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Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli
PLASMID
22,275280
(1989)
Analysis of Relative Reversion Frequencies for IS2 Insertion Mutations in the Regulatory Region of the ga/OPETK Operon of Escherichia co/i E. MUSSO’
RICHARD
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 Received August 2 I, 1989; revised November 22, I989 Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP3 and galOPE-490, contain IS2 insertions only I bp apart in the gul regulatory region; yet only the former yields Gal+ phenotypic revertants at a detectable frequency. We have shown that the gafOPE-490 allele comprises two mutations-an IS2(1) insertion at bp+(2-6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490*) which retains the IS2 of galOPE-490 but is galE+ reveals a reversion frequency approximately 30-fold higher than that of ga/OP-3. Reversion of galOPE-490 is at least lO,OOO-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems. 0 1989 Academic
Press, Inc.
The galactose operon (galOPETK) of Escherichia coli comprises three structural genes for enzymes involved in both catabolic and anabolic metabolism of D-galactose (Fig. 1). Transcription can be initiated at either bp+ 1 or at bp-5 using overlapping promoters Pl or P2, respectively, each of which is regulated by both the cyclic AMP:CRP complex and the gal2 repressor. The former stimulates Pl and inhibits P2 and serves to monitor intracellular cyclic AMP levels which respond to the availability of glucose (Aiba et al., 198 1; Irani et al., 1989; Muss.0 et al., 1977). The gal repressor acts at two operator loci, Or (centered at bp-60) and Or (centered at bp+53) to regulate each of the promoters (DiLauro et al., 1979; Fritz et al., 1983; Irani et al., 1983a; Kunke et al., 1986; Majumdar and Adhya, 1984). Three independent pleiotrophic Gal- mutations have been previously isolated and characterized as IS2 insertions in orientation (I) (i.e., gal transcription proceeds from left ’ To whom correspondence should be addressed. ’ Abbreviations used: gal, gene(s) for galactose metabolism: Gal+, galactose fermenting; Gal-, galactose nonfermenting; IS, insertion sequence; Tn, transposon. 215
(L) to right (R) through the IS2) within the gal mRNA leader region before the galE gene (Fig. 1). The resulting severe polarity has been attributed to Rho-dependent transcription termination within the IS2 sequence (de Crombrugghe et al., 1973). Two of the mutations, galOP-3 : : IS2 (hereafter ga13) (Ahmed et al., 198 1) and galOP-308 : : IS2 (hereafter ga1308) (Ghosal ef al., 1979) appear to be identical with IS2 inserted at bp+(l to 5); the third, galOPE-490 : : IS2 (hereafter ga1490), has an IS2 at bp+(2 to 6), displaced 1 bp toward galE (Muss0 and Rosenberg, 1977). The latter was originally thought to be an insertion within galE (de Crombrugghe et al., 1973) contrary to the position determined subsequently by sequence analyses. In agreement with the genetic assignment, gal490 behaved differently than the other two (ga13/308) in each of three genetic tests (Dr. Sankar Adhya, personal communication and cited references): (a) whereas ga13lga1308spontaneously revert (by IS2 excision or by other pseudoreversion events) to Gal+ at a frequency of about lo-’ (Ahmed et al., 198 I ; Hill and Echols, 1966; Saedler et al., 1972), gal490 rarely yields revertants; (b) whereas gal3 is suppressed to a Gal+ phenotype by the rhol5(Ts) allele at 0147-619X/89
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Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form resewed.
276
SHORT COMMUNICATIONS EcoR I I
/
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bp-100
(p,Jy
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/
/
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HiTaYf /
/
-.
-.
pi
-0, PP
L
I
I -.
-.
-.
-.
+l c -5
gal T
. .
-.
N-s
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IS2
I gal K
-.
-. 0 I --... I E 49O(oc)
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FIG. 1. A schematic map of the gal operon. The relative positions of restriction enzyme sites, regulatory loci, and the IS2 insertion sites (H indicates the location of the 5-bp target site for each IS2 insertion) in ga13/308 and gal490/490* are shown. The left and right termini and the Rho-dependent transcription terminator of IS2 are indicated by L, R, and t, respectively. The deletion of bacterial DNA upstream of the galoperon in phage Xpgal, is designated A320; galE490(0c) is the transversion mutation present in ga1490.
