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Bioscience Reports i, 299-307 (1981)
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299
A l i g n m e n t of c l o n e d amiE g e n t of Pseudomonas
aeruginosa
with the N-terminal sequence of amidase
P.H. CLARKE,* R.E. DREW,* C. TURBERVILLE,*
W.3. BRAMMAR~% R.P. AMBLERsw and A.D, AUFFRET~w182
*Department of Biochemistry, University College London,
London WCIE 6BT, U.K.; %Department of Biochemistry,
University of Leicester, Leicester LEI 7RH, U.K.; and
w
of Molecular Biology, University of
Edinburgh, Edinburgh EH9 3JR, U.K.
(Received 4 March 1981)
A restriction enzyme map was constructed for a 5.1-kb
f r a g m e n t of Pseudomonas aeruginosa DNA inserted into
plasmid pBR322. Restriction enzym e sites were matched
to the N-terminal amino acid s e q u e n c e of a m i d a s e to
o b t a i n a l i g n m e n t of t h e amiE gent within the cloned
fragment.
Pseudomonas a e r u g i n o s a strain PAC1 produces an inducible aliphatic
amidase whose synthesis is under positive control by a regulator gent ,
am iR~ c l o s e l y l i nked to the structural gene~ amiE (Brammar et al.,
1967; Farin & Clarke~ 1978).
Amidase is also subject to c a t a b o l i t e
r e p r e s s i o n by s u c c i n a t e and other i nt erm edi at es of the tricarboxylic
acid cycle (Smyth & Clarke, 1975a~b).
We h a v e c l o n e d a m i d a s e g e n e s in E s c h e r i c h i a
c o l i using a
derivative of bacteriophage lambda as the cloning vect or (Drew et al.,
1980) and confirmed that the enzyme synthesized in phage-infected E.
c o l i is identical to t hat of P. a e r u g i n o s a .
Preliminary r e s t r i c t i o n
e n z y m e m a p p i n g of l a m i R D 1 DNA show ed the loss of one of the
HindIII sites used to construct the r e c o m b i n a n t and r e v e a l e d si ngl e
Kpr~ and SalI sites within the inserted DNA.
Digestion with KpnI and
//indIII in c o m b i n a t i o n g a v e DNA f r a g m e n t s c o r r e s p o n d i n g to an
insertion of about 9 kb of P. a e r u g i n o s a DNA (Drew et a l , 1980).
Cleavage of t a m i DNA w i t h HindIII and S a l I g e n e r a t e d a 5.1-kb
f r a g m e n t c o n t a i n i n g amidase genes, which was subcloned in plasmid
pBR322 for restriction enzyme mapping.
A m i d a s e is a h e x a m e r i c protein with identical subunits of about
33 000 in m o l e c u l a r w e i g h t .
The a m i n o a c i d s e q u e n c e has b e e n
e s t a b l i s h e d for a major part of the enzyme protein (Auffret~ 1977;
R.P. Ambler and A.D. A u f f r e t , u n p u b l i s h e d ) .
The d i s t r i b u t i o n of
restriction e nz ym e sites suggested that it would be possible to identify
the position of the amiE structural g e n t in t h e c l o n e d s e g m e n t by
' t r a n s l a t i n g ' th'e n u c l e o t i d e s e q u e n c e s of t he r e s t r i c t i o n e n z y m e
recognition sites and matching all t h e p o s s i b l e s e q u e n c e s with t h e
known amino acid sequence of the N-terminal region. The method was
applied subsequently to published data for o t h e r g e n e s and p r o t e i n s
where the amino acid sequences~ DNA sequences~ and restriction maps
were already known.
82
address: Department of Biochemistry, University of Leeds,
Leeds LS2 9JT, U.K.
9
The Biochemical
Society
300
Materials
CLARKE ET AL.
and Methods
Bacteria and bacteriophages
E. c o l t C600 (Appleyard, 195#) was used as the standard host for
growth of lambdoid phages.
