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Divergent orientation of transcription from the biotin locus of Escherichia coli
J. Mol. Biol. (1971) !%,53-62
Divergent Orientation of Transcription from the Biotin Locus of Escherichia coli ARABINDA GUHA of Microbiology, Erindate College and Department of Medical Cell Biology, University of Toronto, Toronto 181, Ontario, Canada
Dqmrtment
Y. SATUREN AND W. SZYBALSKI
McArdle Laboratory, University of Wisconsin, Wk. 53706, U.S.A.
Madison
(Received 12 May 1970) Pulse-labeled [3H]RNA produced by the bio locus of Escherichia coli grown in biotin-free medium hybridizes with both DNA strands of %iot124, a bio-transducing phage that contains the entire bio locus, with an I: r strand ratio of 25 : 75 (fully derepressed in bio- mutants) or 40 : 60 (partially repressed by endogenous hiotin in bio + cells). When employing DNA strands of Atiol, which contains only genes A and B, the latter 1: r ratio becomes 89 :ll, with the amount of r3H]RNA hybridizing with the E strand remaining unchanged. These results indicate that both strands of the tie locus are transcribed and the orientation of the two transcriptions is divergent, with cistron A belonging to the leftward (counterclockwise) transcribed operon, and the other bio cistrons forming a rightward (clockwise) oriented operon. The bio transcription from both strands is subject to co-ordinate repression by biotin. The Ko[~H]RNA amounts to as much as 0.6% of the total pulse-labeled RNA in fully derepressed E. coli bio- cells, and is reduced to approximately 0.2% in bio+ cells grown in biotin-free medium, and to undetectable amounts in medium supplemented with more than 2 x low3 g biotin/ml. Desthiobiotin does not act as a corepressor unless converted to biotin.
1. Introduction synthesis in Eschmichia coli is controlled by the genes of the biotin locus (denoted bio throughout this communication and bioA by Taylor & Trotter, 1967), which are situated to the right (clockwise) of the attachment site, &bb’, for the h prophage. The locus is composed of a cluster of genes, the order of which, in relation to the neighbouring bacterial markers, is shown in Figures 1 and 2 (de1 CampilloCampbell, Kayajanian, Campbell & Adhya, 1967; Rolfe, 1970). The pathways of biotin biosynthesis follow the sequence. Biotin
I b PA c
II 7KAP -
F
III DAP -
A
IV DTB -
D
biotin, B
where PA is pimelic acid, 7KAP is L-7-oxo4Laminopelargonic acid, DAP is DL-7,8diaminopelargonic acid, and DTB is desthiobiotin (Rolfe & Eisenberg, 1968; P. Cleary, personal communication), Roman numerals I to IV and capital letters A to 53
A.
64
GUHA,
Y.
SATUREN
AND
chl D
blu
bb’ A.bi0.D
w-5
5. 201i
chlA
--*
r
*-----;----. J
A __________
SZYBALSKI
ab” 1*-
1 +gal
W.
----
-----
b.?
-~---..+ aa’
------*
exo,3 c,N
c,OP
Q R
I
3.
-----+.
Ah/o 1
QR
i bio 10
A
J
Fibio t124
QR
FIU. 1. Genetic and molecul8r maps of E. coli bacteriophage h and of three X&o transducing phages containing various regions of the E. coli bio locus. The maps of h, Xbiol and XbiolO are drawn approximately to scale according to the electron micrographic maps (Hradecna & Szybalski, 1969) and the genetic data for the content of bio genes in hbiol (G. Kettner & A. Campbell, personal communication), XbiolO (K. F. Manly, personal communication) and Xbiot124 (C. R. Fuerst, personal communication). The dotted lines connect the right ends of the deletions on the XZ& genomes with the corresponding sites on the genome of A. The E. c& genome is indicated by the double lines and the A genome by single lines. The map of Xiot124, in which region a’-cl4 is replaoed by bio genes, is based on the electron micrographioally determined position of gene CI (cut in cIB in X&030-7; Fiandt et d., 1971) and the buoyant density of this transducing phage. The duoyant densities in the CsCl gradient are 1-508(X), 1*502(XbioZ), 1.501 (hbiolO) and 1.498 g/cm3(Xbiot124). As shown in the upper map, the bio locus (denoted bioA on the map of Taylor & Trotter, 1987) is located between gal-chZD-bZu(pgZ)-att”bb’ to the left and uvrB-&A to the right (de1 Campillo-Campbell et al., 1967; Rolfe t Eisenberg, 1968; Adhya, Cleary & Campbell, 1968; Kupor & Fraenkel, 1969; Taylor & Trotter, 1967; Szybalski, 1970). The orientation of transcription wherever known, is indicated by the dashed arrows on the E. coli and h maps. For 8 more detailed map of the A-D region of the bio loci8 see Fig. 2.
-----
1 strand 3 66’ aif”
OAPA ~--A
EEFGC
D
WB
r strand
I
---
FIG. 2. Transcriptional map of the bio locus. Genes ABFCD are arranged according to de1 Campillo-Campbell et aZ. (1967) and P. Cleary (personal communication), and genes EFB according to Rolfe (1970). The existence of genes E( =B?) and a( = F?) is not llrmly established (Rolfe, 1970). Symbols (p and o denote promoters and operators, respectively. The arrows indicate the orientation of transcription from the Z and T strands of the DNA. The map is drawn approximately to scale, based on the electron micrographic data of Hradecna & Szybalski (1969), on the buoyant densities of the Xbio phages (see Fig. l), and on the present transcription81 data. The distance between attl and the right terminus of the bio operon corresponds to 5000 to 6000 nucleotide pairs, i.e. to 10 to 12% of the length of the AP8pBgenome.
DIVERGENT
BIOTIN
55
LOCUS
D and F indicate the genes controlling the particular steps, according to Rolfe & Eisenberg (1968), de1 Campillo-Campbell et al. (1967), Rolfe (1970) and P. Cleary (personal communication), respectively. These papers list earlier references on the biosynthesis of biotin. We have studied the patterns and regulations of transcription of the bio locus in E. coli K12. The method, which makes use of biotin-transducing mutants of E. coli phage h, is similar in principle to that previously used by Guha, Tabaczynski & Szybalski (1968) for the analysis of transcription in the gal operon. ft consists of the hybridization of bio-specific E. coli [3H]RNA, pulse-labeled in the derepressed and repressed conditions, with the isolated complementary DNA strands of several hbio phages, which contain various regions of the bio locus (Fig. 1). The present results indicate that the bio locus consists of two co-ordinately controlled operons, one containing cistron A and transcribed leftward (counterclockwise) from strand 1 and the other comprising cistrons B to D, and transcribed rightward (clockwise) from strand r. The preliminary results of this study were reported by A. Guha, Y. Saturen and W. Szybalski at the 1969 Bacteriophage Meetings in Cold Spring Harbor, N.Y., U.S.A. and were cited in the review of Szybalski et al. (1969).
