Copyight 0 1996 by the Genetics Society of America
A Search for a General Phenomenon of Adaptive Mutability Timothy Galitski and John R. Roth Departmat of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received January2, 1996 Accepted for publication March 14, 1996 ABSTRACT The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and theF plasmid. Searching for general adaptive mutability, wehave investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. Wehave studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced 210 late revertant colonies (appearing threeto seven days after plating onselective medium). For the prolific mutants, the number of late revertants showed rank correlation with the with residual &galactosidase activity; for the same mutants, revertant number showed no correlation the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertantswas not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene.
T
HE orthodox neo-Darwinian view of mutability postulates that all mutations occur without regard to their fitness value; in this sense they are “random”. This view excludes the notion of “adaptive” mutability, the preferential production of fitness-enhancing mutations. Classical experimentsdemonstratedthat some 1943; LEDmutability is random ( L u m and DELBRUCK ERBERG and LEDERBERG 1952). However,DELBRUCK (1946), SWIRO (1984), and CAIRNS et aL (1988) have pointed out the lack of evidence to exclude the possibility that another fraction of total mutability is adaptive. Reviving this question, SHAPIRO(1984), CAIRNS et al. (1988), and CAIRNS and FOSTER(1991) described experimental systems that appearedto demonstrate adaptive mutability. However, the participation of complex genetic elementslike bacteriophage Mu and theF plasmid has complicated these prominent bacterial examples. In these systems, the locus under selection shows increased mutability during starvation because it is experimentally coupled to the transposasedependent excision of Mu in one case (SHAPIROand LEACH1990; MAENHAUT-MICHEL and SHAPIRO1994;FOSTER and CAIRNS 1994) or mutationally derepressed transfer functions of F in the other (FOSTERand TRIMARCHI 1995; GALITSKI and ROTH 1995; PETERSand BENSON 1995; RADICELLA et al. 1995a). While these systems have provided examples of adaptive mutability, their singular complexity makes one suspect that they may be examples of limited generality. The evolutionary relevance Curre~pondingauthor:Timothy Galitski, Department of Biology, University of Utah, Salt Lake City, UT 84112. E-mail:
[email protected] Genetics 143: 645-659 Uune, 1996)
of these novel systems is the subject oflively debate (BRIDGES 1995a; CAIRNS 1995; LENSKI and SNIEGOWSKI 1995; 1995; RADICELLA et al. 1995b; ROTHand GALITSKI SHAPIRO 1995a,b). Because of the great attention received by the F-based system, some of its most striking characteristics have become associated with adaptive mutability. In particular, mutability of nongrowing cells (CAIRNS and FOSTER 1991; FOSTER1994) and dependence on homologous recombinationfunctions (CAIRNS and FOSTER1991; HARRls et aL 1994) are cited. Neither of these properties is, a priori, a characteristic of adaptive mutability. We suggest that adaptive mutability should be a more general phenomenon. It should be easily detectable without resorting to complex genetic systems that require transposon or conjugation functions, and it should operateunder naturally relevant conditions of slow growth, not only in nongrowing cells. The possibility ofadaptive mutability has been investigated in various bacterial and yeast systems (reviewed in FOSTER1991, 1992, 1993; HALL 1992; STAHL1992; LENSKI and MITTLER 1993; ROSENBERG 1994; SYMONDS 1994; BRIDGES 199513). Since organisms typically exist under relatively adverse or selective conditions, the potential importance of adaptive mutability is great. Most of what we know of mutational mechanisms involves the study of microorganisms growing rapidly under favorable conditions. A thorough investigation of mutational mechanisms under conditions of strong selection and slow growth may reveal new aspects of mutability with evolutionary implications. We have undertaken a systematic study of the rever-
