfor individual colonies to be screened for mutants as de-scribed below.UV survival and mutagenesis. (i) UV survival measure-

ments. Cells were grown to a density of 2 x 108 CFU/ml.Portions (50 RA) of a series of 10-fold dilutions were spreadon TYE plates, and the plates were UV irradiated. Dosedelivery was based on different exposure times to a UVsource kept at a constant height. The UV source was

calibrated with a Blak-Ray UV light meter. After overnightgrowth at 320C for P. putida or 370C for P. aeruginosa,individual colonies were counted, and the numbers were

used to calculate the percentage of survivors at each UVdose tested. Each experiment was repeated three times, andsurvivals are expressed as the mean and standard error ofthe mean for each UV dose tested.

(ii) Mutagenesis measurements. To quantitate reversion ofauxotrophic mutations, 2 x 107 to 5 x 107 cells fromundiluted exponential-phase cultures were spread on PAplates with glucose as the carbon source. Plates were UVirradiated with various doses and incubated for 2 days beforecounting of revertants. Reversion to carbenicillin resistance(Cbr cells) of strains carrying the bla(Am) mutation inpMRP119 was quantitated similarly except that cells were

plated on TYE plates with carbenicillin. To measure forwardmutation to rifampin resistance (Rif' cells), cultures were

grown in TYE at 320C for P. putida and 370C for P.aeruginosa to 1 x 108 to 2 x 108 cells per ml. Portions of 5ml were UV irradiated at various doses, and then thecultures were incubated for 1 h with shaking at the sametemperature as before. Then 0.1-ml portions were plated onTYE plates containing rifampin. After overnight incubation,mutants were counted. Mutation frequencies are expressedas mutants per surviving CFU at each UV dose tested.Tables represent data from three experiments expressed as

the mean reversion frequency and standard error of themean.Mutant isolation. (i) UV-sensitive mutants. Immediately

after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis ofPpS102, cells suspended in TYE were divided into 25 equal

portions and incubated for 3 h at 32TC. Each culture wasplated for isolated colonies on TYE plates. Individual colo-nies were transferred with toothpicks to TYE plates, incu-bated overnight, and replica plated to TYE plates, whichwere then UV irradiated (40 J/m2). Patches that survivedirradiation less well than did control patches on the sameplates were restreaked for isolated colonies, retested, andthen streaked once again. From about 25,000 coloniesscreened, 82 UVS mutants were isolated. Since all mutantsused in this study came from different initial samples, theyshould not have been sibling strains. Seven mutants werechosen at random for analysis of the effects of CAM-OCT.One additional strain, PpM10, was chosen because it alsoexhibits a temperature-sensitive phenotype at 370C. Rever-tants selected for methyl methanesulfonate resistance andthen purified and retested always regained UV resistance,although not always at wild-type levels (data not shown).This finding suggests that the strains carry single mutationsin UV response genes.

(ii) UV-inducible mutagenesis mutants. UV-inducible mu-tagenesis mutants were isolated by screening for mutantswith altered abilities to revert the bla(Am) mutation carriedon pMRP119. Samples of the 25 small cultures grown forscreening UV' mutants were mixed with exponential-phasecells of MRP119. Matings were performed as describedabove, and then neomycin-resistant PpS102 transconjugantscontaining the pMRP119 plasmid were selected. Mutantswhich failed to revert the bla(Am) mutation or reverted athigher levels were identified by replica plating of individualpatches to TYE containing carbenicillin, UV irradiation (10J/m2), incubation for 48 h, and then screening for patches inwhich the relative numbers of Cbr revertants differed fromthe numbers for control strains present on the same plates.All such mutants were restreaked and retested at least twotimes. Again, all mutants used came from different smallcultures and should therefore have been independent, non-sibling isolates. From the 25,000 colonies screened, 8 mu-tants with elevated levels of mutagenesis and 30 with lowmutagenesis rates were isolated. Of these, two of the formerand six of the latter were chosen at random to test the effectsof CAM-OCT on their mutant phenotypes.

