Bioscience Reports, Vol. 8, No. 2, 1988
REVIEW
An Experiment in Enzyme Evolution.
Studies with Pseudomonas aeruginosa
amidase
Patricia H. Clarke* and Robert Drew
Received January 22, 1988
The regulation of amidase synthesis in P. aeruginosa is under positive control. This review describes
the experimental evolution of amidase and its regulator protein for the hydrolysisof novel substrates
and experiments to elucidate the mechanismof the control system.
KEY WORDS: Pseudomonas aeruginosa; enzyme evolution; amidase
INTRODUCTION
Bacteria offer many advantages for comparative studies in molecular evolution.
The optimal and tolerated physicochemical conditions for growth (temperature,
salt concentration and pH) can be determined very simply; nutritional and
end-product tests can provide information about metabolic pathways and
enzymes; physicochemical properties can indicate similarities or differences
between enzymes; the sequences of proteins and nucleic acids can be compared.
Such observations, ranging from the conventional methods of classical bacteriology to the sophisticated methods of modern molecular biology, have been used
for both classification and the construction of evolutionary trees. It is also
possible to observe the evolution of new metabolic activities under laboratory
,conditions.
In the early days of bacteriology, reports of changes in the properties of
strains maintained in laboratory culture cast doubt upon the criteria used for
identification and classification. In some cases the original culture had included
more than one type of organism, or a pure culture had become contaminated but,
when contamination could be ruled out, it became clear that both phenotypic and
genotypic changes could be followed under laboratory conditions. The study of
Department of Biochemistry,UniversityCollege London, Gower Street, London WCIE 6BT, UK.
* Present address: Glebe House, School Hill, Cirencester, Gloucestershire, GL7 2LS (Address for
correspondence).
103
0144-8463/88/0400-0103506.00/0~) 1988 Plenum Publishing Corporation
104
Clarke and Drew
inducible (adaptive) enzymes, synthesized in response to the presence of their
substrates in the growth medium, opened up investigations on the mechanism of
gene expression. At the same time, studies on the genetics of bacteria and their
viruses led to the analysis of the genes themselves. It became recognised that
bacteria, and other microorganisms, could be used as model systems for studies in
experimental evolution (1).
BIOCHEMICAL ACTIVITIES OF PSEUDOMONAS SPECIES
We started work on the amidase of Pseudomonas aeruginosa in 1958, a few
years before the Jacob-Monod operon hypothesis was first published (2). It was
already clear that enzyme induction involved the de novo synthesis of enzyme
protein and that this was a phenotypic and not a genetic response to environmental conditions. In any analysis of the origin of novel metabolic activities it is
essential to take into account changes in the regulation of enzyme synthesis as
well as changes in enzyme properties. There were several reasons for choosing P.
aeruginosa rather than Escherichia coli, which had already become the organism
preferred by most microbial geneticists.
The pseudomonads are aerobic organisms that are able to grow in simple salt
media at the expense of many different organic compounds. Several complex
catabolic pathways had been studied in detail and it had been shown that many of
their enzymes were inducible. One of the questions to ask was whether the
enzymes of these biochemically-versatile organisms were regulated in the same
way as those of E. coli. Some Pseudomonas isolates had been found to utilize
synthetic organic chemicals, such as halogenated aliphatic and aromatic compounds, and it could be asked how such activities had originated. Were the
natural enzymes of pseudomonads able to attack these compounds or were new
enzymes evolving in the natural environment in response to the products of the
chemical industry? Finally, would it be possible to observe the evolution of new
enzyme activities in the laboratory? These questions could not be answered by
biochemistry alone but fortunately Bruce Holloway had recently begun to work
on the genetics of P. aeruginosa (3) and his results were to be an invaluable asset
for our own work.
Very little was known at that time about microbial amidases, but Den
Dooren de Jong had reported that some pseudomonads could grow on acetamide
(4). The major pathway for terminal respiration in pseudomonads is the
tricarboxylic acid cycle so that if acetamide could be hydrolysed there would be
no problem about its further metabolism (5). Further, an amide could be a
potential nitrogen source as well as a carbon source and this would give flexibility
in devising growth media. Monod and his colleagues had used a range of different
~-galactosides (lactose analogues) to elucidate the salient features of the E. coli
/3-galactosidase system and we hoped to exploit a series of aliphatic amides
(analogues of acetamide) in a similar way.
