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Direct Selection and Analysis of Mutational Events Which Diminish

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					JOURNAL OF BACTERIOLOGY, Jan. 1980, p. 422-426                                                      Vol. 141, No. 1
0021-9193/80/01-0422/05$02.00/0


   Direct Selection and Analysis of Mutational Events Which
      Diminish the Level of Expression of the trp Operont
                         JOHN C.    ANDREWSt       AND RONALD L.      SOMERVILLE*
               Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

            A new procedure was devised for selecting, from lac+ galE strains of Esche-
          richia coli, mutants resistant to galactoside-induced lysis. When applied to tip-
          lac fusions, our method yields down mutations in the trp promoter.
  The metabolism of lactose and related ,B-D-                 into the tip-lac fusion W205 (23) (Fig. 1). We
galactosides in Escherichia coli is mediated by               then perfected a selection procedure which gen-
the gene products of the lac operon. The hy-                  erated several galactose-sensitive, galactoside-
drolysis of such disaccharides, catalyzed by ,B-              resistant mutants, one of which was character-
galactosidase (lacZ gene product), produces in-               ized as a promoter mutation. The bacterial
tracellular D-galactose, which is further metab-              strains utilized (Tables 1 and 2) were all sub-
olized by the enzymes of the gal operon. In the               strains of E. coli K-12. The F'tip+ episome (5)
presence of a neutral carbon source, exposure of              carries the chromosomal markers att80, tonB+,
a galE strain (deficient in uridinediphosphoga-               trpPOEDCBA+, and cysB+.
lactose 4-epimerase) to galactose or a hydrolyz-                 Because galactoside-induced lysis of sensitive
able ,B-D-galactoside causes cell lysis (21), owing           cells ultimately leads to the release of free galac-
to the accumulation of UDP-galactose (6). Bac-                tose, precautions must be taken in any selective
teriolysis induced in this manner constitutes se-             scheme to prevent the subsequent lysis of the
lective pressure suitable for examining a range               desired galactoside-resistant, galactose-sensitive
of genetic events that reduce lac operon expres-              cells. Seeding selective medium with an auxo-
sion (14). The acquisition of resistance to f8-D-             trophic gal+ gaiR strain prevents such undesir-
galactosides by galE mutants may be attribut-                 able lysis. Strain B78AHis, a histidine auxotroph
able to primary events which generate galE+                   of a gaiR strain first isolated by Buttin (4),
revertants or to secondary mutations within the               fulfills this "scavenger" function.
lac and gal operons. By screening a collection of                Selections (Table 2) were performed in both
,B-D-galactoside-resistant galE mutants for re-               tipR (A8-W205) and trpR+ (A7-W205) back-
tention of galactose sensitivity, Malamy identi-              grounds. Independent L-broth cultures of each
fied several lac operon-specific mutational                   parental strain were grown overnight at 37°C.
events (14).                                                  The samples were centrifuged, washed twice,
   In strains carrying compound operons, such                 and resuspended in twice the original volume of
as the trp-lac fusions X7713 (18, 20, 22) and                 saline. Samples (0.1 ml) of these suspensions
W205 (24), lac gene expression is directly con-               were spread on selective medium. Each plate
trolled by the tryptophan regulatory elements;                was also seeded with 5 x 109 washed cells of
repression by exogenous tryptophan results in                 mutant B78AHis. The plates were incubated at
low-level synthesis of both tip- and lac-specific             370C for 4 days. Ten colonies from each selection
gene products (16, 23). Transcription initiated at            plate were picked and screened for galactose
the trp promoter produces a compound trp-lac                  sensitivity by streaking on medium with 0.2%
message whose translation results in the coor-                galactose. From this initial screening, we ob-
dinate synthesis of fully active tip- and lac-spe-            tained a collection of phenylgalactoside (PG)-
cific enzymes (23). In a galE derivative of such              resistant, galactose-sensitive derivatives of A8-
a fusion strain, resistance to f8-D-galactosides              W205 and A7-W205. On the basis of fi-galacto-
should include tip-linked mutations, such as trp              sidase activities and zone size upon exposure to
polar mutations and down mutations in the pro-                PG, representative mutant stocks were pre-
moter, which reduce the efficiency of expression              served for further study. fi-Galactosidase levels
 of the trp operon.                                           were precisely determined for each of the se-
    In this study, a galE lesion was introduced lected isolates, as were galactose- and PG-in-
                                                              duced zones of lysis. It was evident that PG
   t Journal paper no. 7675 of the Purdue University Agricul- resistance can result from several distinct mu-
tural Experiment Station.