RESULTS

Survival after UV irradiation. Survival after UV irradiationwas compared in two sets of isogenic strains. Each setconsisted of derivatives carrying no plasmid or CAM-OCT.In the PpS102 background, the presence of CAM-OCTsignificantly aided in cell survival in comparison with resultsfor bacteria with no plasmid (Fig. 1). UV protection con-ferred by CAM-OCT resulted in survival increases of 2- to10-fold, depending on the UV dose. Similar results wereobtained with other P. putida derivatives in the PpS445background (data not shown). P. aeruginosa also was pro-tected against UV lethality by CAM-OCT, but the degree ofprotection conferred was much greater than that seen in P.putida (Fig. 1). In the case of the PAC5 derivatives, theeffect of CAM-OCT resulted in survival increases of 20- to500-fold along the range of UV doses analyzed. Similarresults were seen in the PAS106 background (data notshown). The differences between the two species in themagnitude of the protective effect reflected a greater UVsensitivity of P. aeruginosa strains without CAM-OCT thanof P. putida strains without CAM-OCT.

Mutagenesis after UV irradiation. Recovery from UV-induced DNA damage in some bacteria is accompanied by

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FIG. 1. Survival after UV irradiation of P. putida PpS102 (A), P. aeruginosa PAC5 (B), and derivatives carrying CAM-OCT. Symbols:0, cells carrying no plasmid; *, cells carrying CAM-OCT.

increased mutation frequencies due to the action of muta-genic repair pathways (26). Reversion to prototrophy ofauxotrophic P. putida and P. aeruginosa strains was ana-lyzed as a measure of mutagenesis and thus of the presenceof error-prone repair. UV treatment did increase the rever-sion frequency of P. putida mutants, and CAM-OCT in-creased this frequency 2- to 10-fold (Table 2). The presenceof CAM-OCT also increased UV-induced reversion of P.aeruginosa auxotrophic mutants. In fact, UV-induced rever-

TABLE 2. Reversion to prototrophy

Protrophic revertants/Parental Mutant CAM- 107 survivors + SEMWstrain allele OCT

Spontaneous +UV

PpS102 trp-102 - 0.13 ± 0.01 2.93 ± 0.08+ 0.19 ± 0.03 6.45 ± 0.94

PpS445 trp445 - <0.40 ± 0.01b 1.32 ± 0.04+ 0.30 ± 0.03 11.1 ± 1.3

PAC5 his-5 - 0.16 ± 0.05 <0.07 ± 0.02b+ 1.63 ± 0.20 79.1 ± 11.1

PAS106 his-106 - 0.40 ± 0.10 0.30 ± 0.10+ 0.82 ± 0.08 19.3 ± 5.2

a Data from three experiments were used to calculate the reported values.Cells were irradiated at 10 JRm2.

b Lack of detectable reversion; value is that obtainable from a singlereversion event.

sion to prototrophy of P. aeruginosa strains was detectedonly when CAM-OCT was present. In the presence of theplasmid, reversion frequencies of P. aeruginosa strains wereas much as 10-fold higher than those seen in P. putida strainscarrying CAM-OCT.To enable more accurate comparison of reversion frequen-

cies and to circumvent the problems inherent in comparingmutation frequencies in different strains and different spe-cies, reversion of the same amber mutation in the bla gene ofan RP1 derivative, pMRP119, was tested in all four sets ofstrains described above. This novel assay showed that both

TABLE 3. Reversion to Cb' of strains carrying pMRP119

CbY revertants/Parental CAM-OCT 10' survivors + SEMastrain

Spontaneous +UV

PpS102 - 0.25 + 0.03 1.34 + 0.12+ 0.43 + 0.10 15.9 ± 2.5

PpS445 - 0.10 + 0.02 1.10 ± 0.20+ 0.36 ± 0.12 17.5 ± 3.2

PAC5 - 0.11 ± 0.01 0.07 ± 0.01+ 0.15 + 0.01 31.4 ± 3.0

PAS106 - 0.22 + 0.03 0.20 ± 0.02+ 0.34 ± 0.10 36.9 ± 4.1

a Data from three experiments were used to calculate the reported values.Cells were irradiated at 10 J/m2.