Enzyme Evolution
105
AMIDASE OF PSEUDOMONAS AERUGINOSA PAC1
Michael Kelly found that P. aeruginosa NCTC 8602, later designated strain
PAC1, could grow in a minimal salt medium with acetamide as carbon and
nitrogen source (6). It turned out later that growth on acetamide is a
characteristic of most P. aeruginosa strains and of some strains of other
Pseudomonas species (7). Kelly established that acetamidase activity was induced
in cultures grown in minimal salt medium containing acetamide. The amidase
activity of whole cells, or cell extracts, was assayed by the rate of ammonia
production by hydrolysis of amide substrates. He examined a range of aliphatic
amides, some of which are shown in Table 1. Kelly found that the optimal
substrate for hydrolysis was not acetamide, but propionamide and that the
enzyme acted on a very narrow range of aliphatic amides. There was only trace
:hydrolytic activity with butyramide as substrate and although formamide was
:hydrolysed at a significant rate, it could not be used as a carbon source, since P.
aeruginosa cannot assimilate C1 compounds. The substituted amides, glycollamide and acrylamide, were good substrates for the enzyme. GlycoUamide could
be used as a carbon source but acrylamide progressively inhibited the enzyme and
was also a powerful growth inhibitor (6).
The amidase inducer specificity is not the same as the enzyme specificity.
Acetamide is a more effective inducer than propionamide, while formamide is a
poor inducer. An important finding at that time was that many N-substituted
amides were non-substrate inducers and could be used to study the kinetics of
amidase synthesis under gratuitous conditions in the way in which the thiogalactosides had been used to study the synthesis of fl-galactosidase (2). However,
Table 1.
Amides as substrates, inducers and analogue repressors of the
amidase of Pseudornonas aeruginosa PAC1
Amide
Formamide
Acetamide
Propionamide
Butyramide
Lactamide
N-acetylacetamide
Glycollamide*
Phenylacetamide
Cyanoacetamide*
Thioacetamide
Acetanilide
Acrylamide*
Fluoroacetamide*
Iodoacetamide*
Urea*
Chloroacetone
Substrate
Inhibitor
Inducer
Analogue
repressor
+
+ +
+ +
Tr
+
Tr
+ +
0
0
0
0
+
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
+
+
+
+
+ +
+ +
0
+ +
+ +
+ +
0
0
0
0
+
+
nd
0
nd
+
0
0
+
0
0
0
+
+
+
nd
nd
nd
nd
nd
nd
The relative activities are shown as + + , + ; Tr, trace; 0, no activity
detected; rid, not determined. Amides marked * inhibited growth on
certain media as described in the text (6, 8, 11, 12, 31, 32, 36).
106
Clarke and Drew
although thioacetamide was neither a substrate nor an inducer, it was not without
effect on the system and Kelly showed that thioacetamide and cyanoacetamide
prevented amidase induction by inducing amides (amide analogue repression).
Eventually, over 100 acetamide analogues were tested for activity as substrates,
enzyme inhibitors, inducers, or amide analogue repressors.
The observation that arnidase possessed transferase activity with hydroxylamine as acceptor aided the investigations. The amount of acylhydroxamate
produced can be measured by a colour reaction with ferric chloride and this
became the standard method for routine enzyme assays. Acetamide is a better
substrate than propionamide in the transferase reaction. Bill Brammar examined
the rate of amidase induction in exponentially-growing cultures and showed that
cyanoacetamide and thioacetamide competed with both substrate and nonsubstrate inducers (Fig. 1). This suggested that, while all these amides were able
to bind to the amide-binding site of a regulator protein, only the inducing amides
produced the correct response for induction to occur. He also showed, with
cultures growing exponentially in pyruvate medium, that amidase synthesis was
severely repressed by succinate and other intermediates of the TCA cycle
(catabolite repression). Nevertheless, if cultures were grown overnight in
succinate, in the presence of an amide inducer, a significant level of amidase was
produced although less than with pyruvate or lactate as the carbon source. It
appeared that, as observed with other Pseudomonas enzymes, compounds that
supported high growth rates caused the most severe catabolite repression of
amidase synthesis (8).
1.5-
E 1.0
/
C
~ 0.5
E
0.10
0.15
0.20
0-25
Bacterial concentration
(equiv. rag. dry wt/ml.)