   t Present address: Department of Microbiology, Bur- tational events as follows.
roughs-Wellcome Co., Research Triangle Park, NC 27709.           (i) lacY lesions prevent galactoside transport
                                                      422
VOL. 141, 1980                                                                                                                   NOTES          423
       trpE       trpD     trpC trpB trpA lcZ lacY lacA                               TABLE 1. Derivatives of trp-lac fusion strain W205
p,                                __                                trp-loc   mRNA
                                                                                          Bacterial strain              Pertinent genetic markersa
                                                                                     W205                           A(tonB-lacIPO)
              CH20H                                 CH20H                            W205 R-                          (tonB-lacIPO) trpR
                                                                                     cysB W205                      A(tonB-lacIPO) cysB trpR
              OHKOH
                  P-galoctosidose
                                                                 H.OH
                                                                                     cysB W205 R+                   A(tonB-lacIPO) cysB
                      OH                                    OH                       thr W205                       I(tonB-lacIPO) thr
                                               D-goloctose                           A7-W205                        A(tonB-lacIPO) galE
                                                                                     A8-W205                          Q(tonB-lacIPO) trpR galE
UDPG              -   UDPGal          -   Gal-I-P                                    PG3.lb                         A(tonB-lacIPO) trpR galE
              E                   T                  K                                                                 trpP0l1
   FIG. 1. The W205 trp-lac fusion (23) and its rela-                                W205 R- (PG3-1)/F'tipc A(tonB-lacIPO) trpR trpPIOI/
tionship to selection for diminished exprcssion of the                                                                 F'trp+ tonB+
                                                                                     W205 R+ (PG3-1)C               A(tonB-lacIPO) trpPlOl
trp operon. The lac end of the deletion removes lacP                                 W205 R- (PG3-1)c               A(tonB-lacIPO) trpR trpPlOl
and lacO but leaves the lacZ gene intact. The trp
functions are indistinguishable from the wild type                                      aW205 and all its derivatives carry (proAB-lac) deletion
(20). Depression of the trp operon, as in trpR mutants,                              XIII (24). trpPlOl is our allele number for the down mutation
results in a 12- to 13-fold increase in fl-galactosidase                             in the promoter carried by strain PG3-1 (see text).
                                                                                        b Selected in this study as PGr Gal mutants.
synthesis (16). Galactose liberated by the hydrolysis                                   c
                                                                                          Constructed to characterize PG3-1 mutation.