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TABLE 4. Forward mutation to Rif'

Parental CAM-OCT Rif' CFU/108 survivors ± SEMWPtarenta CAM-OCTSpontaneous +UV

PpS102 - 0.39 ± 0.06 1.77 ± 0.29+ 0.77 ± 0.14 5.01 ± 0.36

PpS445 - 0.20 ± 0.02 2.14 ± 0.42+ 0.64 ± 0.22 6.52 ± 1.15

PAC5 - 0.18 ± 0.05 0.82 ± 0.15+ 1.07 ± 0.10 9.44 ± 1.33

PAS106 - 0.25 ± 0.04 1.23 ± 0.27+ 0.82 ± 0.12 8.23 ± 1.42

a Data from three experiments were used to calculate the reported values.Cells were irradiated at 10 J/m2.

sets of P. putida strains exhibited similar reversion frequen-cies; in both cases, CAM-OCT-containing cells producedmore than 10 times more revertants per survivor than didplasmidless cells (Table 3). Similar to results of the auxo-trophic reversion assay, P. aeruginosa strains with CAM-OCT exhibited high levels of UV-induced reversion tocarbenecillin resistance even in comparison with levels seenin P. putida strains carrying CAM-OCT (Table 3). In theabsence of CAM-OCT, no UV-induced Cb' revertants weredetectable in either of the P. aeruginosa strains.A forward mutation assay to rifampin resistance was

developed to compare in yet another way the UV-inducedmutagenesis of P. aeruginosa, P. putida, and their CAM-OCT-carrying derivatives (Table 4). Results for P. putidawere similar to those seen in the other mutagenesis assays.That is, plasmidless cells were easily mutagenized, exhibit-ing approximately fivefold increases in Rif' colony formationafter UV irradiation. CAM-OCT increased the UV-inducedmutation frequency in these strains by about 2.5-fold. Inter-estingly, plasmidless P. aeruginosa cells also showed a

fivefold increase in Rif' colony formation after UV irradia-tion. The UV-induced mutation frequency in these strainswas 7- to 10-fold higher in the presence of CAM-OCT. Thereasons for the obvious difference in the mutability ofplasmidless P. aeruginosa in this forward mutation assaycompared with results obtained from the two reversionassays is not clear. The results do, however, suggest thatboth species encode error-prone repair pathways. The path-ways may differ in either level of expression or specificity.

Analysis of UVS mutant strains. A series of P. putidachromosomal UV response mutants was isolated, and theeffects of plasmid CAM-OCT on the mutant phenotypeswere measured. The mutants have not yet been fully char-acterized. For purposes of these studies, they were used totest the range of the effects of CAM-OCT in the absence ofnormal chromosomal DNA repair functions. CAM-OCT wastransferred to all mutant strains by conjugation with strainPpS145. UV survival was quantitated, and comparisonswere made between strains with and without CAM-OCT. Inthe absence of CAM-OCT, all eight mutants examinedexhibited marked increases in UV sensitivity compared withcontrol PpS102, although the levels of sensitivity varied frommutant to mutant (Fig. 2). The presence of CAM-OCTincreased the UV resistance of all mutants to levels notsignificantly different (P > 0.05) from those seen withPpS102(CAM-OCT). Thus, CAM-OCT suppressed pheno-typic expression of the chromosomal mutation in 100% ofthe UVS mutants analyzed. In addition, one of the mutants,PpM10, was temperature sensitive for growth at 37°C. Thepresence of CAM-OCT overcame the temperature sensitiv-ity and also markedly improved growth at the permissivetemperature (Fig. 3). This result suggests that in this case,CAM-OCT suppressed a mutation in a gene essential for cellgrowth.

FIG. 2. Survival after UV irradiation of P. putida UV' mutants and derivatives carrying CAM-OCT. Mutant strain numbers are indicatedon each bar graph. All mutants are derivatives of PpS102. Bars: a, UV dose of 10 J/m2; b, UV dose of 20 J/m2. Data from three experimentswere used to calculate means and standard errors.

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320C 370CFIG. 3. Effect of temperature on UV' mutant PpM10 and a derivative carrying CAM-OCT. Cells were grown in complete medium at 32°C,

and appropriate dilutions were plated in duplicate on TYE plates. One plate was incubated at 32'C, and the other was incubated at 37°C.