W3110 polA] (Gross & Gross, 1969) and
N3098, ( l i g t s 7, supF) (Pauling & Hamm, 1968) were used to test
phages for the Fec + phenotype (Zissler et al., 1971). E. c o l t L910
( metB, hsdS, supE, s u p F , r e c B , r e c C ) , a d e r i v a t i v e of the nonrestricting strain 803 (Wood, 1966), was used as the host for transformation.
The p l a q u e - f o r m i n g lambdoid phages carrying the amiE
gene from P. aeruginosa strain PAC433, lamiRDI (Xami ( a t t - x i s ) A
imm~1 n i n S ) and l a m i 3 1 4 ( l a m i ( a t t - x i s ) A imm z ci857 n i n +) have
been described (Drew et al., 1980).
lDB43 (XtrpE (DC)502 BA/lacZ
cIAninS)
( D . W . B ur r , u n p u b l i s h e d ) was used as a s o u r c e of a
Hina]II-Kpnl linker f r agm e nt carrying the t r p A and l acZ genes.
Transfer of the ami gene to a plasmid:
Construction of pJB950
The amiE gene was transferred from ),ami314 to the plasmid vect or
pBR322 ( B o l i v a r et al., 1977) as part of the fragm ent of P. aerug in o s a DNA flanked by the HindlII and SalI targets.
Samples of 0.5
IJg of Xami314 and pBR322 DNAs were digested with Hin4III and SalI
simultaneously in HindIII buffer as previously described (Drew et al.,
1980). Digested DNAs (1.0 lJg total DNA) were mixed and ligated in
a volume of 65 IJl for 16 h at 10~ as described elsewhere (Drew et
al., [980). Samples from the ligation mixture were used to transform
E. c o l t strain Lg10 made c o m p e t e n t by the method of Mandel and
Higa ( 1 9 7 0 ) .
A f t e r p r e - i n c u b a t i o n for 1 h at 37~
in L - b r o t h
(Lennox, 1955), the t r a n s f o r m a t i o n m i x t u r e was p l a t e d on L - a g a r
c o n t a i n i n g a m p i c i l l i n (20 p g / m l ) .
A p p r o x i m a t e l y 8000 ampicillinresistant transformants per lJg of total DNA were obtained, of which
about 10% were shown to be sensitive to t e t r a c y c l i n e (20 t~g/ml) on
subsequent testing.
T h e p l a s m i d DNA f r o m t h e p a r e n t a l
s t r a i n and f r o m 10
tetr acy clin e - s e ns i t i ve transformants was subjected to electrophoresis on
a 1% agarose gel, using the rapid screening method of Barnes (1977).
One of the ten isolates contained enlarged p l a s m i d DNA, which on
s u b s e q u e n t p u r i f i c a t i o n and a n a l y s i s p r o v e d to c a r r y t h e 5. l - k b
f r ag men t bounded by SalI and Hin4III s i t e s .
St ai ns h a r b o u r i n g t h e
r e c o m b i n a n t plasmid p3B950 were shown to have low but d e t e c t a b l e
amidase activities.
D e l e t i o n o f DNA from kami
D e r i v a t i v e s of Xami314 with deletions were obtained by selecting
for resistance to the chelating agent sodium pyrophosphate (Parkinson
& Huskey, 1971; Shulman & Gottesman, 1971). Dilutions of stocks of
Xami314 were adsorbed to indicator st rai n C600 on BBL T r y p t i c a s e
agar plates (Parkinson, 1968) containing 10 mM P2OTNa4, pH 7.0, and
incubated for 16 h at 37~
P y r o p h o s p h a t e - r e s i s t a n t phages, which
a p p e a r e d at a f r e q u e n c y of a b o u t 3"10 -s, were plaque-purified on
selective plates and propagated by lytic infection for the isolation of
phage DNA as described by Murray et al. (1977).