Fig. 1. The effect of cyanoacetamide and thioacetamide on induction of the amidase of Pseudornonas aeruginosa PAC1. N-acetylacetamide was added at the time indicated by the first arrow. At the
time indicated by the second arrow the culture was divided into three parts and the following
additions made: O, no additions; A, cyanoacetamide; El, thioacetamide (8),
Enzyme Evolution
107
ISOLATION OF MUTANTS
The next requirement was to obtain mutants and, as with all such
investigations, the first mutant was the most difficult to isolate. Methods used for
isolating constitutive mutants of E. coli were unsuccessful and Brammar decided
to exploit the substrate and inducer specificities. He observed that PAC1 grew
very slowly on plates containing succinate as carbon source and formamide as
nitrogen source. He concluded that the rate of growth was limited by the rate of
hydrolysis of formamide which depended, in turn, on the rate of amidase
induction by formamide. He argued that a constitutive mutant would not be
limited in this way and demonstrated that constitutive mutants could be isolated
from succinate/formamide (S/F) plates. Succinate was a good choice as carbon
:source since it was also acting as a catabolite repressor. There were several
,different classes of constitutive mutants; some were magno-constitutive, producing high levels of amidase; some were semi-constitutive and could be induced to
higher levels by amide inducers. Of these two classes some retained high
sensitivity to repression by amide analogues, such as cyanoacetamide, while
others were resistant. A few mutants grew well on S/F plates but were inducible
and these proved to be altered in inducer specificity. One, in particular, had a
rate of induction by formamide that was greater than that by acetamide. This
provided a wide variety of regulatory mutants and large numbers could be
selected with ease (9). Examples are shown in Table 2 and Fig. 2.
P. aeruginosa
S/L
CB6
/
Cll
B6
I
PhB3
F6
C1
LI1
LIO
__/____ __/____~ P/FI_.
RF17
TSl
432
I
433
I X Am7
IBIO
_ J_ P/F I_
FI B29
Fig. 2. Family tree of amidase mutants with altered regulatory properties. Details of the selective
media, B, S/F, S/L and P/FI and of the phenotypes of mutant strains are given in Table 2. Am7 and
LAml were obtained by negative selection. 433 is a constitutive strain producing a high level of
amidase and was obtained as a revertant of the acetamide-negative strain 432.
108
Clarke and Drew
Table 2. Amide media used for mutant selection
Carbon
source
S/F
S/B
S/L
P/F1
B
Succinate
Succinate
Succinate
Nitrogen
source
Formamide
Butyramide
Lactamide
Pyruvate
(fluoroacetamide)
Ammonium
Butyramide
Ammonium
V
Valeramide
Ammonium
S/Ph
Succinate
Phenylacetarnide
Mutants isolated
Constitutive
Butyramide-sensitive
Butyramide-resistant
Semiconstitutive
Formamide-inducible
Constitutive
Butyramide-resistant
Catabolite-resistant
Altered enzyme
Butyramide-sensitive
Catabolite-sensitive
Catabolite-repression
resistant
Constitutive
Inducible
Acetamide-negative
Enzyme defect
Activator defect
Constitutive
Butyramide-resistant
Altered enzyme
Butyramide-inducible
Constitutive
Altered enzyme
Constitutive
Altered enzyme
C11
C1
C5
F6
SB1
SB63
L10
L11
FIB1
TS1, RF17
FIB29, 432
CB6
B6
BB1
V9
PhB3, PhF1
Examples of amide media used for the selection of mutants with altered amidases or with
regulatory mutations. See text for details and Figs. 2 and 3 for lines of descent of mutant
strains (9, 10, 11, 12, 17, 18, 21, 24, 25, 28, 29, 31, 32).
The first amidase-negative mutants were isolated by the traditional methods
of replica plating or selection of minute colonies f r o m acetamide plates containing
trace amounts of succinate. These rather tedious methods also gave mutants with
defects in enzymes of the T C A and glyoxylate cycles which provided an
interesting diversion (i0). Later, when we knew m o r e about amidase and the
regulation of its synthesis, we were able to devise a m o r e efficient method. The
hydrolysis of fluoroacetamide produces fluoroacetate, which is a potential
inhibitor of the T C A cycle. The constitutive mutants are m o r e sensitive than the
wild type and the extent of growth inhibition depends on the level of amidase
activity of the inoculum and the carbon source for growth. The selection medium
finally adopted contained pyruvate or lactate as carbon sources, compounds
which give minimal catabolite repression. The inoculum from an inducible or
constitutive parent was grown overnight in a m e d i u m that would give a high
amidase activity (11). Most of the colonies appearing on these plates were
Enzyme Evolution
109
acetamide-negative and included both structural gene and regulatory mutants.
Recently, Paul Brown found that glycollate inhibited growth on lactate and
devised a method for isolating amidase-negative mutants from lactae/
glycollamide medium (12).