of appropriate 3- D -galactosides enters central metab-
olism following phosphorylation catalyzed by galac-
tokinase (K), transglycosylation catalyzed by galac-                                      TABLE 2. Miscellaneous bacterial strainsa
tose 1-phosphate uridyl transferase (T), and epimer-                                        Strain                   Relevant genetic markers
ization of the 4'-hydroxyl of UDP-galactose, cata-
lyzed by epimerase (E). Cells unable to catalyze the                                 W3110                      Wild type
final reaction lose viability, owing to the accumula-                                YS57/F'trp                 Ftrp+ tonB/his pro A(trpE-A-
                                                                                                                  tonB)
tion of UDP-galactose. Strain W205 lacks the tonB                                    His- B78A                  F- galR lacY str leu thi his
locus, which specifies a protein involved in phage                                   YS57/F'(PG3-1)             his pro A(trpE-A-tonB)/
penetration and iron transport (29). This leads to a                                                              F'trpP101
requirement for Fe. To allow maximum growth,                                         ALD102                     &(tipE-D)
FeSO4 (25 ,uM) was routinely added. Our basal mini-                                  SP-333                     tna-2 trpR AtrpLD102
mal medium was the forrnulation of Vogel and Bon-                                    W3110 (PG3-1)b             trpPlOl
ner (28). The Fe present in Difco agar, tryptone, or                                 W3110 (PG3-1)/F'lO1"       trpPlOl/F'tipB
yeast extract is sufficient to allow normal growth (16).                             W3110 (PG3-1)/FVA2b        trpPlOl/F'tipA
                                                                                     1I1/F'(PG3-1)b             trpB/F'tipPlOl
                                                                                     A2/F'(PG3-1)b              trpA/F'trpPlOl
and are therefore unable to utilize melibiose at                                     W3110/F'101                cysB/F'trpB
                                                                                     W3110/F'A2                  cysB/F'trpA
420C (18).                                                                           101/F'trp+                  cysB t7pB/F'trp+
   (ii) lacZ lesions prevent galactoside hydrolysis                                  A2/F'trp+                   cysB trpA2/F'trp+
and were resistant to PG- and lactose-induced                                           a Unless otherwise specified, media are as described by
bacteriolysis but sensitive to galactose and mel-                                    Miller (17). Vogel and Bonner (28) salts solution (medium E)
ibiose. Activity determinations directly verified                                    supplemented with 0.2% glucose was used as minimal medium.
the absence of ,B-galactosidase. Normal expres-                                      In some cases glucose was omitted and a neutral carbon
sion ofthe trpAB genes was observed. This                                            source, 0.2% sodium succinate or glycerol, was substituted.
                                                                                     For cultivation of strain W205 and its derivatives (all of which
evidence, coupled with the demonstration of                                          harbor a pro-lac deletion), L-proline (20 1&g/ml) and thiamine
wild-type 5-methyl-DL-tryptophan (5-MT) re-                                          hydrochloride (1 jig/ml) were routinely added. Repression of
sistance (data not shown), proved that the ab-                                       the tip operon was achieved at 20 jig of L-tryptophan per ml.
sence of lacZ gene expression was not a trp                                          L-broth is described by Lennox (13). Selective minimal me-
                                                                                     dium was supplemented, for A8W205, with 2 mg of sodium
operon-specific phenomenon.                                                          succinate per ml, 1 mg of phenylthiogalactoside per ml, 50 Ag
  To conclusively identify such lesions as lacZ                                      of PG per ml, L-prolne, L-tryptophan, and thiamine hydro-
mutations, W205 R+ Lac- isolates were trans-                                         chloride; for A7W205, medium was supplemented with 2 mg
duced to Lac' with X plac5. This phage carries                                       of sodium succinate per ml, 0.5 mg of phenylthiogalactoside
                                                                                     per ml, 100 mg of PG per ml, L-proline, L-tryptophan, and
only the lacZ+ locus (17), and hence the appear-                                     thiamine hydrochloride.
ance of Lac' transductants restricts the lac le-                                       b Strains constructed in this study to characterize the trp
sion to a sequence intemal to the lacZ gene.                                         mutation of the PG3-1 isolate.
   (iii) Some cysB-linked, trp-specific lesions
were predicted to diminish gene expression                                           mulates no pathway intermediates (determined
across the entire trp-lac fusion, thereby reducing                                   by specific color tests for anthranilic acid, indole,
lac activities. Strain PG3-1, isolated in the W205                                   and indoleglycerolphosphate), it grows slowly in
trpR galE background, displays properties con-                                       the absence of tryptophan. When the galE le-
sistent with a mutation of this type (Tables 3                                       sion was overcome by transduction with A gall8,
and 4). Although the strain is Trp+ and accu-                                        the strain failed to grow on lactose as the sole