2. Materials and Methods (a) Ractem’al
and bacteriophage
strairts
The following strains of E. coli were used: W3350, gal-bio+pm; PlO-1, gal+bio+pm+ ; T5-2, guZ+, region att-bio-uvr.B deleted; and double lysogen T50-1 (Abiot124&1857), all region from the collection of Dr C. R. Fuerst; C224, gal+lac-trp-leu-thr-bioA+B+F+C+, bioD-uurB-chlA deleted, from Dr P. Cleary; Bio310, gal+ bioA+B-C’+D+uvrB, described by Rolfe & Eisenberg (1968). Biotin-transducing phages Xbiol, XbiolO (Signer, Manly & Brunstetter,l969) and hbiot124 were obtained from Drs E. R. Signer, K. F. Manly, and C. R. Fuerst, respectively. The extent of MO genes present or deleted is shown in Fig. 1. To avoid any uncontrolled production of new Xbio mutants, the phages were grown in E. coli strain T5-2, in which the bio locus we.3 deleted. Stocks of hbiot124 and hcI857 were prepared by thermal induction of T50-l(Ac1857,hbiot124) and T50-l(/\cIS57), respectively. Particles of Xbiot124 were separated from those of helper phage by CsCl density-gradient centrifugation, which was also used for final purification of all other phages. (b) Isolation
of DNA
and of the cowqdementary
DNA
strand8
The phenol extraction procedure, as described by Mandell & Hershey (1960), was used to isolate phage DNA. The poly(U,G)-binding technique was employed to isolate the complementary DNA strands directly from the X and htio phages (Hiadecna & Szybalski, pH 8, containing 0.1% Sarkosyl was 1967). The phage suspension in 10V3 M-EDTA, heated in a boiling water bath for 3 min in the presence of poly (U,G) and immediately chilled. A saturated CsCl solution was then added to adjust the density of the solution to 1.72 g/ml., the solution was spun at 30,000 rov./min. in the I.E.C. rotor no SB405 (International Equipment Corp.) at 4°C for 48 hr, and the fractions were collected. The 0.D.2e0 reading showed the presence of two bands separated by a density difference of approximately 20 mg/cm3. The DNA strands in the denser band were designated r strands and those in the lighter one 1 strands (Szybalski, 1969). The r and 1 strands of the ;\bio phages correspond to the r and 2 strands of h DNA, as shown by (i) DNA-DNA hybridization and subsequent electron microscopic studies (Hradenca & Szybalski, 1969), and (ii) the annealing efficiency of [3H]RNA specific for hbio DNA (Kumar et al., 1969). The Eand r fractions of the strands were separately self-annealed at 65% for 2 hr after treatment for 2 min in a boiling water bath. This treatment removes any cross-contamination by the complementary strands, which form bihelical structures and thus are unable to participate in DNA-RNA hybridization reactions (Taylor, Hradecna & Szybalski, 1967).
56
A. GUHA,
Y. SATUREN (c) I.sokktion
AND
IV. SZYBALSKI
of [3H]IixA
The M9 buffer (3.0 g KH,PO,, 7.0 g Na2HP04, 1.0 g NH&l, 0.6 g N&l, 0.12 g MgSO, and 0.01 g CaCl,, 1000 ml. water, pH 7*2), supplemented with 0.2% glycerol and 0.25% vitamin-free Casamino acids (Difco), pH 7.2, referred to hereafter as growth medium, was used for propagation of bio+ cells and for isolation of bio-specific mRNA under the derepressed condition. For studies of repression the growth medium was supplemented with biotin or desthiobiotin (Mann Research Laboratories) in varying concentrations. When bio- strains were used as the source of labeled RNA, cultures were first grown to the desired optical density in growth medium containing biotin (2 x 10m4 pg/ml.), and then washed twice with M9 buffer. The cells were subsequently starved for biotin by incubating in biotin-free growth medium for various periods. In all experiments the cells were grown at 37°C. W’hen the O.D.650 reached 0.15 (about 3 x lo* cells/ml.), [3H]uridine (25 PC/ml.) was added and 1 or 2 min later the culture was poured over 5 ml. of frozen buffer (0.01 rcr-Tris-HCI, pH 7.2). The cells were spun down at 4”C, and the sediment was resuspended in 5 ml. “protoplasting” medium (0.015 M-Tris-HCl, 0.45 M-sucrose, 0.008 M-EDTA, 80 pg lysozyme/ml., pH 8) and kept for 15 min at 4°C. Protoplasts were sedimented and resuspended at room temperature in a lysing medium containing 0.02 M-potassium acetate and 1.5% sodium dodecyl sulfato, pH 5.2. To the lysed solution, 0.1 ml. of 2yb Macaloid was added and the RNA extracted by the phenol method (see Taylor, Hradecna & Szybalski, 1967). The radioactivity after trichloroacetic acid precipitation of 1 ~1. samples was determined. RNA concentrations were determined spectrophotometrically (1 O.D.ZBOnm unit = 40 pg RNA/ml., 1-cmlightpath) after removing phenol by ether extraction and ethanol precipitation of the RNA. The specific activit.ies of RNA were 1.26 to 1.4 x lOa cts/min/pg of RNA. (d) Hybridization The techniques of hybridization with denatured DNA, subsequent dution, and secondstep hybridization with the separated strands were described by Taylor et al. (1967) and Guha et al. (1968). All hybridizations were performed under conditions of DNA excess. For the first-step hybridization (“prehybridization”) about lOa cts/min of [3H]RNA was placed in a scintillation vial containing 1 ml. of 2 x standard saline citrate (1 x = O-1 M-NaCl + 0.015 M-sodium citrate, pH 7.2) and 50 pg of denatured hbiot124 DNA immobilized on a nitrocellulose filter (B6, 24 mm, Schleicher & Schuell Co., Keene, N.H.), according to the method of Gillespie & Spiegelman (1965). Annealing was carried out at 60°C for 20 hr, after which the filter was washed with 2 x standard saline citrate, treated with 2 ml. pancreatic RNase (20 pg/ml.) and T, RNase (10 units/ml.), washed again with 2 x standard saline citrate and then treated with 0.015 M-sodium-iodoacetate, pH 5.2, at 54°C for 40 min. After washing, the filter was transferred to another scintillation vial containing 1 ml. of 0.01 x standard saline citrate. The RNA was eluted from the filter by heating the vial for 15 min in boiling water. The filter was removed and the eluted RNA was treated with electrophoretically purified DNase (Worthington Biochemical Corp.) for 30 mm at 37°C. The DNase activity was destroyed by heating the solution in boiling water for 10 min. The efficiency of elution was over 90%. In the second step, the sample or the total eluted RNA was hybridized with 3 pg of the isolated I or T strands of hbio DNA immobilized on B6 filters. Three filters, one carrying strand I, another r, and one DNA-free filter (control), were annealed in the same vial with the [3H]RNA in 2 x standard saline citrate for 18 hr at 60%. After DNA-RNA annealing under conditions of DNA excess, the filters were treated with pancreatic and T, RNases at 37’C for 30 mm, washed on both sides and dried, and the radioactivity was determined. When the same Abio DNA was employed in the first and second hybridization steps, the sum of the counts hybridizing in the second step to both separated DNA strands was as high as 94 to 98% of the input counts in the eluted, bio-specific [3H]RNA. For “liquid” hybridization, the technique of Nygaard & Hall (1964) was adapted. Radioactive RNA and the isolated strands of hbiot124 DNA (3 pg) were added to phenolsaturated 2 x standard saline citrate bringing tho total volume to O-4 ml. The mixture was annealed at 60°C for 5 hr, diluted with 15 ml. of chilled 2 x standard saline citrate, and then slowly passed (1 to 2 ml./min) through the 2 x standard saline citrate-equilibrated
DIVERGENT B6 filter. The filter T, RNasos, washed,
BIOTIN
57
LOCUS
containing the DNA-RNA hybrid was treated dried and counted, as described in the previous
with pancreatic section.
