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The mom gene of bacteriophage Mu: a unique regulatory scheme to control a lethal function
G
61
Elsevier GENE
1408
The mom gene of bacteriophage (Recombinant
DNA;
acetamido
Mu: a unique adenine;
regulatory
DNA modification;
scheme
to control
a lethal function
gene corn; Mom protein;
post-transcriptional
activation)
R. Kahmann*, A. Seiler, F.G. Wulczyn and E. Pfaff** Max-Piunck-Institut ,fir molekulare Genetik, Otto- Warburg-Laboratorium. Ihnestrasse 63. D-1000 Berlin 33 (DuhlemJ Tel. (30)8307-253, and **Institut_fir Mikrobiologie der UnivevPitiit Heidelberg, D-6900 Heidelberg (F.R. G.) Tel. (06221)564-294 (Received
April 23rd,
(Revision
received
(Accepted
1985)
June 7th, 1985)
July 17th, 1985)
SlJMMARY
The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation : gene expression requires methylation by the Escherichiu coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene corn (control of mom, previously termed ORF-x) whose coding region overlaps the 5’-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in corn completely abolish expression of the mom gene. Fragments lacking the 5’ end of corn can be cloned downstream of constitutive plasmid promoters. The corn gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDa1, has been visualized on gels. Efficient expression of Mom from pL requires gene corn. A fusion between MS-2 polymerase and corn has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit. We show that the defect associated with corn deletions can be complemented in tram, and propose that corn acts as a positive regulator on a post-transcriptional level.
INTRODUCTION
Bacteriophage
* To whom addressed. Abbreviations: pair(s);
EOP.
Mu encodes
correspondence
and
function, Mom (Toussaint, 1976), which is unusual in several aspects. The modification converts aden-
a DNA modification
reprint
requests
should
be
kanamycin; PAGE,
aa.
amino
efficiency
0378-I 119/85/‘$03.30
0
acid(s);
of plating:
1985 Elsevier
Ap,
bp,
base
R, resistance;
pair(s);
Km,
nates prophage
ampicillin;
kb, kilobase
Science
ORF,
Publishers
open
reading
PA gel electrophoresis;
[ 1, designates in lysogen.
p,
frame;
PA, polyacrylamide;
, major leftward I promoter;
plasmid-carrier
state;
( ), desig-
ine to acetamido
adenine
(Swinton
et al.,
1983)
I (Plasterk
et al., 1983) and can be separated
from
within the sequence
region I (Kahmann, 1983). The finding that the transacting Mu function is required for transcription
5’ -GAGNPy-3’ c c
(Hattman and Ives, 1984; Hattman et al., 1985) and that the need for activation is alleviated when the
(Kahmann,
1984). This rather
broad
mom gene is fused to foreign
modification
promoters
affects about 15 “/, of all adenine residues and renders
et al., 1983; Kahmann,
DNA
makes it likely that the activator positively influences
resistant
restriction basis
to a variety
systems,
for several
Mom-specific Toussaint, al feature
of type I and type II
a property
which provides
biological and physical modification (Hattman,
the interaction
the
1983; Plasterk
(Hattman
between
RNA
et al., 1983)
polymerase
and the
mom
promoter, possibly by binding to the -35 region. How and why this transcription-initiation
tests for 1979;
complex upstream
1976; Kahmann, 1984). A second unusuof the mom gene lies in its regulation.
Transcription of the mom gene requires the E. coli Dam-methylase (Hattman, 1982) and this requirement is linked with the presence of three Dam-sites in region I (Hattman, 1983; Kahmann, 1983; Plasterk et al., 1983 ; see Fig. 1). Internal deletions in region I which remove either one or two Dam sites render mom expression Dam-independent and the same effect is observed when the mom gene is cloned without region I (Kahmann, 1983; Plasterk et al., 1983). From S 1 nuclease mapping studies, the 5’ end of the mom mRNA maps downstream of region I at position 974 (Plasterk et al., 1984) or 963 (Hattman and Ives, 1984; see Fig. 1). It is clear that region I does not coincide with either of the two putative overlapping mom promoters (Hattman and Ives, 1984). When cloned on plasmid vectors the mom gene is usually not expressed and requires a transacting Mu function (Chaconas et al., 198 1) presumably the product of gene C (Plasterk et al., 1983; Hattman et al., 1985). The site required for acti-
could be influenced by methylation sequences is still a mystery.
of
In the present communication we present evidence that in addition to this unique regulatory situation on the transcriptional level, mom expression is subject to post-transcriptional regulation as well. We implicate the gene product of gene corn (Kahmann, 1984), whose reading frame partially overlaps the mom coding region (Fig. l), in this process.
MATERIALS
AND METHODS
(a) Bacterial
and phage strains
C600 (Appleyard, 1954); C600(PlCm) (Toussaint, 1976); K-12dHldtrp (Remaut et al., 1981); K5219 (Remaut et al., 1981); WM648 = CM987(asnA 3 1, asnB32, recA 1, spoT1, thi-1; Meyenburg et from W. Messer); and al., 1978; obtained
NlOO (Gottesman and Yarmolinski, 1968) were employed. Mucts62,,, s (Chow et al., 1978) phage
vation maps to the right but in close vicinity to region
transortlvotlon SI te Dam-dependence region I II/
162aol
1
*
I
r*
I
1100
1
PVUI
Hindll
Fig. I. Schematic
overview
of the regulatory
or 840 (Kahmann,
1983; Plasterk and
Dam-methylase. (Kahmann,
Ives,
N_N___N___v_v__c_MMN region of the mom gene. Numbers
et al., 1984), position
The site recognized
give distances
as open bars. ORF-.Y = corn. The translational
1984). Asterisks
1983; Plastcrk
800
t?clI
1983). ORFs are indicated
or 963 (Hattman
I
900
Pvul I Clal [-iaI mRNA
bp (Kahmann,
I
I
1000
I
known
I
ORF-XI
I
mark
1 (start point) ofthe the three
by the Mu-specific ct al., 1983). Several
GATC
transactivator restriction
mRNA
sequences
from the right end of the Mu genomc
start of the mom gene is either at position
(wavy line) is at position in region
(gene C product)
sites are indicated
I which
is bracketed,
as landmarks.
974 (Plastcrk
are methylated
in XIO
et al., 1984) by the E. co[i
the extent of the region is not
63
carries
a substitution
in its fi region;
region of mom up to position sequences, Kahmann
which causes
by IS2
a Mom - phenotype
and D. Kamp, unpublished).
ClaI site of pBR322 and here the fragment
the regulatory
851 is replaced
(R.
The substi-
tution has destroyed ORF-x. Mucts42morn~~~~ has been described (Toussaint, 1976) and will be referred to as Mucts62momin the text. Mucts62mom+ described by Howe (1973).
is
(b) Plasmids Plasmids
in clockwise
included
pBR322 (Bolivar et al., 1977);
(c) Biological
assay for Mom-specific
modification
Plasmids for which the Mom phenotype was to be determined were tr~sformed into a host strain lysogenie either for Mucts62mom,,,, or Mucts62,,, _ 5. The residing prophage was induced and the resulting phage lysates were plated on C600 and C6OO(P 1Cm) indicators. The EOP [titer on C6OO(Pl~m)~titer on C600] was determined. An EOP of < 10 - ’ indicates a Mom-, and EOP of > lop4 a Mom+ phenotype (Toussaint, 1976).