424     NOTES                                                                                       J. BACTERIOL.
                                 TABLE 3. Characterization of PG3-1 mutationa

      Strain                 16-Galactoaidase activity        Tryptophan syn-       Zone of growth inhibition (mm)'
                             0         +trp         +IA          a                   Gal      PG 5-MTd IAe
A7W205                       450        120        2,500         -       -           46       41       33        0
A8W205                     1,350      1,000        4,900        5.2     5.2          50       48        0        0
PG3-1                        170        119         -           1.1     1.3          38       32       44       30
(PG3-1)/F'tip +              113        104         -            -       -           37       32        0       -
W205 R+ (PG3-1)              170        125        140           -       -           -        -        58       36
W205 R- (PG3-1)              170        160        250          1.2     1.2          -        -        45       30
W3110                          -         -          -           2.0     2.0          -        -        25        -
W3110 (PG3-1)                  -         -          -           0.9     0.9          -        -       64       20
YS57/F'trp+                    -         -          -           6.7     6.3          -        -       25       -
YS57/F' (PG3-1)                -         -          -           1.6     2.5          -        -       46
   a Earlier work (16) had demonstrated that the f?-galactosidase of stationary E. coli cells was metabolically
stable, thus allowing appreciable latitude in the choice of cell growth times before assay. To ensure that the
cultures had achieved balanced growth at the time of assay, the inoculum was always pregrown for 12 to 15 h
under the same conditions used in preparation of the cells for determination of enzymatic activity. The cells to
be assayed were cultivated for 12 to 15 h in minimal liquid medium containing 20,ug of FeSO4 per ml and
required nutrients or acid-hydrolyzed casein (0.1%). jB-Galactosidase was assayed by the procedure of Reznikoff
et al. (24).
   bTryptophan synthetase a and 2 specific activities were assayed in the presence of excess complementing
subunit, according to the method of Smith and Yanofsky (25). A unit of tryptophan synthetase is that amount
of enzyme which will convert 0.1,umol of indole to tryptophan in 20 min at 370C. Crude extracts were prepared
by sonication or in a French pressure cell, and protein concentration was estimated by the method of Lowry et
al. (13).
   '
     Growth inhibition by 5-MT, a semiquantitative indication of the level of trp gene expression, was estimated
by diluting an overnight L-broth culture 1:10 or 1:100 in saline, then plating 0.1 ml on a minimal agar plate
containing required supplements. A sterile filter paper disk (13 mm) on the plate surface was saturated with 20
fd of a 2-mg/ml solution of 5-MT. The plate was incubated at 37°C for 15 to 20 h, or until confluent cell growth
around the perimeter of the plate was apparent. The diameter of any clear zone around the disk was measured.
The sensitivity of strains carrying a galE lesion was demonstrated in a similar way, except that the cells were
plated on a medium containing a neutral carbon source such as succinate or glycerol. The Penassay disk was
saturated with 10 or20 ul of a 2% sugar solution. Sensitivities to galactose (to verify the galE lesion), lactose, or
PG were used to assess the extent of expression of the lac-specific functions. A difference in zone diameter of 4
 mm or greater was considered significant.
    dA
        20-ul portion of a 1-mg/ml solution of 5-MT was used in the same manner as described above.
    'A40-pl   portion of a 4-mg/ml solution of indoleacrylic acid (IA) was used as described above.

carbon source. Both f8-galactosidase and tryp-              strains) activities is apparent. This inability to
tophan synthetase activities were significantly             respond to the tryptophan derepression signals
lower than the parental activities under condi-             mediated by indoleacrylic acid results in in-
tions of tryptophan starvation. The PG3-1 lesion            creased sensitivity to this compound and to
was transferred into a standard background both             other tryptophan analogs (Table 3).
by cotransduction with cysB+ (supporting a lo-                The location of the lesion in strain PG3-1 was
cation within the trp operon) and by transduc-              established genetically using a test devised by
tion using strains harboring trp deletions (spe-            Zurawski et al. (30) for attenuator mutants.