T. Galitski and J. R. Roth
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sion of a collection of strains carrying various mutant alleles of a single gene, l a d . In this collection, the locus under selection is chromosomally situated; reversion events are not obviously associated with the specialized activities of any extraneous genetic element. The goal was to correlate various characteristics of mutant strains with their ability to revert under selection. We found that leaky lac2 mutants show reversion under selection whereas tight nongrowing mutants generally show no late reversion. Late reversion of leakychromosomal lac2 mutants does not require homologous recombination functions. This behavior is expected if there is no adaptive mutability. However, manyof these strains produce an excess of revertants under selective conditions compared with the number expected based on observed growth and nonselective reversion rates. We suggest that these findings reveal a general phenomenon of adaptive mutability. MATERIALSAND
METHODS
Bacterial strains and growth media: All bacterial strains used in this study (Table 1) are derivatives of Salmonella typhzmurium LT2. The defined minimal medium was NCE salts (BERKOWITZ et al. 1968) with 0.2% of the appropriate carbon source plus auxotrophicsupplements at final concentrations recommended byDAVIS et al. (1980). The complex medium was nutrient broth,NB, (8 g/liter, Difco Laboratories) with added NaCl (5 g/liter). Solid media contained BBL agar at 1.5%. MacConkey agar was obtained from Difco laboratories and prepared according to the manufacturer's instructions. Final concentrations of antibiotics in complex medium were as follows: tetracycline hydrochloride, Tc, 20 pg/ml; and kanamycin sulfate, Km, 50 pg/ml. The @galactosidase chromogenic substrate, 5-bromo-4chlorc-3-indolyl-~-D-galactopyranoside (Xgal), was added to a final concentration of 25 pg/ml. The nonmetabolizable inducer of the lac operon, isopropyl-P-Dthiogalactopyranoside (IPTG), was used at a final concentration of 1 mM.All incubations were at 37" except as noted. Sterile 0.85% NaCl (saline) was used to dilute cultures except as noted. Transductional crosses, mediated by the phage mutantP22 HT105/1 int-201 (SCHMIEGER 1971), were performed as described by ROTH(1970a). Recombinationdeficientderivatives of hcZ mutantswere constructed by first introducing the srl203::TnlWCm mutation by transduction; the linked recAl mutation was cotransduced into these strains selecting a Srl+ phenotype on NCE sorbitol histidine plates. Isolation of lac mutants: To referencestrain SGSC180 (ara9) an insertion of MudF(lad+Z+Y'A+ K m R ) in the hzsC gene was introduced by transduction, resulting in strain TT18519 ( a m 9 hisC10081 ::MudF). The MudF insertion of this strain is oriented such that lac operon transcription is opposite that of the his operon (data not shown; Figure 1). This element is unable to catalyze its own transposition or excision since it lacks all Mu genes, including the Mu A and B transposase genes. Mutant Lac- derivatives of strain TT18519 were isolated after mutagenesis with either DES (diethyl sulfate)or ICR-191 (2-chloro-6-methoxy-9-[3-(2-chloroethyl) aminopropylamino] acridine dihydrochloride). These mutageneses were carried out as described by ROTH (1970a). Insertion mutations were made using Tn IMTc, the transpositiondefective T n l M e -