AND
DISCUSSION
{a) The ability to clone the wont gene is determined by the absence of strong constitutive vector promoters or ORF-x (corn) Studies aimed at understanding the regulatory mechanism that controls mom gene expression required the cloning of the mom gene or parts of it in plasmid vectors. In doing so we realized that certain restriction fragments could be cloned exclusively in one orientation in pBR322 while for other fragments no such orientation bias existed. The most striking examples are plasmid pMuAS 1 and plasmid pMomCla (Fig. 2). In pMuAS 1 the mom gene is cloned into the BamHI site of pBR322 and all clones isolated contained the fragment in an anticlockwise orientation. In pMomCla the mom gene is cloned into the
orientation.
were reported by Hattman
is always
Similar
results
and Ives (1984). Attempts
to invert the respective BamHI or ClaI fragments were unsuccessful (more than 100 clones with inserts were analyzed). In both instances the orientation which could not be isolated would have fused the mom gene to pBR322 the pMuASl-derived pMomCla-derived
pLc2833 and pLc24 (Remaut et al., 1981); pMu1034, pMuI001 and pMu1010 (Kahmann, 1983) and ~~1857 (Remaut et al., 1983). pWS2 is a pBR322 derivative containing a 1.2-kb EcoRI fragment encoding KmR (obtained from W. Messer).
RESULTS
inserted
promoters (p2 in the case of plasmid, pl in case of the
plasmid).
In contrast,
shorter
fragments containing the mom gene, for example on a BclI-BarnHI fragment, can readily be cloned in both orientations into the BanzHI site of pBR322 (pMulOO1 and pMu1010, described by Kahmann, 1983). In pMu 100 1 (Fig. 2) the mom gene is fused to thep2 promoter and this results in a low-level constitutive expression which apparently is tolerated by the host cell. These results suggested that the additional sequences between the BclI and C(a1 site determine whether the mom gene can be cloned downstream of constitutive plasmid promoters p 1 and ~2. A trivial explanation, e.g., homologies between this region and parts of pBR322 which might confer instability to such plasmids can be excluded. In attempts to invert the CluI fragment of pMomCla, one clone was isolated which contained the fragments in clockwise orientation. This clone did not express the mom gene after activation by a helper phage nor was there any constitutive modification detectable. Presumably this clone had acquired a spontaneous mutation in the mom gene (not shown). From these results it was tempting to speculate that the underlying reason for the inability to clone mom on larger fragments downstream of plasmid promoters was the presence of an intact ORF-x (corn) on such fragments. Either transcription or translation of the region 5’ to the mom gene could be implicated in enhancing mom gene expression. (b) High level expression
of Mom protein requires
corn
Experiments presented above suggested that corn is somehow involved in the regulation of mom gene expression. To test the possibility that this additional control might operate on the transcriptional level as Dam and the transactivator do, the mom gene was cloned downstream ofp, on plasmid pLc2833 with and without corn (Fig. 3). In addition corn alone was
64 Barn HI
BamHI
Clai
_----- -- I
pMuAS1
EcoRl
__lL p20
ECDRI
i
----_
-F4
P3e
0P3 P? tlamtll
Clal
ClaI ‘11
_--_-----_
pMomCla
a
PI
G.-
MU
-----_--
pMu:034
_ii P3
IBM---------__
e
pMu1034AromZO
----------*
_---_--_-__-_
pMulOXArom9
--------_-_+
-----------_
pMu103LAcom9KmR
-_------___ EcoRI
pMul03lcomAmom
- --
pMu1001
Fig. 2. Plasmid
maps.
-----------------------------
----11 a
Maps are presented
in a linear form. For construction
cloned into the BamHI
Mu DNA and about 900 bp of bacterial represents
a restriction
pMul034dcom20
Acotn9 extends was cloned
linearization by cleavage BumHI
with BumHI with HueIII,
Bujard,
cleavage
(indicated
from position
into the GoRI
is between
position
892 to 884, Acorn20 extends
direction
a CluI fragment
751 and 692. pMulOOl
toward
contains
the rightmost
of pMul034. by sequencing
position
a BclI-BamHI
was isolated of the deletion
fragment
relevant
to ,n~rn gene expression
the hla gene, 112 is the tetracycline
The third line
pMu1034d~~m9 (Maxam
are indicated promoter.
endpoint,
ORFs
and
and Gilbert, the KmK
from pMulO34
from pMu1034
Mu DNA, wavy lines E. co/i DNA, and broken
linkers
1026 bp of
X93 to 874. In pMu1034Acom9KmK
pMul034comA~~rom
The approximate
the rightmost
of BarnHI
into the C/u1 site of pBR322.
were determined
from position
containing
encompassing
below are derivatives
site of pMu1034Acom9.
before ring closure.
a Hind11 fragment
DNA and after addition
DNA and inserted
1983). All plasmids
sites are shown. pBR322 promoters
indicating
contains
from Mucts62
1983). Solid lines represent
1981). p I and p3 initiate transcription
with arrowhead
pMomCla
of pMuAS1, from Mucts62
by gaps). Deletion endpoints
cleavage
and BAL 31 digestion
site of pBR322 (Kahmann,
Only relevant
DNA dcrivcd
carry BAL 31 deletions
1980). Deletion
DNA was isolated
site of pBR322.
map of Mu DNA (Kahmann,
gene of pWS2
----__
-__---P2
I 110 bp of Mu DNA and about 1100 bp of bacterial (dCGGATCCG)
____
ClaI
after
determined
cloned into the
lines pBR322 sequences.
by open arrows are indicated
(Stiiber
and
by open bars
of transcription.
fused top,. (pLCom1; see legend to Fig. 3). Proteins produced after induction ofp, were analyzed (Fig. 3). While a peptide of 27 kDa1, the size expected for Mom, is overproduced in strains containing plasmid pLMom3, no overproduction is detected in cells harboring plasmid pLMom1 which contains only the C-terminal part of corn and the intact mom gene. A peptide which could correspond to the 7.4-kDa1 product of corn is not visible. Cell growth of
K-12dHIdtrp containing pLMom3 is severely reduced after induction ofp,, while cells containing pLMom1 and pLCom1 show only slightly reduced growth compared to control cells containing pLc2833 (not shown). This effect is even more pronounced when cultures of the respective strains are plated at restrictive and permissive temperatures (Table I). Cells harboring pLMom1 and pLCom1 show the same colony-forming efficiency at both
65
(Bl
(A) Hind11 attR
Fig. 3. Construction to distances broken
scheme
lines vector
pLCom1
were adjusted
were prepared
to represent
about
(M)M,
serum albumin with Coomassie
standards:
lysozyme
(66 kDal), phosphorylase Brilliant
ORFs.
(A) and SDS-PAGE
Solid lines indicate
The thick arrow
indicates
(see Fig. 2) by replacing amount
of protein.
(14 kDal), trypsinogen
described
fragment
-
anhydrase
were separated
on a SDS-IS?,
by Weber and Osborn
(B). Numbers
(1969). Arrows
of transcription. p,. fragment
of
et al., 1984). The volumes analyzed (b) K-12dHIdtrp-
(f) M5219[pLMom3];
(g) M5219-
(29 kDal), ovalbumin
(45 kDal), bovine
PA-gel (Laemmli,
1970) and stained
in (B) mark the Mom protein.