cifically AtrpLD102 and AtrpEDCBA) as recipi-               Phage P1 kc lysates prepared on PG3-1 were
ents. A more complete analysis (Table 3) of the             used to transduce tna-2 trpR AttrpLD102 to pro-
enzyme activities in wild-type and trp-lac fusion           totrophy. Among the trp+ transductants were
backgrounds, coupled with increased sensitivi-              found recombinants fully resistant to high levels
ties to the tryptophan analogs 5-MT and indo-               of 5-MT. The same recombinants were growth-
leacrylic acid, proved that the lesion in PG3-1 is          inhibited on media containing indole (5,ug ml-')
a cis-dominant mutation which reduces the                   and 5-methylanthranilic acid (100 ,ug ml-1).
expression of the trp operon independently of               Since the tipLD102 deletion removes most of
repression mediated by the trpR system.                     the trp leader sequence as well as trpE and trpD,
   In strains harboring the galactoside-insensi-            the wild-type allele of PG3-1 must lie outside
tive mutation PG3- 1, an approximately fivefold             the region of DNA deleted in At7pLD102. PG3-
reduction in derepressed levels of tryptophan               1 cannot be a regulatory mutation in the leader
synthetase and ,B-galactosidase (in fusion                  region similar to those characterized by Zu-
VOL. 141, 1980                                                                                       NOTES          425
    TABLE 4. cis-dominance of PG3-1 mutation               impose tryptophan auxotrophy, but also cause
                          Tryptophan             E/C-
                                                           severe transcription termination, thereby reduc-
       Strain              synthetase                      ing the synthesis of downstream gene products
                                                           (26). Nor did our procedures yield changes in
                            a       ,        a             the tip attenuator (2, 3, 11), similar to those
W3110/F' Olb                4.1      1.6   1.7       -     found by Zurawski et al. (30), or in the tip
W3110/F'A2b                  1.3    2.7     -       1.7    operator (7, 15). In an earlier study, we isolated
101/F'trp+b                  3.4    2.5    2.9       -     a number of E. coli strains displaying enhanced
A2/F'trp+b                   2.6    3.2     -       1.5    sensitivity to 5-MT (12). All had partial defects
W3110 (PG3-1)/F'101C         9.7 4.2       2.3      -
                                                           in one of the trp structural genes. It is not clear
W3110 (PG3-1)F'A2c           2.6    9.0     -       2.5
101/F'(PG3-1)c             10.1      1.1   0.12      -     at present why the aforementioned studies failed
A2/F'(PG3-1)c               0.65 10.9       -       0.06   to yield promoter-down mutations such as that
                                                           in strain PG3-1, but these results, taken together
   aE/C values quantitate the amount of enzyme             with ours, emphasize the desirability of employ-
made from episomal genes relative to that from chro-       ing a variety of selective procedures in mutant
mosomal genes (for details see Stetson and Somerville
[27]). By positioning the PG3-1 mutation at each           hunts directed toward identifying regulatory ele-
location it is demonstrated above that E/C increases       ments.
when PG3-1 is on the chromosome and decreases                 In selective schemes that rest upon galacto-
when on the episome. This phenomenon verifies the          side sensitivity mediated by galE mutations, it
cis-dominance of the PG3-1 mutation. (Tryptophan           is of paramount importance that one avoid con-
synthetase specific activities are defined in footnote b   ditions that might lead to the accumulation in
of Table 3.)                                               the selection media of free galactose, since this
   b These values were determined for cells grown in       compound is readily taken up and converted to
unsupplemented Vogel and Bonner minimal medium.            UDP-galactose. Even moderate levels of galac-
See Stetson and Somerville (27).
   c
     These values were detennined in this study for        tose generated in situ may eliminate or diminish
cells grown in medium supplemented with 0.1% acid-         the survival of the galE Lac- cells that are the
hydrolyzed casein.                                         object of a mutant hunt. Our procedures effec-
                                                           tively overcome the problem of nonspecific kill-
rawski et al. (30), and it is most probably a              ing due to galactose accumulation in the selec-
change that diminishes the normal functioning              tion media. Phenylthiogalactoside, a weak com-
of the trp promoter, based upon the following              petitive inhibitor of ,B-galactosidase, also inhibits
                                                           the induction of the gal operon (4). Both of
lines of evidence. First, it is a cis-dominant mu-         these effects "tighten up" the selection proce-
tation which severely restricts the expression of          dure. The inclusion of a nongrowing scavenger
adjacent trp (and lac, in the case of fusion               strain constitutive for the uptake and catabolism
strains) genes (Table 4). Second, it lies outside          of galactose (in our case, strain B78A his) further
of the trp leader region, because the wild allele          reduces the likelihood that galE Lac- cells will
of PG3-1 is carried by the attenuator deletion             perish during the period of exposure to selection.