nonpermissive temperatures (Das et al., 1976, 1978),gal490 remains Gal- (galK is expressed but not galE); and (c) whereas gal3 is phenotypically suppressedto Gal+ under conditions of escapesynthesis from a X prophage, gal490 remains Gal- (galE is not expressed) (Adhya et al., 1976). Under the latter conditions of escapesynthesis pseudorevertants of gal490 can be detected as colonies that are Gal+ at high temperature but Gal- at low temperature even in the presenceof a gal inducer, i.e., D-galactose (Drs. S. Adhya and M. Gottesman, personal communication). We have characterized one such pseudorevertant, ga1490*, which is indistinguishable from gal490 by restriction enzyme digestion (data not shown). Studies described below show that ga1490* yields Gal+ revertants at a markedly higher frequency than seen for ga1490. To understand these differences we have determined (Maxam and Gilbert, 1980) the sequencesof the IS2 termini and adjacent gal DNA for gal490 and gal490*. The terminal - 100 bp of eachgal: : IS2 match the published IS2 sequence(Ronecker and Rak, 1987) but the galE sequenceof gal490 differs from the gal+ (Muss0 et al., 1974) and ga1490* sequencesby a single base pair
substitution (Fig. 1). At position bp+59 a C/G (gal+ and ga1490*) to A/T (ga1490) transversion converts galE codon 11 from Tyr(U-A-C) to an ochre (U-A-A) termination signal. The presence of this point mutation, designated galE49O(Oc), explains the failure of gal490 to yield Gal+ revertants or phenotypic suppression under Rho- conditions. In support, Gal+ revertants of gal490 can be detected in an ochre suppressor background (S. Adhya, personal communication). The galE49O(Oc) mutation also lies within the Or locus of the bipartite gal operator. We sought to determine if this mutation would interfere with gal repressor binding by using the in vivo repressor titration assay (Irani et al., 1983b). Bacterial strain SA1796 has a superrepressor allele, galR”78, which prevents expression of a wild-type galOPETK operon resulting in white colonies on MacConkey-galactose indicator plates. When transformed with a multicopy plasmid containing a functional gal operator the bacteria produce red colonies since the limiting gal superrepressor is sequesteredby the plasmid-borne operators. The results of our experiments are shown in Table 1. As negative controls we tested the plasmid vectors lacking gal DNA inserts and
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ASSESSMENT OFOPERATORFUNCTIONBY REPRESSOR TITRATION Test plasmid a
Relevant gal loci
Source/reference
Colony color on MGA plates”
Operator phenotype’
pBR322 pBH20 b pASTlOO’ pMIld pAST20 11’ pAST2018’ ~AST2146~ ~AST3563~
None None 0; 0: 0: 0: (490*) OI(490) 0; 0: (490*) 0; 0, (490)
Bolivar et al., 1977 Itakura et al., 1977 Hinton and Musso, 1982 Irani ef al., 1989 This study This study This study This study
W W R W W W R W
+ + -
a The indicated plasmids (at least two independent isolates for each new construct) were transformed into bacterial strain SA1796 [E. coli K12 Fgal(OPETK)+, galRs78, SmR] and two ampicillin-resistant transformants were tested by incubation on MacConkey-galactose-ampicillin (MGA) plates at 30°C for 24-36 h. Red(R), Gal+ colonies indicated the plasmid carried a functional gal operator with sufficient affinity to titrate the galR” repressorand allow expression of the chromosomal galOPETK operon. White (W), Gal- colonies indicated the plasmid-borne operator allele lacked sufficient affinity to sequesterall the superrepressor.On the basis of this Gal phenotype an operator phenotype (+ or -) is assigned,but quantitative rather than qualitative differencesare likely involved. bpBH20 has the 1acOPregion as a 204-bp HaeIII fragment at the EcoRI site of pBR322. c pASTlO has a l.l-kb EcoRI-Hind111 fragment inserted into pBR322. The insert was derived from Xpga& and correspondsto bp-650 through bp+445 relative to the gal mRNA startsite. d pMI1 has a 240-bp insert corresponding to bp-187 through bp+45 of gal located at the Hind111site of pBR322. epAST2011 has a 0.9-kb Hind111fragment inserted into pBH20. The insert is derived from Xgai490* and corresponds to bp87 1 to bp 1331 of IS2 plus bp+2 through bp+445 of galE with the latter oriented as in pASTlO and pM1 I. IpAST is identical to pAST2011 except that the 0.9-kb insert was derived from Xga1490and thus contains the galE49O(Oc) mutation. g pAST2746 was constructed from pM1 I and pAST20 11 by digesting each with BstEII plus PstI and then ligating the small fragment (containing the gal bp- 187to bp+38) of the former with the large fragment (containing gal bp+39 to bp+445) of the latter. h pAST3563 is identical to pAST2746 except that it was constructed from pMI 1 and pAST20 18 and thus contains the galE49O(Oc) mutation.