The first genetic analysis was carried out with the transducing bacteriophage
Fl16, originally isolated by Holloway for strain PAO. The amidase regulatory
and structural genes were co-transduced at high frequencies (9), but there was no
transduction with any other genetic markers available at that time (1967). The sex
factor FP2 did not promote conjugal transfer with PAC strains. We considered
transferring our attentions to PAO but there were technical advantages in staying
with PAC. The amidases of the two strains are similar in physico-chemical
properties and substrate and inducer specificities (7), but PAC strains are more
sensitive to catabolite repression and this made it easier to select certain classes of
mutants from plate cultures. However, amidase-negative mutants of PAO could
be selected by the fluoroacetamide method and, from the frequency of conjugal
transfer with FP2, It appeared (1975) that the amidase genes were in the late
region of the PAO chromosome (13). There are now about 200 genes mapped
and interruped mating and linkage analysis indicated that the ami genes were
located in the 40' to 50' region (14). O'Hoy and Krishnapillai (15) have now
recalibrated the map in time units and this will make it possible to give more
precise gene locations. The chromosome maps of PAC and PAO appear similar
in the regions that have been mapped so far. The drug-resistance factors R68.44
and R68.45 transfer short regions of the chromosome of PAC and were used to
,examine linkage o f ami genes to other chromosomal markers. Complementation
of PAO mutants with R'plasmids carrying regions of the PAC chromosome
indicated homology over the regions examined (16). R'ami plasmids, carrying
'wild type or mutant ami genes, were selected in a P. putida strain which lacks
amidase. In this case the plasmids transferred chromosomal catabolic genes from
one species to another. Plasmid transfer is known to occur in nature and such
events can contribute to the evolution of new metabolic activities in microbial
populations. Horizontal gene transfer, together with structural and regulatory
mutational events, can lead to novel catabolic pathways in which the individual
components may have been acquired from different parental strains.
GROWTH ON A NOVEL SUBSTRATE: BUTYRAMIDE
Neither the wild-type strain PAC1 nor the constitutive mutant C l l grew on
butyramide as carbon source. After treatment with the chemical mutagen,
N-methyl-N'-nitro-N-nitrosoguanidine (NMG), C l l gave rise to. mutant colonies
on butyramide plates. Of these, mutant B6 was shown to produce an amidase
with significant hydrolytic and transferase activity for butyramide as substrate.
We reported in 1969 that it differed from the wild type enzyme in both Km and
Vmax (17). This was the first step in the evolution of a family of novel amidases.
In choosing C l l we had acted more wisely than we knew at the time. There
110
Clarke and Drew
were two reasons why C l l could not grow on butyramide. First, the wild-type (A
amidase) has a very low affinity for butyramide (Kin about 500 mM) and second,
amidase synthesis by C l l was severely repressed by butyramide. Jane Brown
showed that strain C l l could also give rise to a class of mutants that grew on
butyramide plates but produced A amidase, and not the mutant B amidase (18).
These mutants were constitutive and resistant to butyramide repression. Thus,
there were two possible responses to the challenge to strain C l l to use
butyramide for growth; a single mutation in the amidase structural gene could
result in an altered enzyme; a second mutation in the regulator gene could abolish
the sensitivity to butyramide repression. The first class of mutants produced a
more efficient enzyme and, although butyramide still repressed amidase synthesis,
it was hydrolysed rapidly enough for growth to occur. The second class of
mutants produced high levels of the original poor enzyme. The effective in vivo
amidase activity, conferring the butyramide-positive phenotype, could therefore
relate either to the production of a better enzyme or to the production of large
amounts of a poor enzyme. At a symposium on microbial growth in 1969 we
defined the threshold level of amidase activity required for growth on butyramide
as follows:
E•
E is the amount of amidase synthesized in unit time, B is the rate of
hydrolysis of butyramide in unit time and P is the critical activity below which
growth cannot occur (19). The threshold for growth can be reached either by
changes in E or in B, or in both parameters simultaneously. In 1976 Hall and
Clarke (20), working with evolved /3-galactosidase (Ebg) mutants, showed that
the rate of growth on lactose was dependent on both the rate of enzyme synthesis
and the specific activity and that there was a threshold activity for growth. The
acquisition of new metabolic activities frequently involves regulatory mutations
(1).