Analysis of mutagenesis mutants. CAM-OCT-carrying de-rivatives of all mutagenesis mutant strains were constructedso that the effects of the plasmid on mutagenesis in theabsence of normal chromosomal functions involved in thisprocess could be assessed. The high-mutagenesis mutants,PpM82 and PpM88, were affected quite differently by CAM-OCT (Table 5). Both strains showed about fivefold-higherfrequencies of UV-induced carbenecillin resistance than didcontrol strain PpS102(pMRP119). CAM-OCT substantiallydecreased this frequency in PpM82 and increased it to levelssignificantly higher (P < 0.05) than those for PpS

TABLE 5. Reversion to CbF of PpS102 mutagenesis mutantswith and without CAM-OCT

Cb1 revertants/10' survivors ± SEMIStrain CAM-OCT

Spontaneous +UV

PpM82 - 0.06 ± 0.03 7.40 ± 0.82+ 0.32 ± 0.12 1.04 ± 0.12

PpM88 - 0.56 ± 0.20 8.42 ± 0.40+ 0.07 ± 0.01 22.9 ± 2.3

PpM90 - 0.03 ± 0.01 0.02 ± 0.01+ 0.92 ± 0.15 0.47 ± 0.08

PpM94 - 0.02 ± 0.01 0.04 ± 0.01+ 0.76 ± 0.03 3.70 ± 1.00

PpM101 - 0.02 ± 0.01 <0.06 ± 0.01b+ <0.04 ± 0.01b 1.62 ± 0.04

PpM107 - 0.03 ± 0.01 <2.10 ± 0.23b+ 0.04 ± 0.01 <0.03 ± 0.01b

PpM111 - <0.01 ± 0.00b <0.01 ± 0.005b+ 2.11 ± 0.30 12.9 ± 1.8

PpM115 - 0.05 ± 0.01 0.02 ± 0.01+ 0.07 ± 0.02 13.5 ± 2.6

PpS102(pMRP119) - 0.25 ± 0.03 1.34 ± 0.12control + 0.43 ± 0.10 14.9 ± 2.5

a Data from three experiments were used to calculate the reported values.Cells were irradiated at 10 J/m2.

b Lack of detectable reversion; value is that obtainable from a singlereversion event.

102(pMRP119)(CAM-OCT) in strain PpM88 (Fig. 4). Thelow-mutagenesis mutants were affected by CAM-OCT in anumber of ways (Table 5 and Fig. 4). CAM-OCT appeared tohave no effect on mutagenesis of PpM107 and to onlymodestly increase UV-induced mutagenesis in PpM94 andPpM101. PpM90 exhibited a higher spontaneous mutation

FIG. 4. Qualitative analysis comparing the effect of UV irradia-tion on CbY reversion between mutagenesis mutants and controls.Patches were replica plated from complete medium to completemedium with carbenicillin and then UV irradiated at a dose of 10J/m2, followed by incubation at 32°C for 48 h. Patches on the left arethe original mutagenesis mutant isolates, and those on the rightcarry CAM-OCT introduced via conjugation. Letters correspond tostrains: a, PpM82, a high-mutagenesis mutant; b, PpM88, a high-mutagenesis mutant; c, PpS102(pMRP119), control; d through i,low-mutagenesis mutants PpM90 (d), PpM94 (e), PpM101 (f),PpM107 (g), PpM111 (h), PpM115 (i).

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s-J

a0 a

U)

calculatea b aWJ40-Uj

2 b b bb

b~~~~

Ps PpM PM p p - U Pp12 82' B D 9 1 0O7 1I1

FIG. 5. Survival after UV irradiation of P. putida mutagenesis mutants and their derivatives carrying CAM-OCT. Mutant strain numbersare indicated on each bar graph. All mutants are derivatives of PpS102 and were isolated as having high or low rates of UV-induced reversionof a bla(Am) mutation carried on pMRP119.]Bars: a, UV dose of 10 j/m2 ; b, UV dose of 20 j/m2 . Data from three experiments were used tocalculate means and standard errors.

frequency in the presence of the plasmid, as did PpM94 andPpM111. PpM90(CAM-OCT) did not, however, exhibit UV-induced mutagenesis, but its rate of mutagenesis was higherthan that of plasmidless PpM90. Mutagenesis in strainsPpM111 and PpM115 carrying CAM-OCT was raised to alevel that was indistinguishable from that seen inPpS102(pMRP119)(CAM-OCT) (P > 0.05). These resultsindicate that the presence ofCAM-OCT partially suppressedthe mutations in three of the low-mutagenesis mutants(PpM90, PpM94, and PpM101) and fully suppressed themutzltions in PpM111 and PpM115.The UV sensitivity of the mutagenesis mutants and the