and
3. Results and Discussion (a) Transcription
and control of the bio+ locus
About 0.2% of the pulse-labeled E3H]RNA isolated from E. coli W3350 or YlO-1 grown in biotin-free medium hybridizes with Miot124 DNA (Table 1). This material must represent bio-specific mRNA since (i) it is not produced by bacteria repressed for the bio expression, i.e. grown in the presence of exogeneous biotin (Table 1, lines 3 and TABLE 1 Hybridization
specijc for the derepressed and repressed 1ocu.sof E. coli with isolated DNA strands
of r3H]RNA,
[3H]RNA hybridized with hbio DNA ( y0 of tjotal)
Source of [3H]RNA Donor strain
bio locus
(1)
(2)
biotin (bio)
Biotin in medium
l-strand %
r-strand
(4)
w3350
present
0
0.09
0.13
YlO-1
present
0
0.08
0.12
W3360 YlO-1 T5-2
present present deleted
0.00 0.00 0.00
0.00 0.00 0.00
2x10-4 2x10-4 0t02x10-*
i+r %
1 :r
(6)
(7)
0.22 (3400 cts/min) 0.20 (3700 cts/min) 0.00 0.00 O-00
41:59 41:69 -
Approximately 1 to 2 x 10s &s/mm of [3H]RNA (specific act. 1.3 to 1.4 x lOa cts/min/pg RNA) was hybridized with 60 pg of denatured Xbtit124 DNA as described in Materials and Methods. The hybridized [sH]RNA was then eluted and the eluete hybridized with 3 pg of isolated 2or r strands of hbiot124 DNA (columns (4) and (6)). The [*H]RNA eluate hybridized with the separated DNA strands with an efficiency of 94 to 98% (see Materials and Methods). The figures in column (6) are the sum of the figures in columns (4) and (5). The control counts (100 to 200 cts/min) that hybridized with denatured hcI857 DNA, were subtracted from the actual counts (figures in parentheses). before computing the percentages.
4), (ii) it is absent in cells having the bio region entirely deleted (Table 1, line 6), and (iii) it does not hybridize with DNA of h phages which do not carry the bio genes. The bio-specific mRNA (i.e. [3H]RNA that was first hybridized to denatured Abiotl24 DNA and then eluted) was annealed to the isolated 1and r strands of Xbiot124 DNA which contains the entire bio locus. Roughly 40% of this bio mRNA hybridizes with strand 1 and 60% with strand r (Table 1). This result indicates that the bio region must consist of at least two operons, one transcribed leftward (counterclockwise) from strand 1 and one rightward (clockwise) from strand r. To determine which regions of the bio locus are transcribed from the 1 or r strand, the bio-specific mRNA was annealed with the DNA strands of hbio mutants that carry only parts of the bio locus. Their approximate genetic and molecular structures are depicted in Figure 1. As shown in Table 2, the bio-specific mRNA hybridizes with equal efficiency with the l-strands of all three phages. In contrast, the amount of biospecific mRNA that hybridizes with the r strands is rather low for hbiol DNA, which
58
A.
GUHA,
Y,
SATUREN
AND
W.
SZYBALSKI
TABLE 2
of bio-specific E. coli fH]RNA with isolated DNA strands oj hbio bacteriophages containing various parts of the bio locus
Hybridization
Source of DNA
[3H]RNA hybridized with Xbio DNA (% of total) z-strand r-strand 1 :T
strands bio locus
XbiO hbiot124
A+B+F+C+D+
0.09 (452 cts/min)
0.13 (624 cts/min)
41: 59
Xbiol
A+B+; F-D
0.09 (418 cts/min)
0.01 (39 cts/min)
89: 11
0.11 (472 cts/min)
0.09 (390 cts/min)
55: 45
hbiol0
deleted
A+B+C+; D deleted
Approximately 4.2 x 10s cts/min of [3H]RNA (spec. act. 1.26 x 10s cts/min/pg RNA), isolated from E. coli W3350 grown in biotin-free growth medium (see Table 1, line I), was used for each hybridization, using the techniques described in Materials and Methods and in Table 1. The control counts (see Table 1 legend) are subtracted from the actual counts (figures in parentheses).
lacks the bio genes to the right of B (Figs 1 and 2) ; it is only partially reduced for hbiol0, which does not have the terminal part of gene D. These results indicate that only cistron A, and possibly the region to the left of it, are transcribed from the 1 strand since this is the bio segment that these phages have in common, and since the 11 yOof bio RNA that hybridizes with the r strand of Xbiol probably belongs to gene B. The genes to the right of B and most probably also gene B are transcribed from the r strand, i.e. clockwise on the E. coli map of Taylor & Trotter (1967). (b) Transcription
and control of the defective bio locus
Two E. coli strains with defective bio locus were employed. Both produced a 2.5 to 3 times higher amount of bio-specific mRNA than the bio+ strains (Table 3 veTsuB TABLE 3 Effects of mutations and deletions in the bio locus of E. coli on the levels of repressed and derepressed transcription of the bio region Source of [sH] RNA Donor strain
bio locus
Corepressor in the medium
[sH]RNA hybridized with hbio DNA (% of total) l-strand
r-strand
z+r
(5)
(1)
C224
A+B+C+ D deleted
Bio 310
A+B-C+D+
None Desthiobiotin Biotin
0.13 0.00 0.00
0.37 0.00 0.00
0.50 0.00 0.00
26:74 -
None Desthiobiotin Biotin
0.15 0.14 0.00
0.45 0.40 0.00
0.60 0.54 0.00
25:75 26:74 -
The hybridization procedure was the same as described in Materials and Methods legend to Table 10. 2 x 1 -3 pg/ml. of biotin or desthiobiotin was employed.
snd in the
DIVERGENT
BIOTIN
LOCHS
59
Table 1). This result most probably reflects the absence of endogenous biotin and consequently a complete derepression of the bio region. Moreover, comparison of the bio+ and bio- [3H]RNA’s suggests that residual repression of the rightward transcription by endogenous biotin is more pronounced than that of the leftward transcription (Tables 1 and 3). Two other aspects of biotin repression were investigated using the bio- mutants. (1) It was found that five times higher concentrations of biotin have to be employed with the bio- mutants (10e3 pg biotin/ml.) than with the bio+strains (2 x lo-* pg/ml.) in order to attain a similar level of repression of the bio-specific mRNA. This again implicates endogenously synthesized biotin in the repression of the bio+ mutants. Still higher levels (4 x 10e3 rg/ml.) were reported to be required for almost total repression of biotin synthesis in the Crookes strain of E. coli (Pai & Lichstein, 1962). (2) We have examined whether both biotin and its immediate precursor, desthiobiotin, act as corepressors of bio transcription, The results in Table 3 show that exogenous desthiobiotin C&Mot repress the bio operons, unless converted into biotin by the product of gene B (compare lines 2 and 5, Table 3). This has also been observed on
FIG. 3. Kinetics of repression of bio mRNA specific for the 1 and r strands of r\biot124 DNA. E. coli strain C224 (genes A to C present, gene D of the bio locus deleted) was grown to a cell concentration of about 3 x lO*/ml. in growth medium containing 5 x 10T4 pg biotin/ml. The cells were oentrifuged, washed with buffer, resuspended in the same volume of biotin-free growth medium, and incubated for 2 hr at 37°C in a shaker bath; no loss of cell viability was noticed, 10 ml. samples were distributed to seven shaker flasks and all but one was supplemented with 2 x lOa pg biotin/ml. At time zero, a 1-min pulse of [3H]uridine was added to two flasks, one with and one without biotin, and the remaining flasks received 1-min pulses that terminated at the times indicated on the abscissa. The l-step liquid hybridization procedure of Nygaard & Hall (1964) was employed, using 4 to 6 x 10s cts/min of [3H]RNA and 3 pg of I or T strands of hbiot124 DNA, as described by Lozeron & Szybalski (1969). Of the [sH]RNA labeled at time zero, 0.09 and 0.32% (represented as 100% in the diagram) hybridized with the 1 and rstrands, respectively. The control counts (50 to 100 cts/min) that hybridized with denatured XcI857DNA were subtracted.