I
TABLE
Mom synthesis Plasmid
pLc2833
under pL control
and cell viability
Phage titera
Phage titera
on C600
on C600( P 1Cm)
EOP
Cell tite?
Cell tite?
at 28°C
at 42’C
5.6 x 10’
3.0 x lo4
5.4 x 1om6
4.2 x 10’
3.7 x 10”
1.0 X 10’”
6.0 x 10’
6.0 x lo-*
3.6 x 10’
3.0 x IO”
pLMom3
8.8 x 10’
1.0 x lo8
1.1
3.4 x 10’
1.0 x 10”
pLCom 1
4.0 x 10’”
3.0 x 104
7.5 x 10-7
3.8 x 10’
3.9 x 10’
pLMom
refer
DNA, and heavy
and the direction
are: (a) K-lZzlHlzlrrp[pLc2833];
(e) M5219[pLMoml];
__ -- . ..“.^.-
with the respective
(Mertens
(24 kDal), carbonic
b (97.4 kDa1). Proteins
Blue R-250 as described
the pL promoter
Extracts
(d) K-lZdHIdtrp[pLComl];
of total cell extracts
Mu DNA, wavy lines bacterial
the PstI-EcoRI
3 h after the shift to 42°C as previously
the same total
(c) K-lZAHldtrp[pLMom3];
Mom protein
site of Mu (attR).
from pMu1034comdmom
pLc2833. Total cell extracts
[pLComl];
overproducing
DNA. Open bars present
was derived
[pLMoml];
for plasmids
(in bp) from the right attachment
-_
MabcdMefg -------_
I
,’ Phage lysates were prepared 5 x 10s cells/ml, h Cell viability ’ Colonies
heat induced was determined
from K-12dHldtrp(Mucts62,,, at 42°C
and incubated
from overnight
cultures
_ 5) harboring
the indicated
at the same temperature of K-12dHIdtrp
plasmids.
Cells were grown at 28°C to approx.
until lysis.
harboring
the indicated
plasmids.
were ragged.
temperatures; those containing pLMom3 suffer a lOO-fold drop in the colony-forming efficiency at the restrictive temperature. When Mom-expression fromp,-plasmids is monitored biologically by simul-
taneous induction of pi_ and a Mucts62,,, phage (Table I), full modification of progeny is observed only in the presence of pLMom3, sion levels from pLMom1 being about tenfold
5 prophage expreslower.
These experiments
show that pLMom 1 is expressing
mom whenpL
compared
is turned on, albeit at a reduced level with pLMom3. Mom gene expression
from pLMom plasmids was also tested which supplies the )+N antitermination
in M5219 function.
Expression levels were comparable to the K- 12AH ldtrp host which is IV (Fig. 3). Thus fusion to pi_ does not fully compensate
for the lack of corn.
pMu1034Acom9) and 20 bp (pMu1034Acom20) generated (Fig. 2). Both deletion endpoints
sequenced (see legend to Fig. 2). The Acorn20 deletion would cause a frameshift and reduce the size of corn from 62 to 56 aa. A third deletion pMu1034
was isolated but
Amom,
Fig. 2). Mom
biologically
transcription observations
(Table
efftcient synthesis that full synthesis
of the mom gene product and infer of Mom is the direct cause for the
lethal effect to the cell. (c) MSZ-polymerase-Corn pression In pLMom1
fusions and llzom gene ex-
the translation
initiation
signal ofcrim
has been deleted. If the rno~~ gene is subject to translational coupling (Oppenheim and Yanofsky, 1980; Schtimperli et al., 1982), translation of the region upstream of mom could be a prerequisite for full gene expression. To test this, an in-frame fusion between the N-terminal part of MS2-polymerase and the Cterminal part of cam was generated (pLc24-HD2 and pLc24-HD4, Fig. 4). In addition, these plasmids contain an intact mom gene. pLc24-HD5 contains the same Mu fragment employed to generate the fusion but cloned in opposite orientation. When I>,, on these plasmids is induced and proteins are analyzed on SDS-PAGE, high amounts of the fusion product appear (about 200; of the total cellular protein), however, overproduction of the Mom protein cannot be detected on gels (Fig. 4). This demonstrates that translation of the region 5’ to the mom gene cannot restore Mom synthesis to the same high level as in plasmid pLMom3 which carries an intact corn gene. (d) The phenotype of corn deietions
To understand the function of corn, small BAL 3 1 deletions were generated within the corn reading frame in plasmid pMu1034. pMu1034 lacks the region determining Dam-dependence of mm gene expression but still requires the Mu specific transactivator (Kahm~n, 1983). Starting at the only Be11 site in corn deletions of 9 bp (plasmid
parental
derivative
of
in which the ~zoff? gene was
corn remained
deleted
This suggests that corn does not act at the level of initiation. We conclude from these furthermore that corn is required for
were were
intact
expression
(pMul~34~~~~?2was
monitored
in a recA host lysogenic for Mucts62,,,_,
II). After induction plasmid
of the helper phagc the
pMu1034
however, mom expression
yields
full expression,
is completely
abolished
in
pMul~34A~~~m9 and pMuIO34~om20. In the next step we tried to complement the defect of pMul034Acom9 with a plasmid containing only con? and its preceding regulatory region. To be able to introduce both plasmids into the same cell, pMu10341r~om9 was tagged with a Km” gene (pMu 1034Acom9Km”, Fig. 2) and transferred into WM648 (Mucts62,,,_,)[pMucomA~no~~z 1. Neither plasmid alone expresses the mom gene after induction of the helper phage, however, when combined in the same cell the mom gene is partially expressed (Table II). This demonstrates that the defect caused by the corn deletion can be completed in trans. When instead of Murts62,,,_1 (which lacks corn), a Mu~ts62n~o~~ helper phage is used, plasmids carrying a deletion in COM, like pMulO34Ac~m9, show normal levels of mom expression (Table II). This suggests that the latter helper phage is able to complement the con7 defect on the plasmid. The mo~z- mutation is probably C-terminal or not in the region of overlap with corn. Deletion mutants in corn,, similar to pMulO34Ac~~m2~ have been described by other groups as having no effect on Mom expression (Plasterk et al., 1984; Adley and Bukhari, 1984). Such conclusions have not taken into account the possibility that the helper phage used to monitor morn gene expression, Mucts62mom , could complcmcnt the defect in (‘OFV. We have also reinvestigated the possibility that in earlier experiments, where a Mu~ts62in~~m helper phage was used, mom-expression values determined were obscured by corn complementation. Of particular interest in this respect was plasmid pMu1001 (Fig. 2; Kahmann, 1983) in which the morn gene is under control of the p2 promoter of pBR322. In a dam host, pMulOO1 showed about lo-15 times lower expression than pMu1034. To determine
67
(Bl a
b
d
c
M
BamHI
-
29
BclI "GAT TCC TTT:GAT_CAC ..CTA AGG AAA CTA ?jK
Corn
Asp SW 17 18
ATT GAA" TAA CTT..
Phe Asp His Ile Glu.. 19 20 21 22 23 BamHI
"TTG TCA TGG;G_AT_CCG GAT GTT'. ..AAC AGT ACC CTA @C CTA CAA..