ALIGNMENT OF CLONED amiE GENE
301
Restriction endonuclease mapping
AvaI, Avail, Bc!I, Kpnl, Sstl, SstII~ Xbal, XhoI, and Xorll were
purchased from Uniscience L t d . , C a m b r i d g e .
BamHI, Bglll, EcoRI,
Haell, HaeIII, HindlII, HpaI, HpaII, PstI, SalI, and ~ DNA were purchased
from Miles Laboratories Ltd., Stoke P o g e s .
ItindII, SmaI, and TaqI
w e r e p u r c h a s e d from BCL, Lewes, and Sau3A from CP Laboratories
Ltd., Bishop's Stortford.
Digestions were carried out in t h e b u f f e r s
recommended by the manufacturers using 1 IJg of plasmid or bacteriophage DNA in a final volume of 20 Ill. For doubl e d i g e s t i o n s t h e
plasmid DNA was digested first with the enzyme requiring the buffer
of lower ionic strength and subsequently adjusted b e f o r e adding t h e
second enzyme. After addition of 3 Ill of loading buffer (25% Ficoll,
Pharmacia, Uppsala; buffer; 0.025% bromophenol blue; 0.1 M EDTA in
0.04 M T r i s - a c e t a t e buffer; pH 9.3), samples were applied to horizontal agarose gels ( i - 2 . 5 % w/v) using 0.04 M T r i s - a c e t a t e buffer, pH
8.0 (Sharp et al., 1973), and run overnight at 35 mA. Other samples
were desalted with ethanol and applied to vertical polyacrylamide slab
gels ( M a n i a t i s et al., 1975) and run at 10 V per cm.
Gels were
stained for 30 min in i pg/ml ethidium bromide solution, washed for
30 min with distilled water, and photographed. Calculation of the size
of the DNA fragments was based on known s t a n d a r d s of d i g e s t s of
)~cI857 wit h EcoRI and HindIII (Phillipsen et al., 1978) and pBR322
with ttpall (Sutcliffe, 1978).
Converting nucleotide sequences of restriction enzyme sites to
corresponding amino acids
The nucleotide
s e q u e n c e s w e r e ' t r a n s l a t e d ' i n t o a m i n o aci d
sequences in each of the t hr e e reading frames. For example the SstII
sequence can be read as CCG.CGG to code for Pro.Arg; as C.CGC.GG
to code for X.Arg.Gly; as C C . G C G . G to c o d e for ( S e r , Pro, Thr,
A l a ) . A l a . ( G i y , Ala, Asp, Glu, V a l ).
Som e of t h e potential sites
predicted for individual restriction enzymes could be eliminated on the
basis of the restriction data alone.
One of the al t ernat i ve sequences
for AvaI recognition could be eliminated for sites not also targets for
Sinai and a n o t h e r could be eliminated for sites not also targets for
XhoI. Similar considerations applied to o v e r l a p p i n g s p e c i f i c i t i e s of
Hindll, Sail, and Hpal.
It should be emphasized that the restriction targets predicted from
the amino acid sequence are only potential sites.
A restriction site
identified by mapping must correspond to a particular DNA sequence
but some of the potential sites identified by e x a m i n a t i o n of ' t r a n s lations' need not exist at all.
Results
and Discussion
Localization of the amiE gene within the cloned DNA
The recombinant phage XaraiRD1 contains a p p r o x i m a t e l y
9 kb of
D N A derived from P. aeruginosa PACTS33, located between a Kpnl site
and a HindlIl site of a vector phage ( D r e w et al., 1980).
Information
on t h e a p p r o x i f n a t e l o c a t i o n of t h e amiE gene was o b t a i n e d by
302
C L A R K E ET AL.
A
kami314
J
KK
| I
K
H
I
|,,.(a.tt -xi s)A
N cl
Q SR
"~176176176176176176176176176176
~'~
.....................