00
A.
GUHA,
Y.
SATUREN
AND
W.
SZYBALSKI
the enzymic level by Eisenberg & Krell (1969 and personal communiccttion) Pai & Lichstein (1967). (c) Kinetics
and by
of the repression of bio operons
Addition of biotin results in repression of both the l- and r-specific bio mRNA’s. One may ask whether biotin affects the 1 and r transcription in the same manner, or biotin repression of transcription from one DNA strand controls in some indirect way transcription from the other strand. The present data show that after addition of biotin the repression of transcription from both strands shows similar kinetics and practically the same short lag period (Fig. 3), indicating that the mechanisms of biotin repression are probably the same for both the leftward and rightward oriented bio operons. 4. Conclusions The simplest model based on the present data postulates that the bio locus consists of two operons, one oriented leftward (counterclockwise) and probably containing gene A, and the other oriented rightward (clockwise) and consisting of genes B-D, tm shown in Figure 2. In this manner promoters pA and p)Bof the A and B-D operons can be closely linked and, as shown in Figure 3, are both under the control of biotin repression. It is not known at present whether there is only one operator site that controls both operons or whether there are two operators, oA and or,, each probably “downstream” from the corresponding pA and pB promoters. It should be relatively easy to distinguish between these alternatives by isolating operator-constitutive mutants and determining whether they are constitutive for both or only one of the bio operons. One should also map out the relative positions of the p, and p, promoters and ascertain whether p, is to the left of pB, as shown in Figure 2, or the opposite relationship holds true, with resulting divergent overlap of the two bio operons, a rather unlikely possibility. If there are two operators, ss postulated in Figure 2, they both should be about equivalent and bind the same aporepressor-corepressor complex, in order to explain the simultaneous onset of repression after addition of the biotin. The twooperator model is favored by the finding that the leftward transcription appears to be somewhat less sensitive to the residual endogenous repression than the rightward transcription (compare Tables 1 and 3). Either biotin itself, or its unknown metabolite, play the role of the corepressor. Desthiobiotin is not the corepressor, unless converted to biotin. The rightward (clockwise) orientation of the B-D genes can also be inferred from the current genetic data of P. Cleary (personal communication) obtained with various mutants and partial deletions in the bio region, and by the experiments of K. Krell ancl M. Gottesman (manuscript in preparation), in which it was shown that the synthesis of desthiobiotin synthetase (enzyme D), when directed by the transducing phage hbZbio[AA-B]C+D+ grown in the bio-deleted host, depends on the Q-dependent rightward transcription of the coupled segment of the h genome and is not repressed by exogenous biotin. The bio operons are fully derepressed only in mutant strains that cannot produce any endogenous biotin. Under these conditions about 0.15% of the total pulse-labeled [3H]RNA is transcribed from the leftward p,-A operon and 0.45% from the rightward p&-D operon. If the activities of the pA and p)B promoters are similar, this result might suggest that the B-D operon is three times longer than the A operon,
DIVERGENT
BIOTIN
LOCUS
61
which is reflected in the dimensions adopted in Figure 2, partially based on the electron-micrographic maps of Hradeona & Szybalski (1969). However, the present data are insuBkient to provide any conclusive evidence on the comparative sizes of the pl-A and the pB-B-D operons. The architecture of the bio locus, as elucidated in the present study, is to the best of our knowledge the first example of divergent orientation of two operons that control a common pathway. Although such an arrangement does not appear to offer any obvious advantage over a single operon, one may speculate that it permits two promoters to be closely linked and thus perhaps under control of one operator either presently or sometime during the evolution of this locus. It might, or might not be sign&ant, that the transcriptional map of prophage A, which is the nearest neighbour to the left of the bio locus, involves similar divergent orientations of its leftward and rightward transcriptional units. This research was supported by grants from the Medical Research Council of Canada (MT-2700), the National Science Foundation (GB-2096), and the National Cancer Institute (CA-07175). This study was initiated in 1967 in the McArdle Laboratory in co-operation with Dr K. Taylor, presently at the Biochemistry Department, Teachers College W.S.P., Gdynia, Poland. We would also like to acknowledge with gratitude the help and advice provided by Dr C. R. Fuerst, who supplied us with several strains and directly psrticipated in preparation of phage Xbiot124. His unselfish help and guidance were invaluable to this study. Drs B. Rolfe and P. Cleary provided counsel and strains, for which we are grateful. We are also thankful to Drs A. Campbell, P. Cleary, M. Gottesman, G. Kettner and H. C. Lichstein for their helpful comments and unpublished data. Mrs H. L. Bingham assisted greatly by growing the phage stocks.
REFERENCES Adhya, S., Cleary, P. & Campbell, A. (1968). Proc. Nat. Acud. Sci., Wad. 61, 956. de1 Campillo-Campbell, A., Kayajanian, G., Campbell, A. & Adhya, S. (1967). J. Bad. 94, 2065. Eisenberg, M. A. BEKrell, K. (1969). J. Biol. Chern. 244, 5503. Fiandt, M., Hradecna, Z., Lozeron, H. A. & Szybalski, W. (1971). In Th,e Bacteriophqe timbda, ed. by A. D. Hershey. Cold Spring Harbor: Cold Spring Harbor Laboratories, in the press. Fuerst, C. R. (1966). Virology, 30, 581. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Guha, A., Tabaczynski, M. & Szybalski, W. (1968). J. Mol. Biol. 35, 207. Hradecna, Z. & Szybalski, W. (1967). Virology. 32, 633. Hradecna, Z. & Szybalski, W. (1969). Virology, 38, 473 and 40, 178. Kumar, S., Bevre, K., Guha, A., Hradecna, Z., Maher, V. M. Sr., & Szybalski, W. (1969). Nature, 221, 823. Kupor, S. R. & Frankel, D. G. (1969). J Bact. 100, 1296. Lozeron, H. A. & Szybalski, W. (1969). Virology, 39, 373. Mandell, J. D. & Hershey, A. D. (1960). Adyt Biochem. 1, 66. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. Biol. 9, 125. Pai, C. H. & Lichstein, H. C. (1962). Biochim. biophys. Acta, 65, 159. Pai, C. H. & Lichstein, H. C. (1967). J. Bact. 94, 1930. Rolfe, B. (1970). Virology, 42, 643. Rolfe, B. & Eisenberg, M. A. (1968). J. Box?. 96, 515. Signer, E. R., Manly, K. F. & Brunstetter, M. (1969). Virology, 39, 137. Szybalski, W. (1969). In Can&ion Cancer Conferelzce, vol. 8, p. 183. Oxford: Pergamon Press.