Some constitutive mutants isolated from S/F plates were already resistant to
butyramide repression, indicating that this phenotype could result from a single
regulatory mutation (C1, Fig. 2). Constitutive butyramide-resistant mutants
(CB6, Fig. 2) could also be isolated directly from the wild type on butyramide
plates, although they took longer to appear than those on S/F plates. A further
class of regulatory mutants came from selection on plates containing succinate
and lactamide (S/L). Succinate exerts strong catabolite repression and lactamide
is an inducing amide, but not a good enzyme substrate. PAC1 grows on lactamide
plates, since lactamide induces amidase activity well over the threshold level. On
S/L plates growth is poor but catabolite-repression resistant mutants were readily
isolated. Some were inducible and others were constitutive. Mutant L10
(PAC142) is a very derepressed strain. It is constitutive, resistant to butyramide
repression and also resistant to catabolite repression. Strain L10 grows well on
butyramide and since it produces high levels of amidase in most media it was
invaluable for obtaining the wild-type amidase in quantity (19, 21) In spite of
being highly derepressed it is a very stable strain.
Enzyme Evolution
111
POSITIVE CONTROL OF A M I D A S E SYNTHESIS
The Jacob-Monod model of negative control of gene expression became so
widely accepted that the positive control of the L-arabinose and other operons
took several years to receive recognition (22). We had assumed initially that
amidase would also be under negative control and Farrokh Farin attempted to
:isolate mutants with thermolabile repressors that would be inducible at low
temperatures, but constitutive at higher temperatures, similar to those isolated
:for the /3-galactosidase of E. coli (23). No temperature-sensitive constitutive
:mutants were found for amidase, although over a thousand constitutive mutants
'were screened. Farin argued that if amidase were under positive control then
mutants producing a thermolabile regulator protein would be negative at high
temperatures and positive at lower temperature. The fluoroacetamide method
]had allowed large numbers of acetamide-negative mutants to be isolated and
when these were screened, mutants derived from C l l and F6 (TS1 and RF17,
]Fig. 2) were found to be acetamide-negative at 37~ but positive at 30~ (24).
The wild-type amidase is very thermostable and is unaffected by heating to 60~
for 15 min. The enzyme produced by the two temperature-sensitive mutants was
no less stable and no differences were detected in substrate specificities or
electrophoretic properties. The temperature-sensitivity could be ascribed to a
mutation in a regulatory protein. Only trace amounts of amidase were produced
at 43~ and when the cultures were transferred to 28~ there was a short lag
before amidase was synthesized at a normal rate. A shift-up from 28~ to 43~
resulted in cessation of amidase synthesis. The parental strain C l l is sensitive to
both butyramide repression and catabolite repression. Figure 3 shows that, with
6-0
B
RTS 1
/
E
Shift up ///Control
4.c
C
B
o-
[
E
~ 2-0
-//
l
0 -4
28 "C
/
~Succinate
I
0.6
I
0.8
Bacteria (mg dry wt./ml)
1.0
Fig. 3. Effect of a temperature shift from 28~ to 43~ on the rate of amidase synthesis by strain
TS1. At the time indicated by the arrow the culture growing at 28~ was split into four parts. One part
was retained as the control; 0 , 28~ II, 43~ lk, succinate added; O, butyramide added (24).
112
Clarke and Drew
TS1, the cut-off resulting from the temperature shift followed similar kinetics to
those for repression by butyramide and succinate. Further support for the positive
control model came from the analysis of revertants of amidase-negative mutants
derived from strain IB10 which carries the B6 mutation but is inducible. Two of
the acetamide-negative mutants gave rise to revertants all of which produced B
amidase but which exhibited a variety of regulatory phenotypes. We concluded
that these two acetamide-negative mutants had regulatory defects and that the
amidase regulator gene coded for an activator protein (24). Table 2 and Fig. 2
show the derivation of some of the regulatory mutants which have been
discussed.
AMIDASES WITH NOVEL SUBSTRATE SPECIFICITIES
Meanwhile the search for novel enzymes had continued. The B6 enzyme (B
amidase) was thermostable and retained high activity for acetamide and propionamide as well as its novel butyramidase activity. The B amidase had a 10-fold
higher Vm~x and a 10-fold lower K~ for butyramide than the A amidase and could
be considered to be 100-fold more effective (17). Other mutants isolated on the
same plates produced enzymes with similar properties and may have been sibling
clones. The wild-type strain could not be induced by butyramide so that a
regulatory mutation was essential for the B amidase to be produced. It was
theoretically possible for such mutants to be obtained directly from the wild type
by simultaneous double mutation and this proved to be the case. Smyth (21, 25)
used succinate/butyramide plates to select various classes of regulatory mutants
and among the colonies appearing on this medium were some that were
magno-constitutive, still sensitive to butyramide repression, but able to grow
since they produced a B6-type enzyme. This double mutation resembles the
selection of the evolved /3-galactosidase (Ebg) mutants of E. coli on lactose
medium, which have both regulatory and structural gene mutations (26). We have
only identified one type of evolved B amidase but the methods used for screening
might well have missed novel enzymes that had only slightly enhanced butyramidase activity.