MS2 p0lflerase
..Leu Ser Trp Asp Pro Asp Val 97 98 99 100 101 102 103 MS2 polylneraseCorn-fusion
Fig. 4. Construction
scheme
"TTG TCA TGG GAT CAC ATT GAA“ ..AAC AGT ACC CTA GTG TAA CTT.. ..Leu Ser Trp Asp His Ile Glu.. 97 98 99 20 21 22 23
for MS2-polymerase-Corn
-14
fusions (A), and SDS-PAGE
of total cell extracts
the pLc24 vector part; x x x , the corn gene; dots, the MOWIgene; striped areas, the N-terminal parts of conz and MS2-polymerase ChOO[pc1857] Proteins
are indicated,
with the sequence
3 h after the shift to 42°C. (a) pLc24-HDS;
were separated
on an SDS-containing
lo-20’1,,
of the joint shown below. Panel B: extracts
(b) pLc24-HD4; PA-gradient
whether this reduced level of mom expression can be attributed solely to transcription originating from p2 or might be affected by corn, Mom expression from pMulOO1 was monitored with a Mucts62mom (corn + ) and a Mucts62,,,_,(com ) helper phage respectively (Table II). Mom synthesis was not detectable with corn- helper phage while levels comparable to those published were obtained with corn+ helper. This demonstrates that corn in
(B). Panel A: double
portion of MS2-polymerase.
(c) pLc24-HD2;
gel. The arrow marks
(d) pLc24;
wcrc prepared
(M) M, standards
the MSZ-polymerase-Corn
line,
The connected from
as in Fig. 3. fusion pcptidc.
creases the level of Mom specific modification
even
when the genuine mom promoter is absent and transcription is driven by a foreign promoter. (e) How does corn function? Without knowing its exact mode of action we can at least exclude several possibilities. Corn cannot act on the level of transcription initiation as Dam and
6X
TABLE
II
Mom synthesis
in plasmid
pMul034
and derivatives
of pMu1034
Plasmid
Strain
EOP
Phage titer”
Phage titer”
on C600
on C600( P I Cm)
-
4.3 x 10’
9.0 x 10”
2.1 x 10
pMu1034
3.2 x 10”
X.0 x lox
2.5 x 10 1.x x 10
WM64X(Mucts62,,,
5)
WM64X(Mucts62,,,
_ 5)
WM648(Mucts62,,,
5)
pMul034romdmom
6.X x 10”
1.2 x 10”
WM64X(Mucts62,,,
5)
pMu 1034.4com20
9.4 x 10’
1.4 x 10”
1.5 x 10
WM648(Mucts62,,,
_ 5)
pMu1034dcomY
7.9 x 10’
2.0 x los
2.5 x 10
pMu 1034d~~71YKm~
7.x x 10’
Y.X x l(F
1.3 x 10
3.3 x IO’
2.0 x lox
S.I x 10
WM648(Mucts62,,,_,)
pMu 1034comdmom
WM64X(Mwts62,,,
5)
pMu1034d~w1YKm~
WM64X(Mucts62mom
)
WM64X(Mucts62mom
)
WM64X(Murts62mom
- )
WM64X(Mucts62mom
)
4.3 x 10”
9.0 x 10J
2.1 x IO
pMu1034
6.5 x 10”
5.3 x 10’
8.2 x 10
pMu 1034comdrwm
7.1) x 10’
Y.0 x lo4
I.1 x 10
pMu1034dcomYKmK
4.2 x lo9
2.2 x 10“
5.2 x 10 6.X x 10
N lOO(Mwts62mom
)
pMu1034
2.2 x 10”’
1.5 x IO”
N 100( Mucts62mom
)
pMulOOl
I.6 x 10”’
3.1 x 10”
1.9 x IO
N lOO(Mucts62,,,
5)
pMu1034
2.0 x IO”’
1.5 x IO”
7.5 x IO
NlOO(Mu(.t~62+,~
5)
pMulOO1
4.0 x 10”’
X.1 x 10’
2.0 x 10
“ Strains depending
were grown
at 32°C to 5 x lO* cells:‘ml in dY.T medium
on the plasmid
the Mu transactivation
state. Cultures
function
were shifted to 42’C
does. If this was the
case, cloning of the mom gene downstream of the p, promoter should alleviate the need for con? and expression of Mom from thep2 promoter in pMu 100 1 should not be influenced by corn. An involvement of corn in translational coupling to mom is excluded by the finding that in plasmids where a fusion product between MS2-polymerase and Corn is made in high amounts, overexpression of the mom gene product is not observed. Furthermore, translational coupling should operate in cis while corn has been shown to function efficiently in trans. Is corn an anti-termination function like the N-gene product of phage i, (see review by Friedman and Gottesman, 1983)? In such a scenario one would have to propose a transcription terminator between the termination codons for corn and the mom gene and a site analogous to a nut site where corn is picked up by RNA polymerase to allow read-through of the termination signal. Since the level of Mom expression in plasmid pLMom1 is unaffected by i N it would have to be a termination signal resistant to N action. Furthermore, a search for terminators using the plasmid pKG1900 (McKenney et al., 1981) has yet proved unsuccessful (F.G.W. and R.K., unpublished). For these reasons we favour the idea that Corn exerts its
(Miller,
lY72) supplemented
for 15 min and incubated
with 60 pg Ap/‘ml and 20 pg Km:ml
at 37’C
until lysx
effect on another level, e.g., stabilization of the mRNA or the Mom protein. Alternatively Corn might increase translation of mom by binding to the message and folding it in a configuration more suitable for binding to the ribosome. These diverse possibilities are currently being tested. One additional remaining question is, however, why the regulation of the mom gene is under such a complex control. The findings that full expression of the mom gene is lethal to the cell and the observation that induction of the mom gene simultaneously with prophage induction reduces the phage burst more that lOO-fold (Table I) may provide the clues to an answer. To assure a normal phage burst and at the same time guarantee full modification of phage progeny the mom gene must be expressed very efficiently at a very late stage in the lytic cycle. This has actually been demonstrated by P. van de Putte (personal communication). The development of a regulatory cascade may be best suited to fulfil these requirements and at the same time allow multiple factors to be sensed: (1) the control on the transcriptional level mediated by the methylation state in region I may be influenced by environmental factors; (2) the positive regulation of transcription through the transacting Mu function links expression to lytic phage develop-
69
ment;
(3) the need for corn further delays the onset
teriophage
Mu. Cold Spring
of efficient modification.
S. and Ives, J.: S I nuclease
Hattman,
mbnr gene promoter: expression. Hattman,
a model
S., Ives, J., Margolin,
We thank W. Messer for strains, W. Szybalski the name corn, and C. Koch, for many
stimulating
for
P. Heisig
discussions.
Part of this work was carried out at the Institute Microbiology
in
Biol. 47
Heidelberg
Schaller for his support.
and
we
We gratefully
thank
Howe,
M.: Prophage
Virology
W. and Howe, M.M.: Regulation
(Dad)
H.
Spring
Mu mom gene: mapping
function
mapping
regulates
function
Harbor
Kahmann,
to the C gene. Gene of bacteriophage
encoded
Maniatis,
Mu-l.
Symp. Quant.
the expression
of a DNA-
by bacteriophage
Mu. Cold
Biol. 47 (1983) 639-646.
R.: The mom gene of bacteriophage
Microbial.
Immunol.