62
A. GUHA,
Y. SATUREN,
AND
W. SZYBALSKI
Szybalski, W. (1970). Ir Handbook of Biochemistry, ed. by H. A. Sober, pp. 1.35-I-38. Cleveland : Chemical Rubber Co. Szybalski, W., Bsvre, K., Fiandt, M., Guha, A., Hradecna, Z., Kumar, S., Lozeron, H. A., Maher, V. M., Sr., Nijkamp, H. J. J., Summers, W. C. & Taylor, K. (1969). J. Cell Physiol. 74, (Suppl. l), 33. Taylor, A. L. & Trotter, C. D. (1967). Bact. Rev. 31, 332. Taylor, K., Hradecna, Z. & Szybalski, W. (1967). Proc. Nat. r2cad. Sci., Wash. 57, 1618.
Divergent Orientation of Transcription from the Biotin Locus of Escherichia coli ARABINDA GUHA of Microbiology, Erindate College and Department of Medical Cell Biology, University of Toronto, Toronto 181, Ontario, Canada
Dqmrtment
Y. SATUREN AND W. SZYBALSKI
McArdle Laboratory, University of Wisconsin, Wk. 53706, U.S.A.
Madison
(Received 12 May 1970) Pulse-labeled [3H]RNA produced by the bio locus of Escherichia coli grown in biotin-free medium hybridizes with both DNA strands of %iot124, a bio-transducing phage that contains the entire bio locus, with an I: r strand ratio of 25 : 75 (fully derepressed in bio- mutants) or 40 : 60 (partially repressed by endogenous hiotin in bio + cells). When employing DNA strands of Atiol, which contains only genes A and B, the latter 1: r ratio becomes 89 :ll, with the amount of r3H]RNA hybridizing with the E strand remaining unchanged. These results indicate that both strands of the tie locus are transcribed and the orientation of the two transcriptions is divergent, with cistron A belonging to the leftward (counterclockwise) transcribed operon, and the other bio cistrons forming a rightward (clockwise) oriented operon. The bio transcription from both strands is subject to co-ordinate repression by biotin. The Ko[~H]RNA amounts to as much as 0.6% of the total pulse-labeled RNA in fully derepressed E. coli bio- cells, and is reduced to approximately 0.2% in bio+ cells grown in biotin-free medium, and to undetectable amounts in medium supplemented with more than 2 x low3 g biotin/ml. Desthiobiotin does not act as a corepressor unless converted to biotin.
1. Introduction synthesis in Eschmichia coli is controlled by the genes of the biotin locus (denoted bio throughout this communication and bioA by Taylor & Trotter, 1967), which are situated to the right (clockwise) of the attachment site, &bb’, for the h prophage. The locus is composed of a cluster of genes, the order of which, in relation to the neighbouring bacterial markers, is shown in Figures 1 and 2 (de1 CampilloCampbell, Kayajanian, Campbell & Adhya, 1967; Rolfe, 1970). The pathways of biotin biosynthesis follow the sequence. Biotin
I b PA c
II 7KAP -
F
III DAP -
A
IV DTB -
D
biotin, B
where PA is pimelic acid, 7KAP is L-7-oxo4Laminopelargonic acid, DAP is DL-7,8diaminopelargonic acid, and DTB is desthiobiotin (Rolfe & Eisenberg, 1968; P. Cleary, personal communication), Roman numerals I to IV and capital letters A to 53
A.
64
GUHA,
Y.
SATUREN
AND
chl D
blu
bb’ A.bi0.D
w-5
5. 201i
chlA
--*
r
*-----;----. J
A __________
SZYBALSKI
ab” 1*-
1 +gal
W.
----
-----
b.?
-~---..+ aa’
------*
exo,3 c,N
c,OP
Q R
I
3.
-----+.
Ah/o 1
QR
i bio 10
A
J
Fibio t124
QR
FIU. 1. Genetic and molecul8r maps of E. coli bacteriophage h and of three X&o transducing phages containing various regions of the E. coli bio locus. The maps of h, Xbiol and XbiolO are drawn approximately to scale according to the electron micrographic maps (Hradecna & Szybalski, 1969) and the genetic data for the content of bio genes in hbiol (G. Kettner & A. Campbell, personal communication), XbiolO (K. F. Manly, personal communication) and Xbiot124 (C. R. Fuerst, personal communication). The dotted lines connect the right ends of the deletions on the XZ& genomes with the corresponding sites on the genome of A. The E. c& genome is indicated by the double lines and the A genome by single lines. The map of Xiot124, in which region a’-cl4 is replaoed by bio genes, is based on the electron micrographioally determined position of gene CI (cut in cIB in X&030-7; Fiandt et d., 1971) and the buoyant density of this transducing phage. The duoyant densities in the CsCl gradient are 1-508(X), 1*502(XbioZ), 1.501 (hbiolO) and 1.498 g/cm3(Xbiot124). As shown in the upper map, the bio locus (denoted bioA on the map of Taylor & Trotter, 1987) is located between gal-chZD-bZu(pgZ)-att”bb’ to the left and uvrB-&A to the right (de1 Campillo-Campbell et al., 1967; Rolfe t Eisenberg, 1968; Adhya, Cleary & Campbell, 1968; Kupor & Fraenkel, 1969; Taylor & Trotter, 1967; Szybalski, 1970). The orientation of transcription wherever known, is indicated by the dashed arrows on the E. coli and h maps. For 8 more detailed map of the A-D region of the bio loci8 see Fig. 2.
-----
1 strand 3 66’ aif”
OAPA ~--A
EEFGC
D
WB
r strand
I
---
FIG. 2. Transcriptional map of the bio locus. Genes ABFCD are arranged according to de1 Campillo-Campbell et aZ. (1967) and P. Cleary (personal communication), and genes EFB according to Rolfe (1970). The existence of genes E( =B?) and a( = F?) is not llrmly established (Rolfe, 1970). Symbols (p and o denote promoters and operators, respectively. The arrows indicate the orientation of transcription from the Z and T strands of the DNA. The map is drawn approximately to scale, based on the electron micrographic data of Hradecna & Szybalski (1969), on the buoyant densities of the Xbio phages (see Fig. l), and on the present transcription81 data. The distance between attl and the right terminus of the bio operon corresponds to 5000 to 6000 nucleotide pairs, i.e. to 10 to 12% of the length of the AP8pBgenome.
DIVERGENT
BIOTIN
55
LOCUS
D and F indicate the genes controlling the particular steps, according to Rolfe & Eisenberg (1968), de1 Campillo-Campbell et al. (1967), Rolfe (1970) and P. Cleary (personal communication), respectively. These papers list earlier references on the biosynthesis of biotin. We have studied the patterns and regulations of transcription of the bio locus in E. coli K12. The method, which makes use of biotin-transducing mutants of E. coli phage h, is similar in principle to that previously used by Guha, Tabaczynski & Szybalski (1968) for the analysis of transcription in the gal operon. ft consists of the hybridization of bio-specific E. coli [3H]RNA, pulse-labeled in the derepressed and repressed conditions, with the isolated complementary DNA strands of several hbio phages, which contain various regions of the bio locus (Fig. 1). The present results indicate that the bio locus consists of two co-ordinately controlled operons, one containing cistron A and transcribed leftward (counterclockwise) from strand 1 and the other comprising cistrons B to D, and transcribed rightward (clockwise) from strand r. The preliminary results of this study were reported by A. Guha, Y. Saturen and W. Szybalski at the 1969 Bacteriophage Meetings in Cold Spring Harbor, N.Y., U.S.A. and were cited in the review of Szybalski et al. (1969).
