JOURNAL OF BACTERIOLOGY, Aug. 1972. p. 383-391 Vol. 111, No. 2 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Characterization of Strong Polar Mutations in a Region Immediately Adjacent to the L-Arabinose Operator in Escherichia coli B/r' MARIANNE ELEUTERIO, BARBARA GRIFFIN, AND DAVID E. SHEPPARD Department of Biological Sciences, University of Delaware, Newark, Delaware 19711 Received for publication 12 April 1972 Seven L-arabinose-negative mutations are described that map in three genet- ically distinct regions immediately adjacent to the araO (operator) region of the L-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dom- inant, trans-recessive polarity effect upon the expression of L-arabinose isom- erase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initi- ator region of the L-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens di- ethyl sulfate and ICR-191. Three of these mutants are suppressible by two in- dependently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara+ revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppres- sors, and their partially polar revertants are discussed in terms of the mecha- nism of initiation of expression of the L-arabinose operon. The initiator region of the L-arabinose op- induction, although in vitro ara DNA-activator eron has been postulated as the site at which binding studies have not yet been performed the activator (the araC gene product) attaches with ara DNA containing a deletion mutation to the deoxyribonucleic acid (DNA) to initiate in the initiator region (31). (ii) L-Arabinose- transcription of the polycistronic message (see positive revertants derived from a strain con- Fig. 1) (7-9, 11, 26, 31, 32, 37). The operator taining a deletion of the araC gene and the region, on the other hand, has been postulated operator (araA OC719 map in the region on the to be the site of interaction with the repressor araB side of the operator (8, 10). These mu- form of the araC gene product. This region, tants, designated initiator-constitutive (araIc), which is genetically defined by araA OC719, is exhibit a low constitutive expression of genes nonessential for full operon induction (9). Two araB, A, and D and are unaffected by inducer classes of mutations have been used to define unless an araC gene is introduced in the trans operationally the initiator region. (i) Deletion position, in which case they are inducible to mutations that encompass the region between the wild-type level. Thus two mutationally al- araB and araO exhibit absolute polarity with tered forms of the initiator have been identi- regard to the expression of genes araB, A, and fied, one in which the initiator is nonfunc- D, even when an araC gene is placed in the tional because it has been removed, and a trans-position to provide a source of activator second type in which the initiator region can (26). Presumably, in these mutants, activator function in some as yet undescribed way to can not make the contact necessary to achieve achieve low levels of operon expression in the absence of activator. 'One portion of this work was submitted by M. E. to the It would appear that the initiator region of Department of Biological Sciences, Univ. of Delaware, in an operon under positive control may have partial fulfillment of the requirements for the Doctor of Phi- many features in common with the promoter losophy degree. A second portion of this work was submitted by B. G. to the same department in partial fulfillment of the region described for negative control systems requirements for the Bachelor of Arts degree, with distinc- (1, 14). It may well be that the initiator con- tion. sists of two binding sites, one specific for the 383 384 ELEUTERIO, GRIFFIN, AND SHEPPARD J. BACTERIOL. activator and one specific for ribonucleic acid strain SB3002 (Hfr-Bl leuBl his-15). Thr+, Leu+, and (RNA) polymerase. If the initiator region is a Ara- recombinants were then crossed with strain promoter-like element, a class of point mu- SB3118 (F araD139/aaD139) to identify recombi- nants the tants should exist that would partially or com- that contained only polar mutation. Map- ping studies were performed with these strains. pletely reduce the affinity of the initiator re- Experimental strain construction. With the use gion for activator, or RNA polymerase, or of P1 transducing phage, each L-arabinose polar both. Such mutants should exhibit a reduced point mutation was transferred by cotransduction level of operon expression. This paper de- with thr-1+ into strain UP1004 (thr-1 leuB1). Thr+ scribes a series of point mutations, lying