rived element No. 11 ofWAY et al. (1984). Methods for use of the TnlCMTc element were those of KLECKNER et al. (1991). Independent Lac- mutants were isolated after each mutagenesis by looking for white mutant colonies among thered colonies on MacConkey agar plates. Each lac mutation was transductionally backcrossed to the unmutagenized parent strain SGSCl8O at least once by selecting the kanamycin resistance of the MudF element on NB Km plates. Transductants from these backcrosses were saved for furtherstudy. For all mutants used, the Lac- phenotype was 100% linked to the kanamycin resistance determinant. This procedure ensured thatonly lacZ or LacYmutants were saved and that otherunlinked mutations were not present in theisolates saved for further study. Qualitative P-galactosidaseassay: To classify lac mutan& as either lacZ or lacy types, qualitative assays of P-galactosidase activity were performed. Mutants of lucZ have little or no 0galactosidase activity whereas lacy mutants have wild-type levels of P-galactosidase activity. Overnight cultures of strains were grown in NCE glycerol histidine IPTG medium. To 0.9 ml of Z buffer (MILLER1972), 50 pl of CHC1:3and 0.1 ml of overnight culture were added. These mixtures were vortex mixed for 10 sec and incubated at room temperature for 10 min. Reactions were started by adding 0.2 ml of 4 rng/ml o nitrophenyl-P-D-galactopyranoside(ONPG). After 10 min at room temperature,thedevelopment of yellow color (nitrophenol production) was noted. Assays of lacy mutants produce strong yellow color whereas assays of l a d mutants produce little orno yellow color. The lac operonstructure (lacZYA) prevents mistaken interpretations dueto polaritylacZ is the first gene of the operon. Classification of DESiduced mutations: Mutants isolated after DES mutagenesis were classified by testing for informational suppression. The hcZmutations were introduced to s u p pressor strains by transduction crosses selecting the K m R determinant of the MudF element on MacConkey Km plates. Transductant colonies were either white like their donor parents (unsuppressed) or red (suppressed). The suppressors (Table l ) included: four amber (UAG) suppressor tRNA (supD, sup& supE and supj), three ochre (UAA) suppressor tRNAS (tyrU, supC, and supG), a recessive opal (UGA) suppressor (supK), as well as a missense suppressor, s u d , that suppresses -25% ofall missense mutations (WHITFIELD et al. 1966; K HUGHES,L. MIESEL, M. MESERW, and E. ALTMAN, personal communications). Strains classified as amber mutants were s u p pressed by all of the amber and ochre suppressors and by no others. Recall that ochre tRNA suppressors suppress both ochre (UAA) andamber (UAG) mutations whereas amber tRNA suppressors suppress only amber mutations. No ochre mutations were found in our set. Mutations classified as opal were suppressed by only the supK suppressor. The missense suppressor, s u d , suppressed one allele, lacZ479m. Four other DESinduced mutations were not suppressed by any of these suppressors. These four, as well as lacZ479m, were found to be nonpolar (see below) and thus are likely to be missense alleles. All DES-induced mutations were checked for temperature sensitivity of growth on NCE lactose Xgal histidine plates. No temperature sensitivity was found at 30, 37, and 42". This spectrum of DES-induced mutations coincides closelywith that reported previously (LANGRIDGE and CAMPBELL 1969) after mutagenesis of hcZwith MNNG (N-methyl-N'-nitro-N-nitrosoguanidine, another alkylating agent). All the h c Z alleles of Table 1 , other than the frameshifts and the TnlOdTc insertions, were induced by DES. Classification of mutations induced by ICR-191: Mutants isolated after treatment withICR-191 (a DNA-intercalating acridine compound) were most likely to be frameshift mutants (AMES and WHITFIELD 1966; BIZAMMAR et d. 1967). MUtants that were induced to revert to Lac+ by ICR-191 were