2. Materials and Methods (a) Ractem’al
and bacteriophage
strairts
The following strains of E. coli were used: W3350, gal-bio+pm; PlO-1, gal+bio+pm+ ; T5-2, guZ+, region att-bio-uvr.B deleted; and double lysogen T50-1 (Abiot124&1857), all region from the collection of Dr C. R. Fuerst; C224, gal+lac-trp-leu-thr-bioA+B+F+C+, bioD-uurB-chlA deleted, from Dr P. Cleary; Bio310, gal+ bioA+B-C’+D+uvrB, described by Rolfe & Eisenberg (1968). Biotin-transducing phages Xbiol, XbiolO (Signer, Manly & Brunstetter,l969) and hbiot124 were obtained from Drs E. R. Signer, K. F. Manly, and C. R. Fuerst, respectively. The extent of MO genes present or deleted is shown in Fig. 1. To avoid any uncontrolled production of new Xbio mutants, the phages were grown in E. coli strain T5-2, in which the bio locus we.3 deleted. Stocks of hbiot124 and hcI857 were prepared by thermal induction of T50-l(Ac1857,hbiot124) and T50-l(/\cIS57), respectively. Particles of Xbiot124 were separated from those of helper phage by CsCl density-gradient centrifugation, which was also used for final purification of all other phages. (b) Isolation
of DNA
and of the cowqdementary
DNA
strand8
The phenol extraction procedure, as described by Mandell & Hershey (1960), was used to isolate phage DNA. The poly(U,G)-binding technique was employed to isolate the complementary DNA strands directly from the X and htio phages (Hiadecna & Szybalski, pH 8, containing 0.1% Sarkosyl was 1967). The phage suspension in 10V3 M-EDTA, heated in a boiling water bath for 3 min in the presence of poly (U,G) and immediately chilled. A saturated CsCl solution was then added to adjust the density of the solution to 1.72 g/ml., the solution was spun at 30,000 rov./min. in the I.E.C. rotor no SB405 (International Equipment Corp.) at 4°C for 48 hr, and the fractions were collected. The 0.D.2e0 reading showed the presence of two bands separated by a density difference of approximately 20 mg/cm3. The DNA strands in the denser band were designated r strands and those in the lighter one 1 strands (Szybalski, 1969). The r and 1 strands of the ;\bio phages correspond to the r and 2 strands of h DNA, as shown by (i) DNA-DNA hybridization and subsequent electron microscopic studies (Hradenca & Szybalski, 1969), and (ii) the annealing efficiency of [3H]RNA specific for hbio DNA (Kumar et al., 1969). The Eand r fractions of the strands were separately self-annealed at 65% for 2 hr after treatment for 2 min in a boiling water bath. This treatment removes any cross-contamination by the complementary strands, which form bihelical structures and thus are unable to participate in DNA-RNA hybridization reactions (Taylor, Hradecna & Szybalski, 1967).
56
A. GUHA,
Y. SATUREN (c) I.sokktion
AND
IV. SZYBALSKI
of [3H]IixA
The M9 buffer (3.0 g KH,PO,, 7.0 g Na2HP04, 1.0 g NH&l, 0.6 g N&l, 0.12 g MgSO, and 0.01 g CaCl,, 1000 ml. water, pH 7*2), supplemented with 0.2% glycerol and 0.25% vitamin-free Casamino acids (Difco), pH 7.2, referred to hereafter as growth medium, was used for propagation of bio+ cells and for isolation of bio-specific mRNA under the derepressed condition. For studies of repression the growth medium was supplemented with biotin or desthiobiotin (Mann Research Laboratories) in varying concentrations. When bio- strains were used as the source of labeled RNA, cultures were first grown to the desired optical density in growth medium containing biotin (2 x 10m4 pg/ml.), and then washed twice with M9 buffer. The cells were subsequently starved for biotin by incubating in biotin-free growth medium for various periods. In all experiments the cells were grown at 37°C. W’hen the O.D.650 reached 0.15 (about 3 x lo* cells/ml.), [3H]uridine (25 PC/ml.) was added and 1 or 2 min later the culture was poured over 5 ml. of frozen buffer (0.01 rcr-Tris-HCI, pH 7.2). The cells were spun down at 4”C, and the sediment was resuspended in 5 ml. “protoplasting” medium (0.015 M-Tris-HCl, 0.45 M-sucrose, 0.008 M-EDTA, 80 pg lysozyme/ml., pH 8) and kept for 15 min at 4°C. Protoplasts were sedimented and resuspended at room temperature in a lysing medium containing 0.02 M-potassium acetate and 1.5% sodium dodecyl sulfato, pH 5.2. To the lysed solution, 0.1 ml. of 2yb Macaloid was added and the RNA extracted by the phenol method (see Taylor, Hradecna & Szybalski, 1967). The radioactivity after trichloroacetic acid precipitation of 1 ~1. samples was determined. RNA concentrations were determined spectrophotometrically (1 O.D.ZBOnm unit = 40 pg RNA/ml., 1-cmlightpath) after removing phenol by ether extraction and ethanol precipitation of the RNA. The specific activit.ies of RNA were 1.26 to 1.4 x lOa cts/min/pg of RNA. (d) Hybridization The techniques of hybridization with denatured DNA, subsequent dution, and secondstep hybridization with the separated strands were described by Taylor et al. (1967) and Guha et al. (1968). All hybridizations were performed under conditions of DNA excess. For the first-step hybridization (“prehybridization”) about lOa cts/min of [3H]RNA was placed in a scintillation vial containing 1 ml. of 2 x standard saline citrate (1 x = O-1 M-NaCl + 0.015 M-sodium citrate, pH 7.2) and 50 pg of denatured hbiot124 DNA immobilized on a nitrocellulose filter (B6, 24 mm, Schleicher & Schuell Co., Keene, N.H.), according to the method of Gillespie & Spiegelman (1965). Annealing was carried out at 60°C for 20 hr, after which the filter was washed with 2 x standard saline citrate, treated with 2 ml. pancreatic RNase (20 pg/ml.) and T, RNase (10 units/ml.), washed again with 2 x standard saline citrate and then treated with 0.015 M-sodium-iodoacetate, pH 5.2, at 54°C for 40 min. After washing, the filter was transferred to another scintillation vial containing 1 ml. of 0.01 x standard saline citrate. The RNA was eluted from the filter by heating the vial for 15 min in boiling water. The filter was removed and the eluted RNA was treated with electrophoretically purified DNase (Worthington Biochemical Corp.) for 30 mm at 37°C. The DNase activity was destroyed by heating the solution in boiling water for 10 min. The efficiency of elution was over 90%. In the second step, the sample or the total eluted RNA was hybridized with 3 pg of the isolated I or T strands of hbio DNA immobilized on B6 filters. Three filters, one carrying strand I, another r, and one DNA-free filter (control), were annealed in the same vial with the [3H]RNA in 2 x standard saline citrate for 18 hr at 60%. After DNA-RNA annealing under conditions of DNA excess, the filters were treated with pancreatic and T, RNases at 37’C for 30 mm, washed on both sides and dried, and the radioactivity was determined. When the same Abio DNA was employed in the first and second hybridization steps, the sum of the counts hybridizing in the second step to both separated DNA strands was as high as 94 to 98% of the input counts in the eluted, bio-specific [3H]RNA. For “liquid” hybridization, the technique of Nygaard & Hall (1964) was adapted. Radioactive RNA and the isolated strands of hbiot124 DNA (3 pg) were added to phenolsaturated 2 x standard saline citrate bringing tho total volume to O-4 ml. The mixture was annealed at 60°C for 5 hr, diluted with 15 ml. of chilled 2 x standard saline citrate, and then slowly passed (1 to 2 ml./min) through the 2 x standard saline citrate-equilibrated
DIVERGENT B6 filter. The filter T, RNasos, washed,
BIOTIN
57
LOCUS
containing the DNA-RNA hybrid was treated dried and counted, as described in the previous
with pancreatic section.