im- transductants were then replica plated to identify mediately to the left of araO (as represented in leuBi recombinants containing the L-arabinose polar Fig. 1), that exhibit properties consistent with point mutation. All enzyme analyses, merodiploid this prediction. analyses, and mutagen reversion studies were carried out with these strains. Because all of the mutations MATERIALS AND METHODS had been transduced into strain UP1004, all of these Media. L broth medium contained 1% tryptone strains should be isogenic except for the thr ara leu (Difco), 0.5% yeast extract, 0.5% NaCl, and 0.1% glu- region. cose. Tryptone broth medium contained 1% tryptone Isolation and classification of nonsense sup- and 0.5% NaCl. Tryptone agar medium was the pressor mutants. Mutations araA4, araB902, and same as tryptone broth medium except that 1.1% araB904 had previously been transduced into strain agar (Difco) was added. For tryptone soft agar, 0.7% UP1004 as described above. Different nonsense sup- agar was added. Mineral base contained 0.7% pressor mutations were then induced in these other- K2HPO4, 0.3% KH2PO4, 0.1% (NH4)2SO4, and wise isogenic strains by selecting for Ara+ revertants 0.01% MgSO4 v 7H20. Mineral-L-arabinose medium on mineral-L-arabinose-leucine agar medium. Non- contained mineral base plus 0.2% L-arabinose. L- sense suppressors were distinguished from primary Arabinose-casein hydrolysate medium was mineral site revertants by their ability to suppress a series of base with 0.2% L-arabinose and 0.1% casein hydroly- T4 amber or UGA phage mutants (Table 2) (22). sate (Nutritional Biochemical Corp., Cleveland, sup-3 (BG5002), derived from a strain carrying ara- Ohio) added. EMB-L-arabinose medium contained B904, was only active against T4 UGA phage, thus 0.45% NaCl, 0.2% K2HPO4, 0.04% eosin Y, 0.006% identifying it as a UGA suppressor (3, 23). sup-1 methylene blue, 0.76% NZ Case (Sheffield Chemical (BG5005) and sup-2 (BG5006), derived from strains Co., Oneota, N.Y.), 0.1% yeast extract, and 1.0% L- carrying araA4 and araB902, respectively, were both arabinose. Amino acids, 0.004% final concentration, active against all of the T4 amber phage mutants, and agar, 1.5% final concentration, were added as thus identifying them as amber or ochre suppressors needed. Casein hydrolysate medium contained min- (4). sup-2 is presumed to be an ochre suppressor for eral base plus 1.0% casein hydrolysate. the following reason. sup-2 relieves the polarity (35) Bacterial and phage strains. Table 1 lists some generated by the nonsense codon in araB14 and of the strains used in this study. phenotypically suppresses araB902 to the Ara+ phe- Isolation of strong polar mutants. All of the notype whereas sup-1 relieves the polarity of only strong polar mutants described in this study were araB14 previously demonstrated to be an amber derived from strain SB5003 (thr-1 araD139) as pre- mutation (16) (see Table 5). This suppression pattern viously described (27). The araD139 mutation was can be most easily explained by assuming that araA4 removed from each mutant by conjugation with and araB14 are amber mutations and araB902 is an TABLE 1. Bacterial strains Strain Matingtype Genotype Source UP1004 F- thr-1 leuBl str-1 (26) SB3002 HfrB1 leuBl his-15 (26) SB5003 F- thr-1 araD139 (17) SB3118 F'ara araD139/araD139 (26) ME1240 F'ara araAlOC2202/araAIOC2202 This paper ME1242 F'ara araAIOC2204/araAIOC2204 This paper ME1247 F'ara araAIOC2209/araAIOC2209 This paper ME1253 F'ara araABIC768/araABIC768 This paper ME1260 F'ara araAOC719/araAOC719 This paper BG2016 F'ara ara 1016/ara 1016 This paper BG2036 F'ara ara 1036/ara 1036 This paper BG5002 F- araB904 leuBl sup-3 (UGA) This paper BG5005 F- araA4 leuBl sup-1 (amber) This paper BG5006 F- araB902 leuBl sup-2 (ochre) This paper BG7002 F- araA4 leuBl sup+ This paper CR63 supD (amber) VOL. 111, 1972 STRONG POLAR MUTATIONS ADJACENT TO araO 385 ochre mutation. Thus sup-2, an ochre suppressor, TABLE 2. T4 amber and UGA phage mutant would suppress both amber and ochre mutations, suppression patternsa whereas sup-i would be limited to amber codons (4). T4 amber mutants were generously supplied by S. BG BG BG Person, Department of Biophysics, Pennsylvania BG 5005 Phage 7002 sup-I 506 02 CR 63 State Univ. (22). T4 UGA mutants were generously sup-2 5up02 sup + (am- sUp-3 supplied by L. Russell, Univ. of California, Los An- ber)(che(UAsup-2e geles. Identification of Ara- mutations immediately T4 amber phage adjacent to the operator. From a large number of N 122 - + + - + Ara- mutants, eight were identified with the fol- N 133 - + + - + lowing properties.