Reversion Under Selection
TABLE 1 Bacterial strains Straina SGSC180 = TR6611 ara-9 TT18518 TT18519 TT18520 'IT18521 'IT18522 'IT18523 'IT18524 'IT18525 'IT18526 'IT18527 'IT18528 'IT18529 TT18.530 'IT18531 'IT18532 'IT18533 'IT18534 ara-9 'IT18535 TT18536 'IT18537 'IT18538 TT18539 TT18540 TT18541 TT18542 TT18543 TT18544 TT18545 TT18546 TT18547 TT18548 TT18549 TT18753 TT18754 TT18758 TT18759 TT18760 TT18761 TT18765 TT18766 TT18767 TT18768 TT18772 TT18773 'IT18774 'IT9167 168 TT9 'IT9169 'IT9 170 'IT2839 'IT4217 'IT13029 supK584 hisD6500 his01242 TR1975 'IT16237 TR6625
Genotypeb ara-9 melB398 metE205 ara-9 hisCl0081: : MudF(laci) ara-9 hisClOO81:: MudF ( lacZ47An) ara-9 hisClOO81::MudF(lacZ48oOp) ara-9 hisC10081: :MudF(lacZ481am) ara-9 hisClOO81: :MudF(lacZ482op) ara-9 hisClOO81: :MudF(lacZ483op) ara-9 hisClOO81: :MudF(lacZ484am) ara-9 hisClOO81: :MudF(lacZ485op) ara-9 hisClOO81: :MudF(lacZ486) ara-9 hisClOO81: :MudF(lacZ487am) ::MudF(lacZ488am) ara-9 hisC1 0081 ara-9 hisClOO81: :MudF(lacZ489am) ara-9MudF(lacZ492am) hisC10081:: ara-9 h i d l 0081:: MudF(lacZ493) ara-9 hisClOO81: : MudF(lacZ494am) hisClOO81: :MudF(lacZ495) ara-9 hisClOO81: :MudF(lacZ497) ara-9 hisClOO81: :MudF( lacZ49Am) ara-9 hisClOO81: :MudF(lacZ500: :Tn 1OdTc) ara-9 hisCl 0081 : :MudF(lacZ501: :Tn 1WTc) ara-9 hisClOO81: :MudF(lacZ502 ::Tn 1WTc) ara-9 hisClOO81: :MudF ( lacZ503: :Tn 1OdTc) ara-9 hisClOO81: :MudF ( lacZ505::Tn 1OdTc) ara-9 hisCl 0081 : :MudF(lacZ507: :Tn 1OdTc) ara-9 h i d l 0081: :MudF(lacZ511: :Tn 1WTc) ara-9 h i d l 0081: : MudF(lacZ4632f+ 1) ara-9 hisC10081:: lacZ4633f+l) MudF( ara-9 hisC10081:: MudF(lacZ4634f- 1) ara-9 hisC10081:: MudF(lacZ4635f-1) ara-9 hisC10081:: MudF(lacZ4638f+ 1) ara-9 hisC10081: :MudF(lacZ4639E- 1) ara-9 hisClOO81::MudF(lac+) hisGlOO51: :Tn 1OdTc ara-9 hisCl 0081 ::MudF(lacZ485hm) hisGlOO51: :Tn 1WTc ara-9 hisCl 0081 : : MudF(lacZ485hm) hisGlOO51: :TnlOdTc day 4 Lac' revertant ara-9 hisClOO81::MudF(lacZ485hm) hisGlOO51: :TnlOdTc day 4 Lac+ revertant ara-9 hisClOO81::MudF(lacZ489am) hisGlOO51: :Tn lOdTc day 4 Lac+ revertant ara-9 hisClOO81::MudF(lacZ48Cbp) hisGlOO51: :Tn 1OdTc ara-9 hisCl0081::MudF(lacZ48Cbp) hisGlOO51: :Tn 1OdTc day 5 Lac' revertant ara-9 hisClOO81::MudF ( lacZ48Cbp) hisGlOO51: :Tn 1OdTc day 5 Lac+ revertant ara-9 h i d l 0081: :MudF(lacZ48Cbp) hisGlOO51: :Tn 1OdTc day 5 Lac+ revertant ara-9 h i d l 0081: :MudF ( lacZ4638f+ 1)hisGlOO51: :Tn 1OdTc ara-9 hisClOO81::MudF (lacZ4638F+ 1)hisGlOO51: :Tn lOdTc day 4 Lac' revertant ara-9 hisClOO81::MudF(lacZ4638f+ 1)hisGlOO51: :TnlOdTc day 4 Lac' revertant ara-9 hisC10081: :MudF(lacZ4638f+ 1)hisGlOO51: :TnlOdTc day 4 Lac+ revertant M 4 1#am supDl0 M414am supE20 led414am supF30 lsupJ60 e d 4 14am led414am hisC527am zii-614::TnlO tyrU90 led414am supG50 led414am hisC527am zde605::TnlO supC80 zif3693::TnlOdTc hisC537 sumAl0
"All strains are derivatives of Salmonella typhimurium LT2. Strain SGSCl8O was provided by K. SANDERSON and the Salmonella Genetic Stock Center. Strains TT18518-nl8774 were isolated or constructed during the course of this work. The remainder are from the ROTH laboratory collection. 'Abbreviated allele descriptors following lacZ allele numbers represent the following: am, amber (UAG); op, opal (UGA); f-1, -1 frameshift; f+ 1, +1 frameshift; m, missense; :: TnlOdTc, insertion of a transpositiondefective TnlO. The lacZ mutations lacking descriptors are likely missense alleles. The full genotypes of Lac+ revertant strains are unknown.