and
3. Results and Discussion (a) Transcription
and control of the bio+ locus
About 0.2% of the pulse-labeled E3H]RNA isolated from E. coli W3350 or YlO-1 grown in biotin-free medium hybridizes with Miot124 DNA (Table 1). This material must represent bio-specific mRNA since (i) it is not produced by bacteria repressed for the bio expression, i.e. grown in the presence of exogeneous biotin (Table 1, lines 3 and TABLE 1 Hybridization
specijc for the derepressed and repressed 1ocu.sof E. coli with isolated DNA strands
of r3H]RNA,
[3H]RNA hybridized with hbio DNA ( y0 of tjotal)
Source of [3H]RNA Donor strain
bio locus
(1)
(2)
biotin (bio)
Biotin in medium
l-strand %
r-strand
(4)
w3350
present
0
0.09
0.13
YlO-1
present
0
0.08
0.12
W3360 YlO-1 T5-2
present present deleted
0.00 0.00 0.00
0.00 0.00 0.00
2x10-4 2x10-4 0t02x10-*
i+r %
1 :r
(6)
(7)
0.22 (3400 cts/min) 0.20 (3700 cts/min) 0.00 0.00 O-00
41:59 41:69 -
Approximately 1 to 2 x 10s &s/mm of [3H]RNA (specific act. 1.3 to 1.4 x lOa cts/min/pg RNA) was hybridized with 60 pg of denatured Xbtit124 DNA as described in Materials and Methods. The hybridized [sH]RNA was then eluted and the eluete hybridized with 3 pg of isolated 2or r strands of hbiot124 DNA (columns (4) and (6)). The [*H]RNA eluate hybridized with the separated DNA strands with an efficiency of 94 to 98% (see Materials and Methods). The figures in column (6) are the sum of the figures in columns (4) and (5). The control counts (100 to 200 cts/min) that hybridized with denatured hcI857 DNA, were subtracted from the actual counts (figures in parentheses). before computing the percentages.
4), (ii) it is absent in cells having the bio region entirely deleted (Table 1, line 6), and (iii) it does not hybridize with DNA of h phages which do not carry the bio genes. The bio-specific mRNA (i.e. [3H]RNA that was first hybridized to denatured Abiotl24 DNA and then eluted) was annealed to the isolated 1and r strands of Xbiot124 DNA which contains the entire bio locus. Roughly 40% of this bio mRNA hybridizes with strand 1 and 60% with strand r (Table 1). This result indicates that the bio region must consist of at least two operons, one transcribed leftward (counterclockwise) from strand 1 and one rightward (clockwise) from strand r. To determine which regions of the bio locus are transcribed from the 1 or r strand, the bio-specific mRNA was annealed with the DNA strands of hbio mutants that carry only parts of the bio locus. Their approximate genetic and molecular structures are depicted in Figure 1. As shown in Table 2, the bio-specific mRNA hybridizes with equal efficiency with the l-strands of all three phages. In contrast, the amount of biospecific mRNA that hybridizes with the r strands is rather low for hbiol DNA, which
58
A.
GUHA,
Y,
SATUREN
AND
W.
SZYBALSKI
TABLE 2
of bio-specific E. coli fH]RNA with isolated DNA strands oj hbio bacteriophages containing various parts of the bio locus
Hybridization
Source of DNA
[3H]RNA hybridized with Xbio DNA (% of total) z-strand r-strand 1 :T
strands bio locus
XbiO hbiot124
A+B+F+C+D+
0.09 (452 cts/min)
0.13 (624 cts/min)
41: 59
Xbiol
A+B+; F-D
0.09 (418 cts/min)
0.01 (39 cts/min)
89: 11
0.11 (472 cts/min)
0.09 (390 cts/min)
55: 45
hbiol0
deleted
A+B+C+; D deleted
Approximately 4.2 x 10s cts/min of [3H]RNA (spec. act. 1.26 x 10s cts/min/pg RNA), isolated from E. coli W3350 grown in biotin-free growth medium (see Table 1, line I), was used for each hybridization, using the techniques described in Materials and Methods and in Table 1. The control counts (see Table 1 legend) are subtracted from the actual counts (figures in parentheses).
lacks the bio genes to the right of B (Figs 1 and 2) ; it is only partially reduced for hbiol0, which does not have the terminal part of gene D. These results indicate that only cistron A, and possibly the region to the left of it, are transcribed from the 1 strand since this is the bio segment that these phages have in common, and since the 11 yOof bio RNA that hybridizes with the r strand of Xbiol probably belongs to gene B. The genes to the right of B and most probably also gene B are transcribed from the r strand, i.e. clockwise on the E. coli map of Taylor & Trotter (1967). (b) Transcription
and control of the defective bio locus
Two E. coli strains with defective bio locus were employed. Both produced a 2.5 to 3 times higher amount of bio-specific mRNA than the bio+ strains (Table 3 veTsuB TABLE 3 Effects of mutations and deletions in the bio locus of E. coli on the levels of repressed and derepressed transcription of the bio region Source of [sH] RNA Donor strain
bio locus
Corepressor in the medium
[sH]RNA hybridized with hbio DNA (% of total) l-strand
r-strand
z+r
(5)
(1)
C224
A+B+C+ D deleted
Bio 310
A+B-C+D+
None Desthiobiotin Biotin
0.13 0.00 0.00
0.37 0.00 0.00
0.50 0.00 0.00
26:74 -
None Desthiobiotin Biotin
0.15 0.14 0.00
0.45 0.40 0.00
0.60 0.54 0.00
25:75 26:74 -
The hybridization procedure was the same as described in Materials and Methods legend to Table 10. 2 x 1 -3 pg/ml. of biotin or desthiobiotin was employed.
snd in the
DIVERGENT
BIOTIN
LOCHS
59
Table 1). This result most probably reflects the absence of endogenous biotin and consequently a complete derepression of the bio region. Moreover, comparison of the bio+ and bio- [3H]RNA’s suggests that residual repression of the rightward transcription by endogenous biotin is more pronounced than that of the leftward transcription (Tables 1 and 3). Two other aspects of biotin repression were investigated using the bio- mutants. (1) It was found that five times higher concentrations of biotin have to be employed with the bio- mutants (10e3 pg biotin/ml.) than with the bio+strains (2 x lo-* pg/ml.) in order to attain a similar level of repression of the bio-specific mRNA. This again implicates endogenously synthesized biotin in the repression of the bio+ mutants. Still higher levels (4 x 10e3 rg/ml.) were reported to be required for almost total repression of biotin synthesis in the Crookes strain of E. coli (Pai & Lichstein, 1962). (2) We have examined whether both biotin and its immediate precursor, desthiobiotin, act as corepressors of bio transcription, The results in Table 3 show that exogenous desthiobiotin C&Mot repress the bio operons, unless converted into biotin by the product of gene B (compare lines 2 and 5, Table 3). This has also been observed on
FIG. 3. Kinetics of repression of bio mRNA specific for the 1 and r strands of r\biot124 DNA. E. coli strain C224 (genes A to C present, gene D of the bio locus deleted) was grown to a cell concentration of about 3 x lO*/ml. in growth medium containing 5 x 10T4 pg biotin/ml. The cells were oentrifuged, washed with buffer, resuspended in the same volume of biotin-free growth medium, and incubated for 2 hr at 37°C in a shaker bath; no loss of cell viability was noticed, 10 ml. samples were distributed to seven shaker flasks and all but one was supplemented with 2 x lOa pg biotin/ml. At time zero, a 1-min pulse of [3H]uridine was added to two flasks, one with and one without biotin, and the remaining flasks received 1-min pulses that terminated at the times indicated on the abscissa. The l-step liquid hybridization procedure of Nygaard & Hall (1964) was employed, using 4 to 6 x 10s cts/min of [3H]RNA and 3 pg of I or T strands of hbiot124 DNA, as described by Lozeron & Szybalski (1969). Of the [sH]RNA labeled at time zero, 0.09 and 0.32% (represented as 100% in the diagram) hybridized with the 1 and rstrands, respectively. The control counts (50 to 100 cts/min) that hybridized with denatured XcI857DNA were subtracted.