T., Fritsch,
A Laboratory Spring Maxam,
deletion
R.: Methylation
modification
cript.
of mom
54 (1973) 93-101.
Kahmann,
the expert technical assistance of K. Altmann and H. Fuss and thank H. Markert for typing the manus-
of the phage Mu
39 (1985) 71-76.
for
acknowledge
mapping
for the regulation
of the bacteriophage
of the transactivation
and G. Mertens
Symp. Quant.
Gene 29 (1984) 185-198.
and expression
ACKNOWLEDGEMENTS
suggesting
Harbor
(1983) 647-653.
Mu. Curr. Topics
I08 (1984) 29-47.
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J.: Molecular
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with base-specific
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chemical
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(Eds.), Analysis
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Miller, J.H.: Experiments
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61
Elsevier GENE
1408
The mom gene of bacteriophage (Recombinant
DNA;
acetamido
Mu: a unique adenine;
regulatory
DNA modification;
scheme
to control
a lethal function
gene corn; Mom protein;
post-transcriptional
activation)
R. Kahmann*, A. Seiler, F.G. Wulczyn and E. Pfaff** Max-Piunck-Institut ,fir molekulare Genetik, Otto- Warburg-Laboratorium. Ihnestrasse 63. D-1000 Berlin 33 (DuhlemJ Tel. (30)8307-253, and **Institut_fir Mikrobiologie der UnivevPitiit Heidelberg, D-6900 Heidelberg (F.R. G.) Tel. (06221)564-294 (Received
April 23rd,
(Revision
received
(Accepted
1985)
June 7th, 1985)
July 17th, 1985)
SlJMMARY
The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation : gene expression requires methylation by the Escherichiu coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene corn (control of mom, previously termed ORF-x) whose coding region overlaps the 5’-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in corn completely abolish expression of the mom gene. Fragments lacking the 5’ end of corn can be cloned downstream of constitutive plasmid promoters. The corn gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDa1, has been visualized on gels. Efficient expression of Mom from pL requires gene corn. A fusion between MS-2 polymerase and corn has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit. We show that the defect associated with corn deletions can be complemented in tram, and propose that corn acts as a positive regulator on a post-transcriptional level.
INTRODUCTION
Bacteriophage
* To whom addressed. Abbreviations: pair(s);
EOP.
Mu encodes
correspondence
and
function, Mom (Toussaint, 1976), which is unusual in several aspects. The modification converts aden-
a DNA modification
reprint
requests
should
be
kanamycin; PAGE,
aa.
amino
efficiency
0378-I 119/85/‘$03.30
0
acid(s);
of plating:
1985 Elsevier
Ap,
bp,
base
R, resistance;
pair(s);
Km,
nates prophage
ampicillin;
kb, kilobase
Science
ORF,
Publishers
open
reading
PA gel electrophoresis;
[ 1, designates in lysogen.
p,
frame;
PA, polyacrylamide;
, major leftward I promoter;
plasmid-carrier
state;
( ), desig-
ine to acetamido
adenine
(Swinton
et al.,
1983)
I (Plasterk
et al., 1983) and can be separated
from
within the sequence
region I (Kahmann, 1983). The finding that the transacting Mu function is required for transcription
5’ -GAGNPy-3’ c c
(Hattman and Ives, 1984; Hattman et al., 1985) and that the need for activation is alleviated when the
(Kahmann,
1984). This rather
broad
mom gene is fused to foreign
modification
promoters
affects about 15 “/, of all adenine residues and renders
et al., 1983; Kahmann,
DNA
makes it likely that the activator positively influences
resistant
restriction basis
to a variety
systems,
for several
Mom-specific Toussaint, al feature
of type I and type II
a property
which provides
biological and physical modification (Hattman,
the interaction
the
1983; Plasterk
(Hattman
between
RNA
et al., 1983)
polymerase
and the
mom
promoter, possibly by binding to the -35 region. How and why this transcription-initiation
tests for 1979;
complex upstream
1976; Kahmann, 1984). A second unusuof the mom gene lies in its regulation.
Transcription of the mom gene requires the E. coli Dam-methylase (Hattman, 1982) and this requirement is linked with the presence of three Dam-sites in region I (Hattman, 1983; Kahmann, 1983; Plasterk et al., 1983 ; see Fig. 1). Internal deletions in region I which remove either one or two Dam sites render mom expression Dam-independent and the same effect is observed when the mom gene is cloned without region I (Kahmann, 1983; Plasterk et al., 1983). From S 1 nuclease mapping studies, the 5’ end of the mom mRNA maps downstream of region I at position 974 (Plasterk et al., 1984) or 963 (Hattman and Ives, 1984; see Fig. 1). It is clear that region I does not coincide with either of the two putative overlapping mom promoters (Hattman and Ives, 1984). When cloned on plasmid vectors the mom gene is usually not expressed and requires a transacting Mu function (Chaconas et al., 198 1) presumably the product of gene C (Plasterk et al., 1983; Hattman et al., 1985). The site required for acti-
could be influenced by methylation sequences is still a mystery.
of
In the present communication we present evidence that in addition to this unique regulatory situation on the transcriptional level, mom expression is subject to post-transcriptional regulation as well. We implicate the gene product of gene corn (Kahmann, 1984), whose reading frame partially overlaps the mom coding region (Fig. l), in this process.
MATERIALS
AND METHODS
(a) Bacterial
and phage strains
C600 (Appleyard, 1954); C600(PlCm) (Toussaint, 1976); K-12dHldtrp (Remaut et al., 1981); K5219 (Remaut et al., 1981); WM648 = CM987(asnA 3 1, asnB32, recA 1, spoT1, thi-1; Meyenburg et from W. Messer); and al., 1978; obtained
NlOO (Gottesman and Yarmolinski, 1968) were employed. Mucts62,,, s (Chow et al., 1978) phage
vation maps to the right but in close vicinity to region
transortlvotlon SI te Dam-dependence region I II/
162aol
1
*
I
r*
I
1100
1
PVUI
Hindll
Fig. I. Schematic
overview
of the regulatory
or 840 (Kahmann,
1983; Plasterk and
Dam-methylase. (Kahmann,
Ives,
N_N___N___v_v__c_MMN region of the mom gene. Numbers
et al., 1984), position
The site recognized
give distances
as open bars. ORF-.Y = corn. The translational
1984). Asterisks
1983; Plastcrk
800
t?clI
1983). ORFs are indicated
or 963 (Hattman
I
900
Pvul I Clal [-iaI mRNA
bp (Kahmann,
I
I
1000
I
known
I
ORF-XI
I
mark
1 (start point) ofthe the three
by the Mu-specific ct al., 1983). Several
GATC
transactivator restriction
mRNA
sequences
from the right end of the Mu genomc
start of the mom gene is either at position
(wavy line) is at position in region
(gene C product)
sites are indicated
I which
is bracketed,
as landmarks.
974 (Plastcrk
are methylated
in XIO
et al., 1984) by the E. co[i
the extent of the region is not
63
carries
a substitution
in its fi region;
region of mom up to position sequences, Kahmann
which causes
by IS2
a Mom - phenotype
and D. Kamp, unpublished).
ClaI site of pBR322 and here the fragment
the regulatory
851 is replaced
(R.