as a positive control we tested PAST100 which contains a gal insert with both 0,’ and 0:. Only the latter plasmid titrated galR” to produce a Gal+ phenotype. Although previous studies (Fritz et al., 1983; Irani et al., 1983b) have shown that partial derepression occurs with plasmids containing a single gal0 locus, neither OEnor O1alone in our plasmids would titrate the superrepressor sufficiently to produce a Gal+ phenotype. To test the 0, loci of gaZ490 and gaZ490* we used the BstEII site at bp+39 to remove the IS2 sequencesand to replace them with the normal ga10EPlP2 region from pMI 1. Plasmid pAST2746 (derived from gal490*) has a wild-type gal sequence and a fully functional operator by this assay. In contrast pAST3563 (derived from gal490)
does not titrate gal repressor and transformants remain Gal-. Thus the galE(Oc) mutation results in a operator defect. Although the quantitative effect of this mutation cannot be assessedfrom this type of assay,it does support the importance of bp+59 in the O1locus for gal repressor action. The closely related alleles ga13, ga/308, gaZ490, and ga/490* allowed us to examine how reversion to Gal+ may depend on the exact position or context of IS2 in the gal flanking sequence.To evaluate the possible effects of other IS2 copies, we conducted studies in both E. coli K12 (with eight chromosomal IS2 copies) and E. coli C (with no chromosomal IS2). We constructed isogenic strains from E. co/i K12 (an F-, cured, derivative of W1485)
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and E. coli C prototrophs (Hu and Deonier, 1981) with the four gul::IS2 alleles by general recombination. Using the phage XpgaPA(chlD-attL-int)3 13~1857(Merril et al., 1978), we were able to transfer each gal::IS2 allele from plasmid or bacterial chromosome to the phage and then to the chromosomal gal locus of our E. coli C and K 12 strains essentially as described by Irani et al. (1983b). Quantitative data on reversion for thesestrains were obtained using a papillation assay(Hopkins et al., 1980) to monitor the appearance of Gal+ (red) papillae within Gall (white) parental colonies on MacConkey-galactose agar medium (Fig. 2). For E. coli C gal: : IS2 strains the results after 4 days of incubation at 37°C indicated a reversion frequency (No. visible papillae/No. viable cells per colony) of 1 X IO-’ (average 1.5 papillae per colony) for gal3 or gal308 compared to 3 X 1O-7 for g&490* (average 46 papillae per colony) but gal490 exhibited no papillae (frequency < 10-l’) even after 4 weeks of incubation. Thus we saw no indication of enhanced double reversion or “adap-
tive evolution” with gal490 contrary to observations with other systems as reported by Cairns et al. (1988) and Hall (1988). Nearly all Gal+ revertants in E. coli C arose by excision (deletion) of IS2 based on the following three tests conducted on over 25 independent papillae from each of the gal3 and gal490* strains: (i) all Gal+ revertants behaved as Rho+ basedon the inability of XNam7Nam53cI857 phage to grow lytically (Richardson et al., 1977);(ii) all Gal’ revertants were stable based on the lack of Gall segregantsafter growth for 30 generations;and (iii) all Gal’ isolateslacked IS2 based on colony blot (Miller and Barnes, 1986)hybridization with an IS2-specificprobe. Thus, if Gal+ pseudorevertants occur as rhosuppressors or IS2 sequence perturbations, they account for a minor fraction of the papillae observed in theseexperiments. Previous studies in E. coli K12 with gal3/gu1308 found that IS2 excision can account for 30 to 90% of the Gal+ revertants (Ahmed, 1981; Morse and Pollack, 1969; Saedler eLal., 1974). Even in the E. coli K 12 background gal490 failed to revert at a detectable rate, whereas
FIG. 2. Relative reversion frequencies for different gal: :IS2 alleles. E. coli C with chromosomal alleles ga/490* (A, C, E), gal490 (B, F), or gu/3 (D) were incubated on MacConkey-galactose medium at 37°C and a density of - 10 colonies per 90 cm diam plate for 2 days (A), 4 days (B, C, D), or 6 days (E, F) before photography of typical colonies through a dissecting microscope at 60x magnification; red (Gal+) papillae appear as black spots within the large white (grey) Gal- parental colonies.