647
T. Galitski and J. R. Roth
648
ara-9
FIGURE .-A 1 map of the Salmonella chromosome illustrating relevant markers (see Table 1). The structure of the his locus interrupted by an insertion of the Muderived element, MudF, is magnified. MudF is a transpositionally inert element with Mu ends but n o Mu functions. It includes a wild-type lac locus and a kanamycin resistance determinant (CHACONAS et al. 1981; SONTI 1990). Salmonellaeare naturally Lac-; the lac genes are derived from Esclwn'chia coli K12. saved forfurther study. It was also possible to classify frameshift mutations as likely +1 or -1 shifts by screening for an induction of reversion with MNNG. MNNG tends to revert +1 frameshifts but not -1 frameshifts (RIDDLEand ROTH 1970). Two NCE lactose Xgal histidine plates were spread with 0.1-ml aliquots from an overnight culture of each Lac- strain. On one plate a 2 0 4 drop of a 1 mg/ml solution of MNNG in citrate buffer (MILLER1972) was placed in the center. Similarly, a 1 mg/ml solution of ICR-191 in 70% ethanol was applied to the other. Solutions and plates with ICR191 were handled and incubatedin subdued light. Plates were incubated for 3 days and scored for a pronounced induction of revertants around the zone of killing. Recombinational grouping of TnZOdTcinsertions: Since TnlO element5 show insertion site specificity (KLECKNER et al. 1979), some independent lacZ insertion alleles were likely to have the insertion at the same site. To study only insertions at distinct sites, insertion strains were placed into groups of mutants thatfailed to recombine with each other togive Lac' transductants. Insertion mutants were crossed to each other by transduction selecting Lac+ on NCE lactose Xgal histidine plates. For each strain, a cross with a lac' donor and a selfcross served as positive and negative controls respectively. Seven groups of nonrecombining insertions were found; only one memberof each recombination group was tested further. Tests of polarity: Classification of lac% mutations was extended by testing for polarity on the downstream lacy gene. The Lacy permease can substitute for the MelB melibiose permease (KENNEDY 1970). Accordingly, we crossed the MudF elements from the h c Z mutants into a melB deletion strain and screened for utilization of melibiose as a sole source of carbon and energy. MudF elements with lacZ mutations were crossed into strain TT18518 (ara-9metE205 melB398) selecting K m ' on NB Km plates. For each cross, four transductants
were screened for growth on an NCE melibiose histidine methionine plate. Fluctuation tests: The nonselective reversion rate of each lac% mutant was measured using the fluctuation test of LURIA and DELRR~CK (1943). From a single colony, an overnight culture was grown in NCE glycerol histidinemedium and diluted 10"-fold in fresh medium. At least 30 0.5ml aliquots of this dilution were dispensed to tubes and incubated to saturation. To verify the independence of mutants found in the 0.5-ml cultures, the original overnight culture was sampled (0.1 ml) and plated on an NCE lactose Xgal histidine plate. Revertant colonies were counted after 40 hr of incubation. To determine thetotal number of colony-forming units in the saturated independent cultures, one was chosen at random. Its volume was measured (some was lost due to evap oration). A sample was diluted and plated on nutrient agar plates. To all cultures, 2.5 ml of molten NCE lactose Xgal histidine with 0.7% agar was added.These were mixed, poured onto an NCE lactose Xgal histidine plate, incubated for 40 hr, andscored for the numbersof Lac' colonies. Since some strains continually produce Lac+ revertants with time, reconstruction control experiments were performed to determine the optimal incubation time to score the number of Lac' mutants present at thetime of plating. This time was 40 hr (data not shown). Mutation rates were calculated using the Po method (LURIA andDELBROCK 1943) or the method of the median (LEA and COUISON1949). ModifiedquantitativeP-galactosidase assays: Assays were performed, and units of &qalactosidase activity were calculated, as described by MILLER(1972). The following modifications were employed to allow the reliable measurement of low activities: (1) Cultures of