effects of CAM-OCT on UV sensitivity were assayed.PpM82 and PpM88, the high-mutagenesis mutants, weremore resistant to UV irradiation than was control strainPpS102 (Fig. 5). Interestingly, at a UV dose of 10 J/m2,PpM82 without CAM-OCT exhibited levels ofUV resistancesimilar to those of PpS102(CAM-OCT). The presence ofCAM-OCT in PpM82 decreased UV resistance, which wassimilar to the effect of the plasmid on mutagenesis in thisstrain. CAM-OCT increased the UV resistance of PpM88 tolevels statistically equivalent (P > 0.05) to those ofPpS102(CAM-OCT). UV sensitivity and the effects of CAM-OCT on that sensitivity varied greatly among the six low-mutagenesis mutants. Only PpM101 and PpM107 showedextreme UV sensitivity (greater than a 100-fold increase inUV sensitivity at 20 J/m2). With the addition of CAM-OCTto these strains, UV resistance increased dramatically tonearly control levels at a dose of 10 J/m2 and to those levelsat a dose of 20 J/m2. These results are in stark contrast to thesmall effect ofCAM-OCT on mutagenesis in PpM101 and theabsence of an effect on mutagenesis in PpM107 (Table 5 andFig. 4). PpM90 and PpM94 appeared to be unaffected in UVsensitivity, since both strains responded to UV irradiationsimilarly to PpS102. CAM-OCT increased UV resistance in

both mutants to control [PpS102(CAM-OCT)] levels.PpM111 and PpM115 exhibited higher levels of UV resis-tance than did PpS102, similar to the results obtained for thehigh-mutagenesis mutants. The presence of CAM-OCT fur-ther increased UV resistance in these strains, but only tolevels comparable to those found in PpS102(CAM-OCT).These were the mutants in which CAM-OCT had the great-est effect on mutagenesis (Table 5 and Fig. 4).

DISCUSSION

The CAM-OCT plasmid significantly enhances both sur-vival and mutagenesis after UV irradiation of P. putida andP. aeruginosa. It was previously reported that CAM-OCTencodes UV functions (13), but a comprehensive analysis ofthese functions has not yet been reported. As discussedbelow, the results presented here show that CAM-OCTqualitatively affects the UV response in a manner similar tothat found for other plasmids encoding UV-response-modu-lating proteins. However, the results also suggest that CAM-OCT UV response genes may differ in number, function ofproducts, or regulation from previously described plasmids,such as pKM101 in E. coli and other enteric bacteria (26) andpMG2 in P. aeruginosa (17, 18).

Plasmid-free P. putida and P. aeruginosa were found todiffer markedly in UV response. P. aeruginosa cells weremore UV sensitive (Fig. 1) and much less susceptible to UVmutagenesis than were P. putida cells (Tables 2 through 4).In fact, mutagenesis of P. aeruginosa was detected only in aforward mutation assay and not in two different reversionassays. These results are in contrast to earlier results ofLehrbach et al. (18), who were able to detect UV-stimulatedreversion of a trp mutation in a plasmid-free P. aeruginosastrain. This difference may reflect either strain differences ora difference in the revertability of the mutation used for the

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assays. The obvious differences in mutability of the twospecies is highlighted by the novel carbenecillin resistancereversion assay (Table 3), in which reversion of one muta-tion is assayed in different genetic backgrounds. Thebla(Am) mutation was reverted by UV irradiation of P.putida but not P. aeruginosa.

In contrast to plasmid-free P. aeruginosa and P. putida,CAM-OCT-carrying derivatives of P. aeruginosa and P.putida had similar UV responses. That is, CAM-OCT in-creased survival in P. aeruginosa to levels comparable tothose for P. putida(CAM-OCT) (Fig. 1). Interestingly, theplasmid increased mutability in P. aeruginosa to levels even

higher than those of P. putida(CAM-OCT) (Table 3). Thereare several possible reasons for this result; for example,CAM-OCT UV response genes may be expressed in greaterquantities in P. aeruginosa, or P. putida gene products may

more tightly regulate CAM-OCT expression. Other interpre-tations are possible. In any case, the effects of CAM-OCT on

these cells are very similar to those seen with other plas-mids, such as pKM101 in E. coli and S. typhimurium (26) andpMG2 in P. aeruginosa. pMG2 exerts its effects, at least inpart, by encoding a DNA polymerase activity (17, 18).pKM101 encodes two genes, mucA and mucB, which are

analogs of the E. coli genes umuC and umuD, which are

required for error-prone repair but do not encode a polymer-ase activity (26).The P. putida UV response mutants isolated for this study