SHORT COMMUNICATIONS
the g&490* allele reverted - 15fold more frequently than gal 3 or gu1308. Since the K12 strain has seven to eight additional chromosomal copies of IS2 (Hu and Deonier, 1981) it seems unlikely that differences in IS2-encoded factors are responsible for the different reversion frequencies. The 15 to 30-fold difference in reversion of gal 3/308 and ga1490* is most likely due to the 1-bp shift of IS2 in the gal sequenceand thus resemblesthe results reported for deletion of Tn5 derivatives located 1 bp apart in the ampicillin-resistance gene of pBR322 (DasGupta et al., 1987). In the latter caseit was suggestedthat the drastic effect of position on deletion frequency might be explained by differences in the symmetry of DNA sequences spanning the deletion endpoints. Comparable changes are observed in the symmetry of sequences flanking IS2 in gal31308 and gu1490*, but the relevance of such symmetries has not been determined. ACKNOWLEDGMENTS I thank Drs. Sankar Adhya and Max Gottesman for providing bacterial and phagestrainsincluding the g&490* mutation and for communication of unpublished results. I also thank Ms. Tara Hodam for excellent technical assistance, Mr. Robert Sullivan and Ms. Byrne Burch who helped construct plasmids, and Ms. K. French for typing the manuscript. This work has been supported by the N.I.H. Biomedical ResearchSupport Award S07RR07 160 to the University of South Carolina and by the American Cancer Society Grant MV-2 14. REFERENCES ADHYA, S.,GOTTESMAN,M., DECROMBRUGGHE, B., AND COURT,D. (1976). Transcription termination regulates gene expression. In “RNA Polymerase” (R. Losick and M. Chamberlin, Eds.),pp. 7 19-730. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. AHMED,A. (1977).The ga13mutation of E. co/i. In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. Shapiro, and S. Adhya, Eds.), pp. 37-48. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. AHMED, A., BIDWELL,K., AND Musso, R. E. (1981). Internal rearrangements of IS2 in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 45, 141- 152. AIBA, H., ADHYA, S., ANDDECROMBRUGGHE, B. (198 1). Evidence for two functional gal promoters in intact Escherichia coli cells. J. Biol. Chem. 256, 11,90511,910.
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BOLIVAR,F., RODRIGUEZ~ R., GREENE,P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W., CROSA, J. W., AND FALKOW,S. (1977). Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2, 95-113. CAIRNS,J., OVERBAUGH,J., AND MILLER, S. (1988). The origin of mutants. Nature (London) 335, 142-145. DAS, A., COURT, D., AND ADHYA, S. (1976). Isolation and characterization of conditional lethal mutants of Escherichia coli defective in transcription termination factor rho. Proc. Natl. Acad. Sci. USA 13, 1959-1963. DAS,A., MERRIL,C., AND ADHYA, S. (1978). Interaction of RNA polymerase and rho in transcription termination: Coupled ATPase. Proc. Nat/. Acad. Sci. USA 15, 4828-4832.
DASGUPTA,V., WESTON-HAFER,K., AND BERG,D. E. (1987). Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322. Genetics 115,41-49.
DECROMBRUGGHE, B., ADHYA, S.,GOTTESMAN,M., AND PASTAN,I. (1973). Effect of rho on transcription of bacterial operons. Nature New Biol. 241, 260-264. DILAURO, R., TANIGUCHI, T., Musso, R., AND DE CROMBRUGGHE, B. (1979). Unusual location and function of the operator in the Escherichia coli galactose operon. Nature (London) 279,494-500. FRITZ, H.-J., BICKNASE,H., GLEUMES,B., HEIBACH,C., ROSAHL,S., AND EHRING,R. (1983). Characterization of two mutations in the Escherichia coli gulE gene inactivating the secondgalactoseoperator and comparative studies of repressor binding. EMBO J. 2, 21292135.
GHOSAL,D., SOMMER,H., AND SAEDLER,H. (I 979). NUcleotide sequenceof the transposableDNA element IS2. Nucleic Acids Rex 6, 1I I 1- 1I2 1. HALL, B. G. (1988). Adaptive evolution that requires multiple spontaneousmutations. I. Mutations involving an insertion sequence.Genetics 120, 887-897. HILL, C., AND ECHOLS,H. (1966). Properties of a mutant blocked in inducibility of messengerRNA for the galactoseopcron of Escherichia coli. J. Mol. Biol. 19,3851.
HINTON, D. M., AND Musso, R. E. (1982). Transcription initiation sites within an IS2 insertion in a Gal-constitutive mutant of Escherichia coli. Nucleic Acids Res. 10,5015-5031. HOPKINS,J. D., CLEMENTS,M. B., LIANG, T-Y., ISBERG, R. R., AND SYVANEN,M. (1980). Recombination genes on the Escherichiacoli sexfactor specificfor transposable elements. Proc. Natl. Acad. Sci. USA 77, 2814-28 18. Hu, M., AND DEONIER,R. C. (1981). Comparison ofI%, IS2, and IS3 copy number in Escherichiu coli strains K-12, B, and C. Gene 16, 161-170. IRANI, M., MuSSO,R., AND ADHYA, S. (1989). The cyclic AMP dependent switch in the initiation of transcription from the two promoters of the gal operon of Escherichia co/i: Identification and assayof 5’-triphosphate ends of
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mRNA by GTP: RNA guanyl transferase.J. Bacterial. 171, 1623-1630.