assayed strains were grown in NCE glycerol histidine with or without IPTG to 100 Klett. The use of glycerol increases induced fl-galactosidase levels about sixfold (due to Crp/cAMP control) compared with growth on glucose (data not shown). (2) Higher concentrations of bovine serum albumin and 2-mercaptoethanol were used to stabilize P-galactosidase activity. To Z buffer, we added bovine serum albumin to 100 pg/ml, 2-mercaptoethanol to 200 mM, and sodium dodecyl sulfate to 0.005%. (3) Mixtures of Z buffer andculture were prepared in 15ml polypropylene screw-cap tubes to prevent evaporationduring extendedincubations. Cells were permeabilized by vortex mixing with two drops of chloroform per milliliter of mixture. (4) Reactions were started by adding the substrate, ONPG. Reaction times were extended over as much as 3 days to allow sufficient product development. (5) Reactions were monitored by withdrawing 1-ml samples over time, mixing these with 0.42 ml of 1 M Na2C03 in a microcentrifuge tube, centrifuging the sample for 1 min to remove most cellular debris, removing 1 ml of the supernatant to a cuvette, and measuring absorbances of 550 and 420 nm light. Sample clarification eliminates most of the experimental error associated with the correction for bacterial absorbance of 420 nm light. The ability of the modified assay to reliably measure low activities was tested. A culture of the lac' parentstrain, TT18519, was diluted lo'-, lo'-, and 10:"fold before assay. The undiluted cultureof strain TT18519 had 5700 units of activity, whereas the dilutions were determined to have 5.5,0.42, and 0 units, respectively. All assays that required extended (>40 hr) incubation showed a significant loss of activity (even diluted wild-type extracts); thus assays of strains with a very low reported activity are likely to underestimate total activity. Reversion on selective medium:The ability of h c Z mutants to produce late revertants was measured. Independent cultures (started from different single colonies) of test strains were grown to full density in NCE glycerol histidine medium. Cells were pelleted and resuspended in an equal volume of
9
Selection
Under
Reversion
saline.Fromeach culture, -5 X lo8 tester cells (0.1 ml) were spread ona plate of selective medium,NCE lactose Xgal histidine. Histidinewas included as an essential nutrient for these auxotrophic strains; it does not provide a carbon or energy source (GUTNICKet al. 1969). The chromogenicsubstrate Xgalwas included to facilitate the scoring of Lac+ reat 37" inloosely closed vertant colonies. Plates were incubated plastic sleeves to minimize drying. New Lac+ colonies were counted each day for 7 days of incubation. Ten independent cultures weretestedforeachstrain. In some experiments (noted in RESULTS), strains were platedwith a fivefold excess of scavengercells to consumecarbonandenergysources other than lactose that might be present in the medium or excreted byLac' revertant cells. Scavenger cells of the LacstrainSGSC180 (am-9) orstrain TR6625 (strAI) were prepared in the samewayas the tester cells. The strains tested both in thepresenceandabsenceofscavengersincluded (lacTT18520(lacZ479m),TT18521(lacZ48oOp),TT18522 Z481am),TT18523(lacZ482op),TT18524(lacZ483op), TT18525(lacZ484am),TT18526(lacZ485op),TT18527 (lacZ486), TT18528 (lacZ487am), and TT18529 (lacZ488am). Growth on selective medium: To determine the number agar plugs ofviable Lac- testercellsperplateovertime, were taken (avoiding Lac+ colonies) from selective platings 20 described above for reversion experiments. Bacteria on mm2 agar plugs were suspended in saline by vigorous vortex mixing for 15sec followed by 15 min of benchtop incubation without agitation and another 15-sec vortex mixing. Dilutions were prepared in saline, spread on nutrient agar plates (with kanamycin for assayingtesterstrainsinthepresence of a scavenger strain), and incubated. The numbers of colonies on nutrient agar plates were multiplied by their respective dilution factors and 289 (the ratio of the area of the agar plate to the area of the agar plug) to determine the number of Lac- tester cells on the selective plates. RESULTS