Mutant B6 did not grow on valeramide as carbon source but did give rise to a
series of valeramide-utilizing mutants that produced V-type amidases. These were
a heterogenous group and some of the enzymes were so altered that the mutants
were unable to grow on acetamide. Most of the enzymes were unstable so that it
appeared that this particular set of mutations, allowing valeramide to be
hydrolysed at a sufficient rate to reach the threshold for growth, had a deleterious
effect on enzyme conformation (17). One advantage was that the acetamidenegative mutants could be used in genetic crosses with other classes of
amidase-negative mutants (27).
The next attempt to evolve a new substrate specificity was to select for
growth with phenylacetamide as nitrogen source. P. aeruginosa does not grow on
phenylacetate as a carbon source, although many other pseudomonads are able to
do so. Joan Betz selected the PhB group of mutants directly from strain B6. Some
Enzyme Evolution
113
were spontaneous mutants and others were obtained after mutagenic treatment.
Mutant PhB3 was selected for detailed study but all those examined appeared to
produce the same mutant enzyme. Two phenylacetamide-positive mutants were
obtained from the valeramide-utilizing mutant V9. One of these, PhV1, was
unable to grow on acetamide and its enzyme was much more thermostable than
that of the other strain, PhV2, indicating that the mutations of the two strains
were not identical. The family of mutant V and Ph enzymes were all related to
strain B6 and genetic recombination with other acetamide-negative mutants,
recovered the B6 genotype indicating that the original B6 mutation had been
retained (27, 28).
A few recombinants had the B6 mutation but could not grow on butyramide
because they had the wild-type specificity of induction. IB10 was obtained by
crossing PhB3 with the amidase-negative mutant Am7 (Fig. 2). Most of the
mutants selected from IB10 for growth on butyramide were constitutive but a few
had altered inducer specificity. Strain BB1 produces B amidase and is inducible
by its novel substrate, butyramide (29).
Attempts were also made to select phenylacetamidases by other routes.
Strain CB4, a constitutive butyramide-resistant mutant, gave rise to PhF1 and this
was assumed to be a single site mutation. The catabolite repression-resistant
mutant, L10, had been used previously to select mutants that were unable to
grow on lactamide or butyramide, but produced slight growth on acetamide.
Mutant LAml was one of these leaky mutants which grew very feebly on
acetamide and produced cross-reacting material. LAml gave rise to the
phenylacetamide-utilizing strain, PhA1. The amidases of both PhF1 and PhA1
'were very thermolabile.
Figure 4 shows the family tree of the phenylacetamide mutants. It is clear
that an acetamidase can give rise to a phenylacetamidase by more than one line of
Wild- type Pseudomonas aeruginosa
strain PAC 1
<
Ph A1
PhV1
Ph V2
Fig. 4. Family tree of mutants of Pseudomonas aeruginosa producing amidases with altered substrate
specificities. C l l , CB4 and L10 are regulatory mutants. LAml is an amidase-defective mutant. Other
mutants produce amidases with novel substrate activities which enable growth on butyramide
(B6), valeramide (V9) and phenylacetamide (PbB3, PhV1, PhV2, PhF1, PhA1). Details of selection
media and mutant phenotypes are given in Table 2.
114
Clarke and Drew
A amidase
100
Ph B3
75
c
so
._~
PhV2
,~E 25
Ph F1
Ph A1
0
2
4
6
8
10
12
14
T i m e (rain.)
Fig. 5. Effectof heating at 60~ on the activities of the wild-typePseudornonas aeruginosa amidase
and the mutant phenylacetamidases(28).
descent and that a variety of mutations can allow phenylacetamide to be
accommodated at the substrate-binding site of the enzyme. There were marked
differences in the kinetic constants and the thermal stabilities of the phenylacetamidases (Fig. 5) (28). Paterson confirmed that the amiE16 mutation had been
retained during the subsequent evolution of the B6 sub-family and reported
(1979) that the B6, V9, PhB3 and PhV1 amidases had a phenylalanine residue
substituted for a serine at position 7 from the N-terminus (30).
Paul Brown had isolated another series of mutants that grew on Nphenylacetamide (acetanilide) as carbon source. Mutant A13 hydrolyses acetanilide to acetate and aniline and Brown showed that the change in amidase
substrate specificity was due to the substitution of isoleucine for threonine at
position 103 (31). More recently he has isolated a further series of altered enzyme
mutants from strain A13 (32).