IRANI, M., OROSZ,L., AND ADHYA, S. (1983a). A control element within a structural gene: The gal operon of Escherichia coli. Cell 32, 783-788. IRANI, M., OROSZ,L., BUSBY,S., TANIGUCHI,T., AND ADHYA. S. (1983b). Cyclic AMPdependent constitutive expression of gal operon: Use of repressor titration to isolate operator mutations. Proc. Natl. Acad. Sci. USA 80,4775-4779. ITAKURA, K., HIROSE,T., CREA, R., Rrccs, A. C., HEYNEKER,H. L., BOLIVAR,F., AND BOYER,H. W. (1977). Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198,
MORSE,M. L., AND POLLACK,B. F. ( 1969). Reversion instability in the galactose operon of Escherichia coli. J. Bacterial. 99, 567-569. Musso, R., DE CROMBRUGCHE, B., PASTAN,I., SKLAR, J., YOT, P., AND WEISSMAN,S. (1974). The S-terminal nucleotide sequenceof galactosemessengerribonucleic acid of Escherichia coli. Proc. Natl. Acad. Sci. USA 71, 4940-4944.
Musso, R., DILAURO, R., ADHYA, S., AND DE CROMBRUGGHE,B. (1977). Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell 12, 847854.
Musso, R., AND ROSENBERG, M. (1977). Nucleotide sequences at two sites for IS2 DNA insertion. In “DNA 1056-1063. Insertion Elements, Plasmids, and Episomes” (A. BukKUHNKE, G., KRAUSE, A., HEIBACH, C., GIESKE, V., hari, J. Shapiro, and S. Adhya, Eds.),pp. 597-598. Cold FRITZ, H.-J., AND EHRING, R. (1986). The upstream Spring Harbor Laboratory. Cold Spring Harbor, NY. operator of the Escherichia coli galactoseoperon is suf- RICHARDSON,J. P., FINK, P., BLANCHARD,K., AND ficient for repression of transcription initiated at the MACY, M. (1977). Bacteria with defective rho factors cyclic AMP-stimulated promoter. EMBO J. 5, 167- 173. suppressthe effectsof N mutations in bacteriophage X. MAJUMDAR,A., AND ADHYA, S. (1984). Demonstration Mol. Gen. Genet. 153, 81-85. of two operator elements in gal: In vitro repressorbind- RONECKER,H.-J., AND RAK, B. (1987). Genetic organiing studies. Proc. Natl. Acad. Sci. USA 81,6 100-6 104. zation of insertion element IS2 based on a revised nuMAXAM, A. M., AND GILBERT, W. (1980). Sequencing cleotide sequence.Gene 59, 291-296. end-labeled DNA with base-specificchemical cleavages. SAEDLER,H., BESEMER,J., KEMPER,B., ROSENWIRTH, B., AND STARLINGER,P. (1972). Insertion mutations in In Methods in Enzymology (L. Grossman and K. Molthe control region of the gal operon of E. co/i I. Biological dave, Eds.), Vol. 65, pp. 499-560. Academic Press,San characterization of the mutations. Mol. Gen. Genet.115, Diego, CA. 258-265. MERRIL, C., GOTTESMAN,M., COURT,D., AND ADHYA, S. (1978). Disco-ordinate expression of the Escherichia SAEDLER,H., REIF, H. J., Hu, S., AND DAVIDSON,N. ( 1974). IS2, a genetic element for turn-off and turn-on coli gal operon alter prophage lambda induction. J. Mol. of gene activity in E. coli. Mol. Gen. Genet. 132, 265Biol. 118, 241-245. MILLER, J. K., AND BARNES,W. M. (1986). Colony probing as an alternative to standard sequencingas a means of direct analysis of chromosomal DNA to determine the spectrum of single-basechangesin regionsof known sequence.Proc. Natl. Acad. Sci. USA 83, 1026-1030.
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WESTON-HAFER, K., ANDBERG,D. E. (I 989). Palindromy and the location of deletion endpoints in Escherichia coli. Genetics 121, 651-658. Communicated by Stuart B. Levy
22,275280
(1989)
Analysis of Relative Reversion Frequencies for IS2 Insertion Mutations in the Regulatory Region of the ga/OPETK Operon of Escherichia co/i E. MUSSO’
RICHARD
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 Received August 2 I, 1989; revised November 22, I989 Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP3 and galOPE-490, contain IS2 insertions only I bp apart in the gul regulatory region; yet only the former yields Gal+ phenotypic revertants at a detectable frequency. We have shown that the gafOPE-490 allele comprises two mutations-an IS2(1) insertion at bp+(2-6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490*) which retains the IS2 of galOPE-490 but is galE+ reveals a reversion frequency approximately 30-fold higher than that of ga/OP-3. Reversion of galOPE-490 is at least lO,OOO-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems. 0 1989 Academic
Press, Inc.