00
A.
GUHA,
Y.
SATUREN
AND
W.
SZYBALSKI
the enzymic level by Eisenberg & Krell (1969 and personal communiccttion) Pai & Lichstein (1967). (c) Kinetics
and by
of the repression of bio operons
Addition of biotin results in repression of both the l- and r-specific bio mRNA’s. One may ask whether biotin affects the 1 and r transcription in the same manner, or biotin repression of transcription from one DNA strand controls in some indirect way transcription from the other strand. The present data show that after addition of biotin the repression of transcription from both strands shows similar kinetics and practically the same short lag period (Fig. 3), indicating that the mechanisms of biotin repression are probably the same for both the leftward and rightward oriented bio operons. 4. Conclusions The simplest model based on the present data postulates that the bio locus consists of two operons, one oriented leftward (counterclockwise) and probably containing gene A, and the other oriented rightward (clockwise) and consisting of genes B-D, tm shown in Figure 2. In this manner promoters pA and p)Bof the A and B-D operons can be closely linked and, as shown in Figure 3, are both under the control of biotin repression. It is not known at present whether there is only one operator site that controls both operons or whether there are two operators, oA and or,, each probably “downstream” from the corresponding pA and pB promoters. It should be relatively easy to distinguish between these alternatives by isolating operator-constitutive mutants and determining whether they are constitutive for both or only one of the bio operons. One should also map out the relative positions of the p, and p, promoters and ascertain whether p, is to the left of pB, as shown in Figure 2, or the opposite relationship holds true, with resulting divergent overlap of the two bio operons, a rather unlikely possibility. If there are two operators, ss postulated in Figure 2, they both should be about equivalent and bind the same aporepressor-corepressor complex, in order to explain the simultaneous onset of repression after addition of the biotin. The twooperator model is favored by the finding that the leftward transcription appears to be somewhat less sensitive to the residual endogenous repression than the rightward transcription (compare Tables 1 and 3). Either biotin itself, or its unknown metabolite, play the role of the corepressor. Desthiobiotin is not the corepressor, unless converted to biotin. The rightward (clockwise) orientation of the B-D genes can also be inferred from the current genetic data of P. Cleary (personal communication) obtained with various mutants and partial deletions in the bio region, and by the experiments of K. Krell ancl M. Gottesman (manuscript in preparation), in which it was shown that the synthesis of desthiobiotin synthetase (enzyme D), when directed by the transducing phage hbZbio[AA-B]C+D+ grown in the bio-deleted host, depends on the Q-dependent rightward transcription of the coupled segment of the h genome and is not repressed by exogenous biotin. The bio operons are fully derepressed only in mutant strains that cannot produce any endogenous biotin. Under these conditions about 0.15% of the total pulse-labeled [3H]RNA is transcribed from the leftward p,-A operon and 0.45% from the rightward p&-D operon. If the activities of the pA and p)B promoters are similar, this result might suggest that the B-D operon is three times longer than the A operon,
DIVERGENT
BIOTIN
LOCUS
61
which is reflected in the dimensions adopted in Figure 2, partially based on the electron-micrographic maps of Hradeona & Szybalski (1969). However, the present data are insuBkient to provide any conclusive evidence on the comparative sizes of the pl-A and the pB-B-D operons. The architecture of the bio locus, as elucidated in the present study, is to the best of our knowledge the first example of divergent orientation of two operons that control a common pathway. Although such an arrangement does not appear to offer any obvious advantage over a single operon, one may speculate that it permits two promoters to be closely linked and thus perhaps under control of one operator either presently or sometime during the evolution of this locus. It might, or might not be sign&ant, that the transcriptional map of prophage A, which is the nearest neighbour to the left of the bio locus, involves similar divergent orientations of its leftward and rightward transcriptional units. This research was supported by grants from the Medical Research Council of Canada (MT-2700), the National Science Foundation (GB-2096), and the National Cancer Institute (CA-07175). This study was initiated in 1967 in the McArdle Laboratory in co-operation with Dr K. Taylor, presently at the Biochemistry Department, Teachers College W.S.P., Gdynia, Poland. We would also like to acknowledge with gratitude the help and advice provided by Dr C. R. Fuerst, who supplied us with several strains and directly psrticipated in preparation of phage Xbiot124. His unselfish help and guidance were invaluable to this study. Drs B. Rolfe and P. Cleary provided counsel and strains, for which we are grateful. We are also thankful to Drs A. Campbell, P. Cleary, M. Gottesman, G. Kettner and H. C. Lichstein for their helpful comments and unpublished data. Mrs H. L. Bingham assisted greatly by growing the phage stocks.
REFERENCES Adhya, S., Cleary, P. & Campbell, A. (1968). Proc. Nat. Acud. Sci., Wad. 61, 956. de1 Campillo-Campbell, A., Kayajanian, G., Campbell, A. & Adhya, S. (1967). J. Bad. 94, 2065. Eisenberg, M. A. BEKrell, K. (1969). J. Biol. Chern. 244, 5503. Fiandt, M., Hradecna, Z., Lozeron, H. A. & Szybalski, W. (1971). In Th,e Bacteriophqe timbda, ed. by A. D. Hershey. Cold Spring Harbor: Cold Spring Harbor Laboratories, in the press. Fuerst, C. R. (1966). Virology, 30, 581. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Guha, A., Tabaczynski, M. & Szybalski, W. (1968). J. Mol. Biol. 35, 207. Hradecna, Z. & Szybalski, W. (1967). Virology. 32, 633. Hradecna, Z. & Szybalski, W. (1969). Virology, 38, 473 and 40, 178. Kumar, S., Bevre, K., Guha, A., Hradecna, Z., Maher, V. M. Sr., & Szybalski, W. (1969). Nature, 221, 823. Kupor, S. R. & Frankel, D. G. (1969). J Bact. 100, 1296. Lozeron, H. A. & Szybalski, W. (1969). Virology, 39, 373. Mandell, J. D. & Hershey, A. D. (1960). Adyt Biochem. 1, 66. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. Biol. 9, 125. Pai, C. H. & Lichstein, H. C. (1962). Biochim. biophys. Acta, 65, 159. Pai, C. H. & Lichstein, H. C. (1967). J. Bact. 94, 1930. Rolfe, B. (1970). Virology, 42, 643. Rolfe, B. & Eisenberg, M. A. (1968). J. Box?. 96, 515. Signer, E. R., Manly, K. F. & Brunstetter, M. (1969). Virology, 39, 137. Szybalski, W. (1969). In Can&ion Cancer Conferelzce, vol. 8, p. 183. Oxford: Pergamon Press.
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