The substi-
tution has destroyed ORF-x. Mucts42morn~~~~ has been described (Toussaint, 1976) and will be referred to as Mucts62momin the text. Mucts62mom+ described by Howe (1973).
is
(b) Plasmids Plasmids
in clockwise
included
pBR322 (Bolivar et al., 1977);
(c) Biological
assay for Mom-specific
modification
Plasmids for which the Mom phenotype was to be determined were tr~sformed into a host strain lysogenie either for Mucts62mom,,,, or Mucts62,,, _ 5. The residing prophage was induced and the resulting phage lysates were plated on C600 and C6OO(P 1Cm) indicators. The EOP [titer on C6OO(Pl~m)~titer on C600] was determined. An EOP of < 10 - ’ indicates a Mom-, and EOP of > lop4 a Mom+ phenotype (Toussaint, 1976).
AND
DISCUSSION
{a) The ability to clone the wont gene is determined by the absence of strong constitutive vector promoters or ORF-x (corn) Studies aimed at understanding the regulatory mechanism that controls mom gene expression required the cloning of the mom gene or parts of it in plasmid vectors. In doing so we realized that certain restriction fragments could be cloned exclusively in one orientation in pBR322 while for other fragments no such orientation bias existed. The most striking examples are plasmid pMuAS 1 and plasmid pMomCla (Fig. 2). In pMuAS 1 the mom gene is cloned into the BamHI site of pBR322 and all clones isolated contained the fragment in an anticlockwise orientation. In pMomCla the mom gene is cloned into the
orientation.
were reported by Hattman
is always
Similar
results
and Ives (1984). Attempts
to invert the respective BamHI or ClaI fragments were unsuccessful (more than 100 clones with inserts were analyzed). In both instances the orientation which could not be isolated would have fused the mom gene to pBR322 the pMuASl-derived pMomCla-derived
pLc2833 and pLc24 (Remaut et al., 1981); pMu1034, pMuI001 and pMu1010 (Kahmann, 1983) and ~~1857 (Remaut et al., 1983). pWS2 is a pBR322 derivative containing a 1.2-kb EcoRI fragment encoding KmR (obtained from W. Messer).
RESULTS
inserted
promoters (p2 in the case of plasmid, pl in case of the
plasmid).
In contrast,
shorter
fragments containing the mom gene, for example on a BclI-BarnHI fragment, can readily be cloned in both orientations into the BanzHI site of pBR322 (pMulOO1 and pMu1010, described by Kahmann, 1983). In pMu 100 1 (Fig. 2) the mom gene is fused to thep2 promoter and this results in a low-level constitutive expression which apparently is tolerated by the host cell. These results suggested that the additional sequences between the BclI and C(a1 site determine whether the mom gene can be cloned downstream of constitutive plasmid promoters p 1 and ~2. A trivial explanation, e.g., homologies between this region and parts of pBR322 which might confer instability to such plasmids can be excluded. In attempts to invert the CluI fragment of pMomCla, one clone was isolated which contained the fragments in clockwise orientation. This clone did not express the mom gene after activation by a helper phage nor was there any constitutive modification detectable. Presumably this clone had acquired a spontaneous mutation in the mom gene (not shown). From these results it was tempting to speculate that the underlying reason for the inability to clone mom on larger fragments downstream of plasmid promoters was the presence of an intact ORF-x (corn) on such fragments. Either transcription or translation of the region 5’ to the mom gene could be implicated in enhancing mom gene expression. (b) High level expression
of Mom protein requires
corn
Experiments presented above suggested that corn is somehow involved in the regulation of mom gene expression. To test the possibility that this additional control might operate on the transcriptional level as Dam and the transactivator do, the mom gene was cloned downstream ofp, on plasmid pLc2833 with and without corn (Fig. 3). In addition corn alone was
64 Barn HI
BamHI
Clai
_----- -- I
pMuAS1
EcoRl
__lL p20
ECDRI
i
----_
-F4
P3e
0P3 P? tlamtll
Clal
ClaI ‘11
_--_-----_
pMomCla
a
PI
G.-
MU
-----_--
pMu:034
_ii P3
IBM---------__
e
pMu1034AromZO
----------*
_---_--_-__-_
pMulOXArom9
--------_-_+
-----------_
pMu103LAcom9KmR
-_------___ EcoRI
pMul03lcomAmom
- --
pMu1001
Fig. 2. Plasmid
maps.
-----------------------------
----11 a
Maps are presented
in a linear form. For construction
cloned into the BamHI
Mu DNA and about 900 bp of bacterial represents
a restriction
pMul034dcom20
Acotn9 extends was cloned
linearization by cleavage BumHI
with BumHI with HueIII,
Bujard,
cleavage
(indicated
from position
into the GoRI
is between
position
892 to 884, Acorn20 extends
direction
a CluI fragment
751 and 692. pMulOOl
toward
contains
the rightmost
of pMul034. by sequencing
position
a BclI-BamHI
was isolated of the deletion
fragment
relevant
to ,n~rn gene expression
the hla gene, 112 is the tetracycline
The third line
pMu1034d~~m9 (Maxam
are indicated promoter.
endpoint,
ORFs
and
and Gilbert, the KmK
from pMulO34
from pMu1034
Mu DNA, wavy lines E. co/i DNA, and broken
linkers
1026 bp of
X93 to 874. In pMu1034Acom9KmK
pMul034comA~~rom
The approximate
the rightmost
of BarnHI
into the C/u1 site of pBR322.
were determined
from position
containing
encompassing
below are derivatives
site of pMu1034Acom9.
before ring closure.
a Hind11 fragment
DNA and after addition
DNA and inserted
1983). All plasmids
sites are shown. pBR322 promoters
indicating
contains
from Mucts62
1983). Solid lines represent
1981). p I and p3 initiate transcription
with arrowhead
pMomCla
of pMuAS1, from Mucts62
by gaps). Deletion endpoints
cleavage
and BAL 31 digestion
site of pBR322 (Kahmann,
Only relevant
DNA dcrivcd
carry BAL 31 deletions
1980). Deletion
DNA was isolated
site of pBR322.
map of Mu DNA (Kahmann,
gene of pWS2
----__
-__---P2
I 110 bp of Mu DNA and about 1100 bp of bacterial (dCGGATCCG)
____
ClaI
after
determined
cloned into the
lines pBR322 sequences.
by open arrows are indicated
(Stiiber
and
by open bars
of transcription.
fused top,. (pLCom1; see legend to Fig. 3). Proteins produced after induction ofp, were analyzed (Fig. 3). While a peptide of 27 kDa1, the size expected for Mom, is overproduced in strains containing plasmid pLMom3, no overproduction is detected in cells harboring plasmid pLMom1 which contains only the C-terminal part of corn and the intact mom gene. A peptide which could correspond to the 7.4-kDa1 product of corn is not visible. Cell growth of
K-12dHIdtrp containing pLMom3 is severely reduced after induction ofp,, while cells containing pLMom1 and pLCom1 show only slightly reduced growth compared to control cells containing pLc2833 (not shown). This effect is even more pronounced when cultures of the respective strains are plated at restrictive and permissive temperatures (Table I). Cells harboring pLMom1 and pLCom1 show the same colony-forming efficiency at both
65
(Bl
(A) Hind11 attR
Fig. 3. Construction to distances broken
scheme
lines vector
pLCom1
were adjusted
were prepared
to represent
about
(M)M,
serum albumin with Coomassie
standards:
lysozyme
(66 kDal), phosphorylase Brilliant
ORFs.