The galactose operon (galOPETK) of Escherichia coli comprises three structural genes for enzymes involved in both catabolic and anabolic metabolism of D-galactose (Fig. 1). Transcription can be initiated at either bp+ 1 or at bp-5 using overlapping promoters Pl or P2, respectively, each of which is regulated by both the cyclic AMP:CRP complex and the gal2 repressor. The former stimulates Pl and inhibits P2 and serves to monitor intracellular cyclic AMP levels which respond to the availability of glucose (Aiba et al., 198 1; Irani et al., 1989; Muss.0 et al., 1977). The gal repressor acts at two operator loci, Or (centered at bp-60) and Or (centered at bp+53) to regulate each of the promoters (DiLauro et al., 1979; Fritz et al., 1983; Irani et al., 1983a; Kunke et al., 1986; Majumdar and Adhya, 1984). Three independent pleiotrophic Gal- mutations have been previously isolated and characterized as IS2 insertions in orientation (I) (i.e., gal transcription proceeds from left ’ To whom correspondence should be addressed. ’ Abbreviations used: gal, gene(s) for galactose metabolism: Gal+, galactose fermenting; Gal-, galactose nonfermenting; IS, insertion sequence; Tn, transposon. 215
(L) to right (R) through the IS2) within the gal mRNA leader region before the galE gene (Fig. 1). The resulting severe polarity has been attributed to Rho-dependent transcription termination within the IS2 sequence (de Crombrugghe et al., 1973). Two of the mutations, galOP-3 : : IS2 (hereafter ga13) (Ahmed et al., 198 1) and galOP-308 : : IS2 (hereafter ga1308) (Ghosal ef al., 1979) appear to be identical with IS2 inserted at bp+(l to 5); the third, galOPE-490 : : IS2 (hereafter ga1490), has an IS2 at bp+(2 to 6), displaced 1 bp toward galE (Muss0 and Rosenberg, 1977). The latter was originally thought to be an insertion within galE (de Crombrugghe et al., 1973) contrary to the position determined subsequently by sequence analyses. In agreement with the genetic assignment, gal490 behaved differently than the other two (ga13/308) in each of three genetic tests (Dr. Sankar Adhya, personal communication and cited references): (a) whereas ga13lga1308spontaneously revert (by IS2 excision or by other pseudoreversion events) to Gal+ at a frequency of about lo-’ (Ahmed et al., 198 I ; Hill and Echols, 1966; Saedler et al., 1972), gal490 rarely yields revertants; (b) whereas gal3 is suppressed to a Gal+ phenotype by the rhol5(Ts) allele at 0147-619X/89
$3.00
Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form resewed.
276
SHORT COMMUNICATIONS EcoR I I
/
/
bp-100
(p,Jy
/
/
/
/
/
/
/
/
/
/
/
HiTaYf /
/
-.
-.
pi
-0, PP
L
I
I -.
-.
-.
-.
+l c -5
gal T
. .
-.
N-s
BSIEII
IS2
I gal K
-.
-. 0 I --... I E 49O(oc)
I bp+iOO
R
FIG. 1. A schematic map of the gal operon. The relative positions of restriction enzyme sites, regulatory loci, and the IS2 insertion sites (H indicates the location of the 5-bp target site for each IS2 insertion) in ga13/308 and gal490/490* are shown. The left and right termini and the Rho-dependent transcription terminator of IS2 are indicated by L, R, and t, respectively. The deletion of bacterial DNA upstream of the galoperon in phage Xpgal, is designated A320; galE490(0c) is the transversion mutation present in ga1490.
nonpermissive temperatures (Das et al., 1976, 1978),gal490 remains Gal- (galK is expressed but not galE); and (c) whereas gal3 is phenotypically suppressedto Gal+ under conditions of escapesynthesis from a X prophage, gal490 remains Gal- (galE is not expressed) (Adhya et al., 1976). Under the latter conditions of escapesynthesis pseudorevertants of gal490 can be detected as colonies that are Gal+ at high temperature but Gal- at low temperature even in the presenceof a gal inducer, i.e., D-galactose (Drs. S. Adhya and M. Gottesman, personal communication). We have characterized one such pseudorevertant, ga1490*, which is indistinguishable from gal490 by restriction enzyme digestion (data not shown). Studies described below show that ga1490* yields Gal+ revertants at a markedly higher frequency than seen for ga1490. To understand these differences we have determined (Maxam and Gilbert, 1980) the sequencesof the IS2 termini and adjacent gal DNA for gal490 and gal490*. The terminal - 100 bp of eachgal: : IS2 match the published IS2 sequence(Ronecker and Rak, 1987) but the galE sequenceof gal490 differs from the gal+ (Muss0 et al., 1974) and ga1490* sequencesby a single base pair