~176176
~176176176176176176176
~176176176176176176
"~176176
X
I
1
I
I
3
I
I
5
I
I
7
I
I
9kb
I
Fig. i.
Locating the amiE gene within the DNA of
%ami314. The map at the top of the figure shows the
genome of %ami314, including some of the phage genes.
Below the phage map, on expanded scale~ are shown the
physical maps of the cloned DNA in %ami314 and three
of its derivatives.
The single horizontal line
represents ~ DNA, an open double line represents DNA
originally from P. aeruginosa, and the shaded double
line shows the DNA of the E. coli trp/lac fusion
operon, isolated from trp/lac phage %DB54.
Brackets
show the positions of deletions, and arrows indicate
the positions of targets for restriction enzymes
KpnI(K), HindlII(H), Sail(S), and XhoI(X).
The
kilobase scale at the bottom applies only to the four
expanded maps.
i s o l a t i o n and c h a r a c t e r i z a t i o n of deletions within Xami314.
The only
non-essential phage genes carried by Xami318 are the red, gam, and
cIII genes located between the cloned D N A and the immunity region of
the phage (Fig. I). The presence of the red and gain genes could
readily be verified by testing the ability of phages to give plaques on
polA and ligts strains (Zissler et al., 1971). Pyrophosphate-resistant
phages retaining the red and gain genes, and likely to contain deletions
within the cloned DNA, w e r e p r o p a g a t e d for DNA isolation.
Positions
of t w o of t h e D N A d e l e t i o n s a r e s h o w n in F i g . I .
D e l e t i o n A7
r e m o v e s a p p r o x i m a t e l y 4.9 kb of DNA, including the t a r g e t for S a l I ,
w h i l e l e a v i n g t h e s e c o n d KpnI t a r g e t of X and all the XhoI t a r g e t s
intact.
D e l e t i o n M o v e r l a p s 47, e l i m i n a t i n g 4.5 kb of DNA including
t h e S a / I t a r g e t , and t h e m o s t l e f t w a r d X h o I t a r g e t .
T h e s e two
deletions t o g e t h e r r e m o v e a b o u t 5.3 kb of DNA at the l e f t end of t h e
cloned Pseudomonas fragment.
Since both tami31~A1 and 47 r e m a i n
strongly a m i d a s e - p o s i t i v e in p l a t e t e s t s , t h e s e results l o c a t e t h e amiE
gene in the r i g h t - h a n d half of the cloned DNA.
This was c o n f i r m e d
by finding a m i d a s e a c t i v i t y in E. c o l i
harbouring the recombinant
ALIGNMENT OF CLONED amiE GENE
303
Hin
1
0
3.74 Kb
5 'lOKb
Xh Kpn
H,nUTuSm P
p
im x i~
2"0
6
Xh
,6o
260
xh
lit
(,s.,
3"0
4"0
S~'OKb
a;o
~60
s~o
,~
~o =
i
Fig. 2.
Restriction enzyme map of cloned amidase
genes: (a) map of plasmid pJB950 (pBR322-ami); (b)
map of P. aeruginosa DNA inserted in pJB950; (c) map
of first 700 bp from HindIII site.
The plasmid was
analysed with restriction enzymes AvaI (AI) 9 AvaII
(AII)~ HaeII (HII), HaeIII (HIII), HindII (HinII)~
HindIII (HinIII), HpaII (Hp), KpnI (Kpn), PstI (P),
SalI (Sal) 9 SmaI
(Xh)~ and XorII
(Sm)~ SstII (S)~ TaqI (T)9 XhoI
(Xo)~ and cleavage sites for these
enzymes are indicated.
The arrow indicates the
location of the N-terminal segment of the amiE gene.
See also Table 3.
plasmid pJB950 into which the 5.l-kb HindIII-SalI f r a g m e n t had been
inserted (Fig. 2a).