Enzyme Evolution
115
1o ATGCGT CACGGCGAT ArT TCC AGCAGCAACGACACCGTCGGAGTG
46.
91.
136.
181.
226.
271.
316.
361.
406.
451.
496.
541.
586.
631.
676.
721.
766.
811.
856.
901.
946.
991.
1036.
Met Arg His Gly Asp Ile Ser Set Set Asn Asp Thr Val Gly Val
GCCGTGGTC AAC TAC AAGATGCCGCGCCTGCAC ACCGCGGCGGAG
Ala Val Val Asn Tyr Lys Met Pro Arg Leu His Thr Ala Ala Glu
GTCCTG GAC AAC GCC CGGAAG ATC GCCGAC ATG ATC GTCGGC ATG
Val Leu Asp Asn Ala Arg Lys Ile Ala Asp Met Ile Val Gly Met
AAGCAGGGCCTG CCC GGCATG GAC CTGGTGGTG TTC CCGGAG TAC
Lys Gln Gly Leu Pro Gly Met Asp Leu Val Val Phe Pro Glu Tyr
AGCCTG CAGGGC ATC ATG TAC GATCCGGCGGAGATGATGGAAACC
Set Leu Gin Gly Ile Met Tyr Asp Pro Ala Glu Met Met G|u Thr
GCGGTGGCG ATC CCCGGCGAGGAAACCGAGATA TTC TCC CGCGCC
Ala Val Ala Ile Pro Gly Glu Glu Thr Gin Ile Phe Set Arg Ala
TGCCGCAAGGCC AAC GTCTGGGGCGTATTC TCC CTC ACCGGCGAA
Cys Arg Lys Ala Asn Val Trp Gly Val Phe Ser Leu Thr Gly Glu
CGGCACGAGGAG CAT CCGCGCAAGGCGCCG TAC AACACCCTGGTG
Arg His Glu Glu His Pro Arg Lys Ala Pro Tyr Asn Thr Leu Val
CTG ATC GAC AAC AAC GGCGAG ATC GTC CAGAAG TAC CGCAAG ATC
Leu Ile Asp Asn Asn Gly Glu Ile Val Gin Lys Tyr Arg Lys Ile
ATT CCC TGG TGC CCC ATC GAGGGCTGG TAT CCC GGTGGCCAG ACC
Ile Pro Trp Cys Pro Ile Glu Gly Trp Tyr Pro Gly Gly Gin Thr
TAC GTCAGCGAAGGGCCGAAGGGCATGAAGATC AGCCTG ATC ATC
Tyr Val Ser Glu Gly Pro Lys Gly Met Lys Ile Set Leu Ile Ile
TGCGACGACGGCAAT TAC CCGGAG ATC TGGCGC GACTGCGCG ATG
Cys Asp Asp Gly Asn Tyr Pro Glu Ile Trp Arg Asp Cys Ala Met
AAG GGC GCCGAGCTG ATCGTGCGCTGCCAGGGCTAC ATG TAC CCG
Lys Gly Ala Glu Leu Ile Val Arg Cys Gln Gly Tyr Met Tyr Pro
GCC AAG GAC CAGCAGGTGATG ATG GCC AAG GCC ATG GCCTGGGCC
AIa Lys Asp Gin Gln Va! Met Met Ala Lys Ala Met A]a Trp Ala
AAC AAC TGC TATGTGGCGGTGGCCAACGCGGCCGGCTTCGACGGT
ASh Asn Cys Tyr Val Ala Val AIa Ash Ala Ala Gly Phe Asp Gly
GTC TAT TCC TAC TTCGGCCACTCGGCGATC ATCGGCTTCGACGGC
Val Tyr Ser Tyr Phe Gly His Set Ala Ile Ile Gly Phe Asp Gly
CGT ACC CTCGGTGAGTGCGGCGAGGAGGAAATGGGTkTCCAGTAC
Arg Thr Leu Gly Glu Cys Gly Glu Glu Glu Met Gly Ile Gin Tyr
GCC CAG CTG TCC CTT TCGCAG ATC CGC GATGCG CGC GCC AAC GAT
Ala Gin Leu Set Leu Set Gin Ile Arg Asp Ala Arg A]a Ash Asp
CAG TCG CAGAAC CAC CTG TTC AAG ATC CTC CAC CGCGGC TAC AGC
Gin Set Gin Ash His Leu Phe Lys Ile Leu His Arg Gly Tyr Set
GGC TTG CAGGCGTCCGGCGACGGCGACCGGGGCCTGGCGGAGTGT
Gly Leu Gin Ala Ser Gly Asp Gly Asp Arg Gly Leu Ala Glu Cys
CCG TTC GAGTTC TAC CGC ACCTGGGTC ACC GACGCCGAGAAGGCG
Pro Phe Glu Phe Tyr Arg Thr Trp Val Thr Asp Ala Glu Lys Ala
CGC GAC AATGTCGAGCGACTGACCCGCTCG ACC ACCGGCGTGGCG
Arg Asp Asn Val Glu Arg Leu Thr Arg Ser Thr Thr Gly Va[ Ala
CAA TGC CCGGTCGGCCGGCTGCCC TATGAGGGACTGGAGAAGGAA
Gln Cys Pro Val Gly krg Leu Pro Tyr Glu Gly Leu G]u Lys Glu
GCCTGA
Ala >>>
Fig. 6. The nucleotide sequence of the coding region of the amiE gene with the amino acid sequence
of the enzyme which was determined independently (33, 39).