substitution (Fig. 1). At position bp+59 a C/G (gal+ and ga1490*) to A/T (ga1490) transversion converts galE codon 11 from Tyr(U-A-C) to an ochre (U-A-A) termination signal. The presence of this point mutation, designated galE49O(Oc), explains the failure of gal490 to yield Gal+ revertants or phenotypic suppression under Rho- conditions. In support, Gal+ revertants of gal490 can be detected in an ochre suppressor background (S. Adhya, personal communication). The galE49O(Oc) mutation also lies within the Or locus of the bipartite gal operator. We sought to determine if this mutation would interfere with gal repressor binding by using the in vivo repressor titration assay (Irani et al., 1983b). Bacterial strain SA1796 has a superrepressor allele, galR”78, which prevents expression of a wild-type galOPETK operon resulting in white colonies on MacConkey-galactose indicator plates. When transformed with a multicopy plasmid containing a functional gal operator the bacteria produce red colonies since the limiting gal superrepressor is sequesteredby the plasmid-borne operators. The results of our experiments are shown in Table 1. As negative controls we tested the plasmid vectors lacking gal DNA inserts and
277
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ASSESSMENT OFOPERATORFUNCTIONBY REPRESSOR TITRATION Test plasmid a
Relevant gal loci
Source/reference
Colony color on MGA plates”
Operator phenotype’
pBR322 pBH20 b pASTlOO’ pMIld pAST20 11’ pAST2018’ ~AST2146~ ~AST3563~
None None 0; 0: 0: 0: (490*) OI(490) 0; 0: (490*) 0; 0, (490)
Bolivar et al., 1977 Itakura et al., 1977 Hinton and Musso, 1982 Irani ef al., 1989 This study This study This study This study
W W R W W W R W
+ + -
a The indicated plasmids (at least two independent isolates for each new construct) were transformed into bacterial strain SA1796 [E. coli K12 Fgal(OPETK)+, galRs78, SmR] and two ampicillin-resistant transformants were tested by incubation on MacConkey-galactose-ampicillin (MGA) plates at 30°C for 24-36 h. Red(R), Gal+ colonies indicated the plasmid carried a functional gal operator with sufficient affinity to titrate the galR” repressorand allow expression of the chromosomal galOPETK operon. White (W), Gal- colonies indicated the plasmid-borne operator allele lacked sufficient affinity to sequesterall the superrepressor.On the basis of this Gal phenotype an operator phenotype (+ or -) is assigned,but quantitative rather than qualitative differencesare likely involved. bpBH20 has the 1acOPregion as a 204-bp HaeIII fragment at the EcoRI site of pBR322. c pASTlO has a l.l-kb EcoRI-Hind111 fragment inserted into pBR322. The insert was derived from Xpga& and correspondsto bp-650 through bp+445 relative to the gal mRNA startsite. d pMI1 has a 240-bp insert corresponding to bp-187 through bp+45 of gal located at the Hind111site of pBR322. epAST2011 has a 0.9-kb Hind111fragment inserted into pBH20. The insert is derived from Xgai490* and corresponds to bp87 1 to bp 1331 of IS2 plus bp+2 through bp+445 of galE with the latter oriented as in pASTlO and pM1 I. IpAST is identical to pAST2011 except that the 0.9-kb insert was derived from Xga1490and thus contains the galE49O(Oc) mutation. g pAST2746 was constructed from pM1 I and pAST20 11 by digesting each with BstEII plus PstI and then ligating the small fragment (containing the gal bp- 187to bp+38) of the former with the large fragment (containing gal bp+39 to bp+445) of the latter. h pAST3563 is identical to pAST2746 except that it was constructed from pMI 1 and pAST20 18 and thus contains the galE49O(Oc) mutation.
as a positive control we tested PAST100 which contains a gal insert with both 0,’ and 0:. Only the latter plasmid titrated galR” to produce a Gal+ phenotype. Although previous studies (Fritz et al., 1983; Irani et al., 1983b) have shown that partial derepression occurs with plasmids containing a single gal0 locus, neither OEnor O1alone in our plasmids would titrate the superrepressor sufficiently to produce a Gal+ phenotype. To test the 0, loci of gaZ490 and gaZ490* we used the BstEII site at bp+39 to remove the IS2 sequencesand to replace them with the normal ga10EPlP2 region from pMI 1. Plasmid pAST2746 (derived from gal490*) has a wild-type gal sequence and a fully functional operator by this assay. In contrast pAST3563 (derived from gal490)
does not titrate gal repressor and transformants remain Gal-. Thus the galE(Oc) mutation results in a operator defect. Although the quantitative effect of this mutation cannot be assessedfrom this type of assay,it does support the importance of bp+59 in the O1locus for gal repressor action. The closely related alleles ga13, ga/308, gaZ490, and ga/490* allowed us to examine how reversion to Gal+ may depend on the exact position or context of IS2 in the gal flanking sequence.To evaluate the possible effects of other IS2 copies, we conducted studies in both E. coli K12 (with eight chromosomal IS2 copies) and E. coli C (with no chromosomal IS2). We constructed isogenic strains from E. co/i K12 (an F-, cured, derivative of W1485)
278