More p r e c i s e l o c a t i o n of t h e amiE g e n e was
o b t a i n e d by c o n s t r u c t i o n of p h a g e XDB219 (Fig. 1) containing the
2.#-kb KpnI-gindIII fragment.
This recombinant phage also e x p r e s s e d
the Ami + phenotype, indicating that the amiE gene is located within
the 2.4-kb f r a gm ent of Pseudomonas DNA f l a n k e d by t h e gpnI and
HindIII targets.
Restriction map
Table 1 gives the total number of f r a g m e n t s produced as a result
of digestion of plasmid p3B950ami with restriction endonucleases. The
average values for the sums of fragments obtained was around 9 kb as
expected.
Digestion with enzymes BamHI, BclI, BglII, HyaI, EcoRI,
S s t I , and XbaI did not indicate any sites for these enzymes in the
30#
CLARKE
Table I.
Fragments produced by restriction endonuclease
digestion of pJB950
Enzyme
Fragment size (kb)
A
HindIII
KpnI
SalI
PstI
SmaI
XhoI
XorII
SstII
HindlI
AvaI
9.14
9.12
8.99
7,94
7.55
7.08
3.80
4.66
4.40
3.09
ET AL.
B
C
1.20
1.50
1.48
2.62
1.78
3.26
1.60
.603
1.41
.897
.706
1.51
D
E
.74
.703
.347
1.44
F
.59
.583
.243
.614
Sum of
Sites
fragment
in
sizes
vector
(kb)
G
.162
.133
.468
.228
9.14
9.12
8.99
9.14
9.05
9.16
9.16
8.79
9.07
8.95
1
0
1
1
0
0
1
0
2
1
located
from
4
Sites in vector
Sutcliffe (1979).
(Sutcliffe,
1978)
or visually
DNA insert. The sites for the rest of the restriction enzymes (Table
1) were mapped by a series of double digests using either Kind[[] or
S'alI compared w i t h digests w i t h the enzymes used singly. This
procedure with KpnI, PstI, and Sinai was f o l l o w e d by double digests
employing this group of enzymes in pairs. Fig. 2b,c shows the map of
restriction sites obtained by these methods.
The r e p e t i t i v e double
digest s were continued until the relative positions could be assigned
with confidence.
Matching amino acid sequences to restriction-mapping data
A catalogue of all potential targets w i t h i n the f i r s t 13# a m i n o
acids f r o m the N-terminus was calculated for the enzymes shown in
Fig. 2c using the 'translation' method.
Examples of the numbers of
p o t e n t i a l sites are: none for XorH, 3 for Sma], 6 for PstI, 10 for
HaeHI. The mapping data indicated that the amiE gene was located
near the Kind[[[ end of the cloned fragment. Therefore the tests for
best fits were made around the Sma[ site at 200 bp and the HaeII site
at 56t bp allowing for all positions of the N-terminus from 0 to 560
bp from the KindII] site. For each of the 3 potential Sinai sites and
the 2 HaeH sites the restriction mapping data were used to calculate
the position in the amino acid sequence where the e x p e r i m e n t a l l y
d e t e r m i n e d sites for the o t h e r restriction enzymes might be found
(Table 2). Thus) if the Glu-Arg-His sequence at residues 105 to 107
is assumed to correspond to the HaM! site at map position 561, the
Pstl site at position #30 should correspond to an amino acid sequence
about #2 amino acids distant.
The nearest f i t for the Pst] 'translation' was found for the Leu-Gln sequence at residues 62-63.
Table
27 Set B, shows t h a t the alignment with the best f i t was obtained
from calculations based on placing the HaeII site at residues 105-t07
ALIGNMENT OF CLONED amiE GENE
305
Table 2. Possible alignments of the restriction map
with the sequence of 132 amino acids from the N-terminus
of the amidase protein
a.a. = amino acid; M = amino acid residue number calculated from
m a p d i s t a n c e ; P = n e a r e s t potential site derived from matching
amino acid sequence; D = M - P. Dashes indicate sites outside the
matched sequence.