Experimental system: We have characterized the reversion and residual p-galactosidase levelsof 30 lacZ mutants derived from a parental Lac' derivative of Salmonella typhimurium LT2. Salmonellae are naturally Lac-; the lac operon was introduced as part of a MudF element inserted in the hisC gene (Figure 1).MudF is a transpositionally inert Mu-derived element with Mu ends flanking a wild-type lac locus and a kanamycin resistance ( K m R ) determinant (CHACONAS et al. 1981; SONTI1990). Since it lacks allMu genes, including theA and B transposase genes, it has no transposition activity. None of the Lacf reversion events occurringinthe his::MudF system was associated with excision or transposition of Mu; all revertants tested had the original MudF insertion and no others (T. GALITSKI, unpublished results). The his::MudF construction was used because its His- K m R phenotype facilitates genetic manipulation. The lac genes can be transduced to a new strain by selecting K m R ; they can be removed from a strain by selecting Hisf. The k c 2 mutant set: Independent mutants of the lacZ gene, encodingp-galactosidase, were isolated after mutageneses of strain TT18519 (lac') with one of three different mutagens, DES (diethyl sulfate, an alkylating agent), ICR-191 (a frameshift-inducing acridine com-
pound), andTnlOdTc (a transposition-defective TnlO derivative). Lac- mutants were isolated by screening for white colonies among the red (Lac+) colonies on MacConkey agar plates. Since MacConkey medium indicates acid production from lactose catabolism but allows both Lac' and Lac- colonies to grow, white colonies isolated from MacConkey plates were checked for their ability to use lactose as a sole source of carbon and energy. The chosen mutants failed to grow overnight when patches were replicated to NCE lactose Xgal histidine plates; extremely leaky lac mutants were excluded. Transductional backcrosses (selecting the K m R phenotype of MudF) to strain SGSC180 ( a m 9 his+) were performed to ensure that theLac- phenotype was due to a mutation of the lac operon (lad, lacZYA) and to eliminate any unlinked mutations incurred during mutagenesis. One transductantfromeach cross was saved for further study. Mutants of the lacA gene are Lac'; they were systematicallyexcluded. Mutants of the lacy gene, encodinglactose permease, were eliminated based on theresults of a qualitative p-galactosidase assay (see MATERIALS AND METHODS); lacymutants have wildtype levels of p-galactosidase activity whereas lacZ mutants show a lossofactivity. Rare mutant lacl alleles encoding a noninducible repressor (Lac-) would have been classified in our tests as "polar" missense (most likely) mutations (classifications described below). Since any lad null mutation will suppress these dominant mutations, such strains should show nonselective highest rates reversion rates 2 100 times higher than the observed in our set. We found no mutants resembling this description. Also, nonrevertible lac mutants (deletions, double mutations) were excluded. Thus, the mutant set includes only revertible lac2 alleles. Classification of k c Z mutations: Various tests were employed to classify lacZ mutations without resorting to sequence determination. These tests included screens for informational suppression, induced reversion with different mutagens, polarity on the lacYgene, temperature sensitivity, and recombination (see MATERIALS AND METHODS). The resulting classifications are indicated by allele descriptors (Table 1 and Table 2). Mutagenesis with DES gave a distribution of alleles including all the amber (UAG), opal (UGA), and missense types. None of these showed any temperature sensitivity;they had the same Lac- phenotype at 30,37, and 42". Frameshift mutagenesis with ICR-191 produced both 1 and - 1frameshift alleles. Multiple independent insertions of TnlOdTc were isolated. Insertions at distinct sites were identified by recombination tests and included in this study. To further classify the lac2 alleles, polarity on the downstream lacy gene was assessed (see MATERIALS AND METHODS and Table 2). Insertionmutationsexhibitedstrong polarity. Nonsense and frameshift alleles showed a wide spectrum of polarities. This spectrum is known to correlate with position in the lac2 gene (reviewed in ZIPSER 1970). Suspected missense alleles showed no polarity.