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and E. coli C prototrophs (Hu and Deonier, 1981) with the four gul::IS2 alleles by general recombination. Using the phage XpgaPA(chlD-attL-int)3 13~1857(Merril et al., 1978), we were able to transfer each gal::IS2 allele from plasmid or bacterial chromosome to the phage and then to the chromosomal gal locus of our E. coli C and K 12 strains essentially as described by Irani et al. (1983b). Quantitative data on reversion for thesestrains were obtained using a papillation assay(Hopkins et al., 1980) to monitor the appearance of Gal+ (red) papillae within Gall (white) parental colonies on MacConkey-galactose agar medium (Fig. 2). For E. coli C gal: : IS2 strains the results after 4 days of incubation at 37°C indicated a reversion frequency (No. visible papillae/No. viable cells per colony) of 1 X IO-’ (average 1.5 papillae per colony) for gal3 or gal308 compared to 3 X 1O-7 for g&490* (average 46 papillae per colony) but gal490 exhibited no papillae (frequency < 10-l’) even after 4 weeks of incubation. Thus we saw no indication of enhanced double reversion or “adap-
tive evolution” with gal490 contrary to observations with other systems as reported by Cairns et al. (1988) and Hall (1988). Nearly all Gal+ revertants in E. coli C arose by excision (deletion) of IS2 based on the following three tests conducted on over 25 independent papillae from each of the gal3 and gal490* strains: (i) all Gal+ revertants behaved as Rho+ basedon the inability of XNam7Nam53cI857 phage to grow lytically (Richardson et al., 1977);(ii) all Gal’ revertants were stable based on the lack of Gall segregantsafter growth for 30 generations;and (iii) all Gal’ isolateslacked IS2 based on colony blot (Miller and Barnes, 1986)hybridization with an IS2-specificprobe. Thus, if Gal+ pseudorevertants occur as rhosuppressors or IS2 sequence perturbations, they account for a minor fraction of the papillae observed in theseexperiments. Previous studies in E. coli K12 with gal3/gu1308 found that IS2 excision can account for 30 to 90% of the Gal+ revertants (Ahmed, 1981; Morse and Pollack, 1969; Saedler eLal., 1974). Even in the E. coli K 12 background gal490 failed to revert at a detectable rate, whereas
FIG. 2. Relative reversion frequencies for different gal: :IS2 alleles. E. coli C with chromosomal alleles ga/490* (A, C, E), gal490 (B, F), or gu/3 (D) were incubated on MacConkey-galactose medium at 37°C and a density of - 10 colonies per 90 cm diam plate for 2 days (A), 4 days (B, C, D), or 6 days (E, F) before photography of typical colonies through a dissecting microscope at 60x magnification; red (Gal+) papillae appear as black spots within the large white (grey) Gal- parental colonies.
SHORT COMMUNICATIONS
the g&490* allele reverted - 15fold more frequently than gal 3 or gu1308. Since the K12 strain has seven to eight additional chromosomal copies of IS2 (Hu and Deonier, 1981) it seems unlikely that differences in IS2-encoded factors are responsible for the different reversion frequencies. The 15 to 30-fold difference in reversion of gal 3/308 and ga1490* is most likely due to the 1-bp shift of IS2 in the gal sequenceand thus resemblesthe results reported for deletion of Tn5 derivatives located 1 bp apart in the ampicillin-resistance gene of pBR322 (DasGupta et al., 1987). In the latter caseit was suggestedthat the drastic effect of position on deletion frequency might be explained by differences in the symmetry of DNA sequences spanning the deletion endpoints. Comparable changes are observed in the symmetry of sequences flanking IS2 in gal31308 and gu1490*, but the relevance of such symmetries has not been determined. ACKNOWLEDGMENTS I thank Drs. Sankar Adhya and Max Gottesman for providing bacterial and phagestrainsincluding the g&490* mutation and for communication of unpublished results. I also thank Ms. Tara Hodam for excellent technical assistance, Mr. Robert Sullivan and Ms. Byrne Burch who helped construct plasmids, and Ms. K. French for typing the manuscript. This work has been supported by the N.I.H. Biomedical ResearchSupport Award S07RR07 160 to the University of South Carolina and by the American Cancer Society Grant MV-2 14. REFERENCES ADHYA, S.,GOTTESMAN,M., DECROMBRUGGHE, B., AND COURT,D. (1976). Transcription termination regulates gene expression. In “RNA Polymerase” (R. Losick and M. Chamberlin, Eds.),pp. 7 19-730. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. AHMED,A. (1977).The ga13mutation of E. co/i. In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. Shapiro, and S. Adhya, Eds.), pp. 37-48. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. AHMED, A., BIDWELL,K., AND Musso, R. E. (1981). Internal rearrangements of IS2 in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 45, 141- 152. AIBA, H., ADHYA, S., ANDDECROMBRUGGHE, B. (198 1). Evidence for two functional gal promoters in intact Escherichia coli cells. J. Biol. Chem. 256, 11,90511,910.
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