have provided some initial observations about the interac-tion of CAM-OCT and chromosomal UV response proteins.The mutagenesis mutants represent, to my knowledge, thefirst mutants in Pseudomonas species isolated solely on thebasis of their mutability phenotype. The novel method usedfor isolation should prove generally applicable to a variety ofspecies because the pMRP119 plasmid is a derivative of RP1,a broad-host-range plasmid (12). The initial results on theeffect of CAM-OCT on the UV survival and mutagenesismutants suggests that there are some significant differencesfrom results of similar studies with both pMK101 (21, 24, 25)and pMG2 (18).Plasmid CAM-OCT fully suppressed the UV-sensitive

phenotype in all UVS mutants tested (Fig. 2) and in twomutagenesis mutants, PpM101 and PpM107, which alsoexhibited a UVS phenotype (Fig. 5). This result is quitedifferent from that obtained in similar experiments withpKM101. The expression of mucA and mucB from pKM101partially suppresses some UVs mutants, including strainswith excision repair mutations (e.g., uvrA, uvrB, apd polA)and postreplicative repair mutations (e.g., recB and recF)(21, 24, 25), but in no case does suppression reach thewild-type levels of resistance seen in this study with CAM-OCT. Similarly, pMG2 suppresses many UV-sensitive mu-

tants of P. aeruginosa, including polA-type mutants andexcision-deficient mutants, but suppression does not raisesurvival frequencies to the levels of wild-type cells carryingpMG2 (18).

Although interpretation of the effects of CAM-OCT on themutagenesis mutants is complicated, some clues can begained by comparison with the effects of pKM101 on variousE. coli mutants and by observation of the effects of CAM-OCT on multiple phenotypes exhibited by the same mutant.One high-mutagenesis mutant, PpM88, was stimulated tohigher levels of mutability by CAM-OCT (Table 5). Thisresult is reminiscent of findings for pKM101 in an E. coliuvrB mutant (21) and may reflect higher levels of error-prone

repair when excision repair is absent. Alternatively, theresult could be due to the additive effects of a mutant

chromosomal protein and a CAM-OCT protein, both in-volved in error-prone repair. The depressive effects ofCAM-OCT on PpM82 mutagenesis may indicate a directinteraction between the mutant and plasmid proteins, neitherof which is then able to carry out its proper role in error-prone repair.The effects of CAM-OCT on mutagenesis of the low-

mutagenesis mutants can be divided into three categories: noeffect (PpM107), slight suppression (PpM90, PpM94, andPpM101), and complete suppression (PpM111 and PpM115)(Table 5). This result suggests that CAM-OCT may encodedirect analogs for the mutant proteins present in PpM111 andPpM115. UV survival studies suggest that four of the mu-tants, PpM90, PpM94, PpM111, and PpM115, express theirDNA repair functions, since they are as UV resistant ormore so than nonmutant cells. This would indicate that theirmutations are in genes whose products are mechanisticallyinvolved in error-prone DNA repair and that, in the absenceof these mutations, other DNA repair functions compensatefor the role of the mutations in UV survival. In E. coli,mutagenesis mutants of this type proved to carry umuCDmutations. However, those mutants differed from the mu-tants described here in that none showed increased UVresistance (14, 23). CAM-OCT suppressed the UV sensitiv-ity of PpM101 and PpM107 even though it had little or noeffect on mutagenesis (Fig. 5 and Table 5). These mutationscould be regulatory in nature and, like E. coli mutagenesismutations in recA and lexA (14), fail to express chromosomalgenes involved in UV repair. If this is true, then the effectsof CAM-OCT on UV survival of these strains would suggestthat the CAM-OCT UV response genes are not regulated bythe proteins that regulate chromosomal UV response genes.This mechanism would be quite different from the regulatorycircuits operating on pKM101 (26) and pMG2 (18), both ofwhich require activated RecA protein to induce expressionof their UV response genes.Taken as a whole, the results reported here suggest major

differences between CAM-OCT UV response genes andthose of pMK101 and pMG2 with respect to regulation andexpression level and may in fact reflect differences in thenumbers and types of UV response genes encoded by thevarious plasmids.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grant2-S07-RR07132 from the National Institutes of Health and in part bygrant 667170 from the PSC-CUNY Research Award Program of theCity University of New York.

I thank Massoud Nikkhoy for expert technical assistance and S.Cosloy, A. Garro, and S. Meshnick for critical reading of themanuscript.

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