(A) and SDS-PAGE
Solid lines indicate
The thick arrow
indicates
(see Fig. 2) by replacing amount
of protein.
(14 kDal), trypsinogen
described
fragment
-
anhydrase
were separated
on a SDS-IS?,
by Weber and Osborn
(B). Numbers
(1969). Arrows
of transcription. p,. fragment
of
et al., 1984). The volumes analyzed (b) K-12dHIdtrp-
(f) M5219[pLMom3];
(g) M5219-
(29 kDal), ovalbumin
(45 kDal), bovine
PA-gel (Laemmli,
1970) and stained
in (B) mark the Mom protein.
I
TABLE
Mom synthesis Plasmid
pLc2833
under pL control
and cell viability
Phage titera
Phage titera
on C600
on C600( P 1Cm)
EOP
Cell tite?
Cell tite?
at 28°C
at 42’C
5.6 x 10’
3.0 x lo4
5.4 x 1om6
4.2 x 10’
3.7 x 10”
1.0 X 10’”
6.0 x 10’
6.0 x lo-*
3.6 x 10’
3.0 x IO”
pLMom3
8.8 x 10’
1.0 x lo8
1.1
3.4 x 10’
1.0 x 10”
pLCom 1
4.0 x 10’”
3.0 x 104
7.5 x 10-7
3.8 x 10’
3.9 x 10’
pLMom
refer
DNA, and heavy
and the direction
are: (a) K-lZzlHlzlrrp[pLc2833];
(e) M5219[pLMoml];
__ -- . ..“.^.-
with the respective
(Mertens
(24 kDal), carbonic
b (97.4 kDa1). Proteins
Blue R-250 as described
the pL promoter
Extracts
(d) K-lZdHIdtrp[pLComl];
of total cell extracts
Mu DNA, wavy lines bacterial
the PstI-EcoRI
3 h after the shift to 42°C as previously
the same total
(c) K-lZAHldtrp[pLMom3];
Mom protein
site of Mu (attR).
from pMu1034comdmom
pLc2833. Total cell extracts
[pLComl];
overproducing
DNA. Open bars present
was derived
[pLMoml];
for plasmids
(in bp) from the right attachment
-_
MabcdMefg -------_
I
,’ Phage lysates were prepared 5 x 10s cells/ml, h Cell viability ’ Colonies
heat induced was determined
from K-12dHldtrp(Mucts62,,, at 42°C
and incubated
from overnight
cultures
_ 5) harboring
the indicated
at the same temperature of K-12dHIdtrp
plasmids.
Cells were grown at 28°C to approx.
until lysis.
harboring
the indicated
plasmids.
were ragged.
temperatures; those containing pLMom3 suffer a lOO-fold drop in the colony-forming efficiency at the restrictive temperature. When Mom-expression fromp,-plasmids is monitored biologically by simul-
taneous induction of pi_ and a Mucts62,,, phage (Table I), full modification of progeny is observed only in the presence of pLMom3, sion levels from pLMom1 being about tenfold
5 prophage expreslower.
These experiments
show that pLMom 1 is expressing
mom whenpL
compared
is turned on, albeit at a reduced level with pLMom3. Mom gene expression
from pLMom plasmids was also tested which supplies the )+N antitermination
in M5219 function.
Expression levels were comparable to the K- 12AH ldtrp host which is IV (Fig. 3). Thus fusion to pi_ does not fully compensate
for the lack of corn.
pMu1034Acom9) and 20 bp (pMu1034Acom20) generated (Fig. 2). Both deletion endpoints
sequenced (see legend to Fig. 2). The Acorn20 deletion would cause a frameshift and reduce the size of corn from 62 to 56 aa. A third deletion pMu1034
was isolated but
Amom,
Fig. 2). Mom
biologically
transcription observations
(Table
efftcient synthesis that full synthesis
of the mom gene product and infer of Mom is the direct cause for the
lethal effect to the cell. (c) MSZ-polymerase-Corn pression In pLMom1
fusions and llzom gene ex-
the translation
initiation
signal ofcrim
has been deleted. If the rno~~ gene is subject to translational coupling (Oppenheim and Yanofsky, 1980; Schtimperli et al., 1982), translation of the region upstream of mom could be a prerequisite for full gene expression. To test this, an in-frame fusion between the N-terminal part of MS2-polymerase and the Cterminal part of cam was generated (pLc24-HD2 and pLc24-HD4, Fig. 4). In addition, these plasmids contain an intact mom gene. pLc24-HD5 contains the same Mu fragment employed to generate the fusion but cloned in opposite orientation. When I>,, on these plasmids is induced and proteins are analyzed on SDS-PAGE, high amounts of the fusion product appear (about 200; of the total cellular protein), however, overproduction of the Mom protein cannot be detected on gels (Fig. 4). This demonstrates that translation of the region 5’ to the mom gene cannot restore Mom synthesis to the same high level as in plasmid pLMom3 which carries an intact corn gene. (d) The phenotype of corn deietions
To understand the function of corn, small BAL 3 1 deletions were generated within the corn reading frame in plasmid pMu1034. pMu1034 lacks the region determining Dam-dependence of mm gene expression but still requires the Mu specific transactivator (Kahm~n, 1983). Starting at the only Be11 site in corn deletions of 9 bp (plasmid
parental
derivative
of
in which the ~zoff? gene was
corn remained
deleted
This suggests that corn does not act at the level of initiation. We conclude from these furthermore that corn is required for
were were
intact
expression
(pMul~34~~~~?2was
monitored
in a recA host lysogenic for Mucts62,,,_,
II). After induction plasmid
of the helper phagc the
pMu1034
however, mom expression
yields
full expression,
is completely
abolished
in
pMul~34A~~~m9 and pMuIO34~om20. In the next step we tried to complement the defect of pMul034Acom9 with a plasmid containing only con? and its preceding regulatory region. To be able to introduce both plasmids into the same cell, pMu10341r~om9 was tagged with a Km” gene (pMu 1034Acom9Km”, Fig. 2) and transferred into WM648 (Mucts62,,,_,)[pMucomA~no~~z 1. Neither plasmid alone expresses the mom gene after induction of the helper phage, however, when combined in the same cell the mom gene is partially expressed (Table II). This demonstrates that the defect caused by the corn deletion can be completed in trans. When instead of Murts62,,,_1 (which lacks corn), a Mu~ts62n~o~~ helper phage is used, plasmids carrying a deletion in COM, like pMulO34Ac~m9, show normal levels of mom expression (Table II). This suggests that the latter helper phage is able to complement the con7 defect on the plasmid. The mo~z- mutation is probably C-terminal or not in the region of overlap with corn. Deletion mutants in corn,, similar to pMulO34Ac~~m2~ have been described by other groups as having no effect on Mom expression (Plasterk et al., 1984; Adley and Bukhari, 1984). Such conclusions have not taken into account the possibility that the helper phage used to monitor morn gene expression, Mucts62mom , could complcmcnt the defect in (‘OFV. We have also reinvestigated the possibility that in earlier experiments, where a Mu~ts62in~~m helper phage was used, mom-expression values determined were obscured by corn complementation. Of particular interest in this respect was plasmid pMu1001 (Fig. 2; Kahmann, 1983) in which the morn gene is under control of the p2 promoter of pBR322. In a dam host, pMulOO1 showed about lo-15 times lower expression than pMu1034. To determine
67
(Bl a
b
d
c
M
BamHI
-
29
BclI "GAT TCC TTT:GAT_CAC ..CTA AGG AAA CTA ?jK
Corn
Asp SW 17 18
ATT GAA" TAA CTT..