Numbers under P are those of the central amino
acids of tripeptides, or N + 0.5 for d i p e p t i d e s N to N + I.
Sequence predicts no Xorll site in the first 396 base-pairs.
Set A: S m a I
at a.a.
Site
M
P
2
3
Sau3A
XorII
Set B: HaeII
at a.a. 106
50
D
M
P
Set C: Haell
at a.a. 144
D
M
37.5
-35.5
No site ~3
.
.
.
.
.
.
.
.
.
.
P
D
//pall
HaeII
17
20
23.5
106
-6.5
-86
.
.
.
.
.
.
.
.
.
.
AvaI
Taqll
28
34
31
35.5
-3
-1.5
.
.
.
.
.
.
.
.
.
.
HindlI
SstI
88
90
102
76
14
14
24
26
18.5
28
5.5
2
32
34
18.5
29
13.5
5
AvaI
PstII
122
127
iii
91
ii
36
57
62
58.5 -1.5
62.5 -0.5
65
70
58.5
70
6.5
0
SstII
-
-
-
79
76
87
76
-3
ii
as described above.
(Two of the potential SmaI sites were r e j e c t e d as
they would place the N-terminus outside the cloned f r a g m e n t . )
T h e r e w e r e not enough restriction enzyme sites to test all other
possible alignments within the 2.5-kb fragment.
However, the fit for
s e t B is so good t h a t it is u n l i k e l y to be due to chance.
This
alignment places the translation s t a r t of t h e a m i E g e n e a t 247 bp
f r o m t h e //indIII s i t e and e x c l u d e s all sites leftward from SmaI to
HindIII (Table 3). Preliminary DNA sequencing starting from the SmaI
site indicates that this is c o r r e c t (W.3. Brammer, unpublished).
Application
to o t h e r
genes
and enzymes
The r e l i a b i l i t y of t h i s m e t h o d of aligning r e s t r i c t i o n maps w i t h
amino acid sequences was assessed w i t h t w o o t h e r s y s t e m s w h e r e
r e s t r i c t i o n maps and DNA sequences had been published. These were
the bacteriophage ;k integrase (Hoess et al., 1980) and t h e E n t e r o bacter cloacae
C D F I 3 cloacin immunity protein (Van den Elzen et
al., 19g0).
The potential restriction enzyme targets w e r e e x a m i n e d
f o r all p o s s i b l e positions and for both orientations of the gene.
In
both cases the position and orientation were predicted c o r r e c t l y by our
method.
306
CLARKE ET AL.
Table 3.
Restriction map of amiE and amino acid sequence*
Map distance (bp)
from
Amino acid
calculated
from
Match
N-terminus** found
Hindlll
from
N-terminus
Estimate
247
0
Hindll
315
68
23
18-19
GTC.AAC
Val.Asn
Sstll
320
73
24
27-29
CCG.CGG
Pro.Arg
Aval
415
168
56
58-59
CCC.GAG
Pro.Glu
Pstl
420
183
61
62-63
CTG.CAG
Leu.Gln
Sstll
480
233
74
75-77
CC.GCG.G
Thr.Ala.Val
Haell
561
314
105
105-107
AG.CGC.C
Glu.Arg.His
Enzyme
Amino acid
sequence***
Met
*Recognition sites from Smal leftward could not be aligned with
the amino acid sequence.
**Amino acid number calculated from map distance from estimated
N-terminus.
Base pairs divided by 3.
***Amino acid sequence from Auffret (1977) and R.P. Ambler and
A.D. Auffret (unpublished data).
Acknowledgements
We thank Anne Smith and 3udy Bundick for excellent technical
assistance. We are grateful to the SRC :[or grants to PHC, W3B, and
RPA for research support.
ADA was in receipt of an SRC Research
Training Grant.
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A L I G N M E N T OF CLONED amiE GENE
307
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