+
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T. Galitski and J. R. Roth
TABLE 2 Polarity, nonselective reversion rate, late revertants,and residual P-galactosidase activity of lac2 mutants
ZucZ allele"
Polarity on lacyb
489am 482op 4800~ 483013 485op 4635f- 1 46?8f+ 1 4634f- 1 492am 46??f+ 1 46?2f+ 1 488am 487am 46?%- 1 494am 484am 486 481am 499am 4 7% 505::TnlWTc 500: : Tn 1OdTc 495 493 497 501 : :Tn 1WTc 502::TnlWTc 503::TnlOdTc 507: :Tn 1WTc 51l::TnlOdTc
Polar Polar Polar Polar, weak Polar, weak Polar, weak Polar Nonpolar Nonpolar Polar Polar Polar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Polar Polar Nonpolar Nonpolar Nonpolar Polar Polar Polar Polar Polar
Nonselective reversion rate'
Late revertad
Residual 0-galactosidase activity'
1.4 1.7 1.9 2.1 0.82 0.40 7.7 3.7 3.0 0.55 0.89 7.1 1.1 0.39 0.80 2.4 0.76 2.5 2.7 0.24 0.038 0.33 0.15 0.12 0.11 0.20 0.023
345 2 30 85 2 9 72 % 8 71 2 6 61 2 9 40 2 8 30 2 2 17 2 2 17 2 2 11 -t 1 9 2 1 6 2 2 6 2 1 6?1 5 2 1 3 2 1 3 2 1 2-tl 2 2 1 2 2 1
13 0.83 1.3 0.37 0.57 2.3 0.09 0.11 0.13 0.81 0.17 0.08 0.28 6.7 0.09 0.05 0.45 0.16 0.53 0.08
~~~
121
I 1 0 0 0 0
t 2 2 2 2 2
1 1 1 0 0 0
0.19
o+o
0.19 0.033
0 2 0
o+o
0 0
0.65 0.29 0.55 0 0 0 0 0
ZucZallele number and type. The abbreviations used represent the following: am, amber (UAG); op, opal (UGA); f- 1, - 1 frameshift; f+ 1, + 1 frameshift; m, missense; : :Tn 1WTc, insertion of a transposition-defective TnlO. The mutations lacking descriptors are likely missense alleles. Polarity of lacZ alleles on Lucy gene expression w a s assessed by screening formelibiose utilization in a m l B deletion background (see MATERIALS AND METHODS). 'Reversion rate to Lac+ (revertants per cell division X 10') during growth without selection for Lac+ (from fluctuation analysis, n 2 30). dNumber (mean 2 SEM, n = 10) of Lac+ revertantcolonies appearing from day 3 through day 7 of incubation on NCE lactose Xgal histidine plates. Error values are the standard error of the mean, SEM =