Phe Asp His Ile Glu.. 19 20 21 22 23 BamHI
"TTG TCA TGG;G_AT_CCG GAT GTT'. ..AAC AGT ACC CTA @C CTA CAA..
MS2 p0lflerase
..Leu Ser Trp Asp Pro Asp Val 97 98 99 100 101 102 103 MS2 polylneraseCorn-fusion
Fig. 4. Construction
scheme
"TTG TCA TGG GAT CAC ATT GAA“ ..AAC AGT ACC CTA GTG TAA CTT.. ..Leu Ser Trp Asp His Ile Glu.. 97 98 99 20 21 22 23
for MS2-polymerase-Corn
-14
fusions (A), and SDS-PAGE
of total cell extracts
the pLc24 vector part; x x x , the corn gene; dots, the MOWIgene; striped areas, the N-terminal parts of conz and MS2-polymerase ChOO[pc1857] Proteins
are indicated,
with the sequence
3 h after the shift to 42°C. (a) pLc24-HDS;
were separated
on an SDS-containing
lo-20’1,,
of the joint shown below. Panel B: extracts
(b) pLc24-HD4; PA-gradient
whether this reduced level of mom expression can be attributed solely to transcription originating from p2 or might be affected by corn, Mom expression from pMulOO1 was monitored with a Mucts62mom (corn + ) and a Mucts62,,,_,(com ) helper phage respectively (Table II). Mom synthesis was not detectable with corn- helper phage while levels comparable to those published were obtained with corn+ helper. This demonstrates that corn in
(B). Panel A: double
portion of MS2-polymerase.
(c) pLc24-HD2;
gel. The arrow marks
(d) pLc24;
wcrc prepared
(M) M, standards
the MSZ-polymerase-Corn
line,
The connected from
as in Fig. 3. fusion pcptidc.
creases the level of Mom specific modification
even
when the genuine mom promoter is absent and transcription is driven by a foreign promoter. (e) How does corn function? Without knowing its exact mode of action we can at least exclude several possibilities. Corn cannot act on the level of transcription initiation as Dam and
6X
TABLE
II
Mom synthesis
in plasmid
pMul034
and derivatives
of pMu1034
Plasmid
Strain
EOP
Phage titer”
Phage titer”
on C600
on C600( P I Cm)
-
4.3 x 10’
9.0 x 10”
2.1 x 10
pMu1034
3.2 x 10”
X.0 x lox
2.5 x 10 1.x x 10
WM64X(Mucts62,,,
5)
WM64X(Mucts62,,,
_ 5)
WM648(Mucts62,,,
5)
pMul034romdmom
6.X x 10”
1.2 x 10”
WM64X(Mucts62,,,
5)
pMu 1034.4com20
9.4 x 10’
1.4 x 10”
1.5 x 10
WM648(Mucts62,,,
_ 5)
pMu1034dcomY
7.9 x 10’
2.0 x los
2.5 x 10
pMu 1034d~~71YKm~
7.x x 10’
Y.X x l(F
1.3 x 10
3.3 x IO’
2.0 x lox
S.I x 10
WM648(Mucts62,,,_,)
pMu 1034comdmom
WM64X(Mwts62,,,
5)
pMu1034d~w1YKm~
WM64X(Mucts62mom
)
WM64X(Mucts62mom
)
WM64X(Murts62mom
- )
WM64X(Mucts62mom
)
4.3 x 10”
9.0 x 10J
2.1 x IO
pMu1034
6.5 x 10”
5.3 x 10’
8.2 x 10
pMu 1034comdrwm
7.1) x 10’
Y.0 x lo4
I.1 x 10
pMu1034dcomYKmK
4.2 x lo9
2.2 x 10“
5.2 x 10 6.X x 10
N lOO(Mwts62mom
)
pMu1034
2.2 x 10”’
1.5 x IO”
N 100( Mucts62mom
)
pMulOOl
I.6 x 10”’
3.1 x 10”
1.9 x IO
N lOO(Mucts62,,,
5)
pMu1034
2.0 x IO”’
1.5 x IO”
7.5 x IO
NlOO(Mu(.t~62+,~
5)
pMulOO1
4.0 x 10”’
X.1 x 10’
2.0 x 10
“ Strains depending
were grown
at 32°C to 5 x lO* cells:‘ml in dY.T medium
on the plasmid
the Mu transactivation
state. Cultures
function
were shifted to 42’C
does. If this was the
case, cloning of the mom gene downstream of the p, promoter should alleviate the need for con? and expression of Mom from thep2 promoter in pMu 100 1 should not be influenced by corn. An involvement of corn in translational coupling to mom is excluded by the finding that in plasmids where a fusion product between MS2-polymerase and Corn is made in high amounts, overexpression of the mom gene product is not observed. Furthermore, translational coupling should operate in cis while corn has been shown to function efficiently in trans. Is corn an anti-termination function like the N-gene product of phage i, (see review by Friedman and Gottesman, 1983)? In such a scenario one would have to propose a transcription terminator between the termination codons for corn and the mom gene and a site analogous to a nut site where corn is picked up by RNA polymerase to allow read-through of the termination signal. Since the level of Mom expression in plasmid pLMom1 is unaffected by i N it would have to be a termination signal resistant to N action. Furthermore, a search for terminators using the plasmid pKG1900 (McKenney et al., 1981) has yet proved unsuccessful (F.G.W. and R.K., unpublished). For these reasons we favour the idea that Corn exerts its
(Miller,
lY72) supplemented
for 15 min and incubated
with 60 pg Ap/‘ml and 20 pg Km:ml
at 37’C
until lysx
effect on another level, e.g., stabilization of the mRNA or the Mom protein. Alternatively Corn might increase translation of mom by binding to the message and folding it in a configuration more suitable for binding to the ribosome. These diverse possibilities are currently being tested. One additional remaining question is, however, why the regulation of the mom gene is under such a complex control. The findings that full expression of the mom gene is lethal to the cell and the observation that induction of the mom gene simultaneously with prophage induction reduces the phage burst more that lOO-fold (Table I) may provide the clues to an answer. To assure a normal phage burst and at the same time guarantee full modification of phage progeny the mom gene must be expressed very efficiently at a very late stage in the lytic cycle. This has actually been demonstrated by P. van de Putte (personal communication). The development of a regulatory cascade may be best suited to fulfil these requirements and at the same time allow multiple factors to be sensed: (1) the control on the transcriptional level mediated by the methylation state in region I may be influenced by environmental factors; (2) the positive regulation of transcription through the transacting Mu function links expression to lytic phage develop-
69
ment;
(3) the need for corn further delays the onset
teriophage
Mu. Cold Spring
of efficient modification.
S. and Ives, J.: S I nuclease
Hattman,
mbnr gene promoter: expression. Hattman,
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S., Ives, J., Margolin,
We thank W. Messer for strains, W. Szybalski the name corn, and C. Koch, for many
stimulating
for
P. Heisig
discussions.
Part of this work was carried out at the Institute Microbiology
in
Biol. 47
Heidelberg
Schaller for his support.
and
we
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