AtrpLD102 (8, 9). We have designated the mu-
tation as trpPlOl.                                            This work was supported by Public Health Service grant
   In many respects the PG3-1 lesion resembles             GM-22131 from the National Institute of General Medical
a naturally occurring promoter-down mutation               Sciences.
                                                              We thank C. Yanofsky, W. Reznikoff, M. Feiss, and H.
discovered in Shigella dysenteriae 16 by Mioz-             Echols for bacterial and phage strains. The capable technical
zari and Yanofsky (19). In strain PG3-1, the trp           assistance of Linda Eades and Barbara Nicodemus is grate-
or trp-lac operon functions at a low constitutive          fully acknowledged.
level (about 10% of normal) and is not subject to
further reduction by tip holorepressor. Because                              LITERATURE CITED
a small but significant increase in downstream             1. Beckwith, J. R., E. R. Signer, and W. Epstein. 1966.
expression is seen following tryptophan starva-                 Transposition of the Lac region of E. coli. Cold Spring
                                                                Harbor Symp. Quant. Biol. 31:393-401.
tion (Table 4, line 6), we argue that the normal           2. Bertrand, K., L. Korn, F. Lee, T. Platt, C. L. Squires,
trp attenuation mechanism remains functional.                   C. Squires, and C. Yanofsky. 1975. New features of
The relative inability of strain PG3-1 to undergo               the regulation of the tryptophan operon. Science 189:
derepression is accompanied by increased sen-                   22-26.
                                                           3. Bertrand, K., C. Squires, and C. Yanofsky. 1976. Tran-
sitivity to 5-MT and indoleacrylic acid, as seen                scription termination in vivo in the leader region of the
by enhanced zones of inhibition on solid medium                 tryptophan operon of Escherichia coli. J. Mol. Biol.
(Table 3).                                                      103:319-337.
   Notably absent from our collection (over 100            4. Buttin, G. 1963. Mechanismes regulateurs dans la bio-
                                                                synthese des enzymes du metabolisme du galactose chez
in all) of galactoside-resistant strains are polar              Escherichia coli K,2. II. Le determinisme genetique de
tip mutants. These pleiotropic lesions not only                 la regulation. J. Mol. Biol. 7:183-205.
426         NOTES                                                                                                   J. BACTERIOL.
 5. Fredericq, P. 1969. The recombination of colicinogenic                 Ippen, E. R. Signer, and J. R. Beckwith. 1970.
      factors with other episomes and plasmids, p. 163-178.                Fusions of the lac and trp regions of the Escherichia
      In G. E. W. Wolstenholme and M. O'Connor (ed),                       coli chromosome. J. Bacteriol. 104:1273-1279.
        Bacterial episomes and plasmids. Little, Brown and Co.,    19.   Miozzari, F., and C. Yanofsky. 1978. Naturally occur-
        Boston.                                                            ring promoter down mutation: nucleotide sequence of
 6.   Fukaswa, T., and H. Nilkaido. 1961. Galactose sensi-                 the trp promoter/operator/leader region of Shigella
        tive mutants of SalnoneUa. H. Bacteriolysis induced                dysenteriae 16. Proc. Natl. Acad. Sci. U.S.A. 75:5580-
        by galactose. Biochim. Biophys. Acta 48:470-482.                   5584.
 7.   Hiraga, S. 1969. Operator mutants of the tryptophan          20.   Mitchell, D. H., W. S. Reznikoff, and J. R. Beckwith.
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