116
Clarke and Drew
THE AMINO ACID SEQUENCE
The amino acid sequence of the wild type amidase was determined in
Edinburgh by Richard Ambler and Tony Auffret (33). Their analysis was ~
virturally complete by 1978 and the final ambiguities were resolved when t h e
D N A sequence became available (Fig. 6). The primary structure of amidase is a
single polypeptide chain of 346 residues, giving a molecular weight of 38,400. The
enzyme exists in its native form as a hexamer. Earlier estimates, based on
sedimentation equilibrium analysis, had given a value of about 200,000 for the
native enzyme and SDS-PAGE and gel filtration had indicated a value of
33,000-35,000 for the monomer. Treatment with dimethyl suberimidate gave 6
bands on SDS-PAGE gels, indicating a hexameric structure (34) and this was
confirmed by kinetic studies.
It was found that 6 thiol groups per molecule of the native enzyme reacted
very rapidly with thiol reagents. However, the rapidly-reacting thiol of each
subunit is not concerned with the active site but probably with maintaining the
correct enzyme conformation (35). The amidase is active only in the hexameric
form and the instability of some of the mutant enzymes is probably related to the
dissociation into subunits which can be seen in immunological cross-reactions
(28). The enzymic reaction is thought to proceed via an acyl intermediate and the
observation that the A13 amidase acts on para-substituted N-phenylacetamides is
an indication of the orientation of substrate amides at the catalytic site.
Chloroacetone is an active-site-directed inhibitor but the identity of the reacting
group of the enzyme has not yet been established (36).
Some strains of P. putida biotype A produce an amidase with very similar
properties to those of P. aeruginosa (7). Ambler and Auffret found that the
amidase of P. putida strain A87 differed from the P. aeruginosa PAC amidase in
respect of about 30 amino acids, most of these being conservative changes (33).
The amidases of P. acidovorans and P. cepacia appear to be less closely related to
that of P. aeruginosa and further comparisons of D N A sequences would be of
interest in respect of the evolutionary relationships of these species (7).
CLONING AND DNA SEQUENCE
Strain 433, selected for high constitutive expression of amidase (see Fig. 2),
provided the amidase genes which were cloned in bacteriophage lambda.
Although expression of Pseudomonas genes in E. coli is poor, a strain carrying
the recombinant )~-ami phage was able to utilize acetamide as a nitrogen source
(37). A Hindl11/Sall D N A fragment of 5.3 kbp was subcloned into pBR322 to
give the recombinant plasmid pJB950 (Fig. 7). Comparison of the N-terminal
amino acid sequence of the amidase with a detailed map of restriction sites
located the N-terminus of the amiE gene starting 262 bp from the Hind111 target
(38). The complete D N A sequence of the amiE gene was determined in Leicester
by Brammar and colleagues and is shown in Fig. 6 (39). The pseudomonads are
characterized by having a high GC ratio. For amidase, the codon usage is heavily
]Enzyme Evolution
117
biassed in favour of G or C in the third position; of 346 codons only 33 have A or
T in the third position. This highly biased codon usage is also found in other
Pseudomonas genes that are expressed at high rates.
About 150 bp upstream of the amiE coding sequence there is a sequence
closely resembling that of a strong E. coli promoter. No differences were detected
between the corresponding promoter sequences from strain 433 and the wild type
PAC1. This indicates that 433 is not a promoter mutant as had been thought
earlier (25). Immediately upstream of amiE is a typical ribosome-binding site and
further upstream a possible leader sequence with a transcription terminator (42).
These features suggest that the amiR protein acts as a transcription antiterminator. This unusual mechanism for the regulation of an inducible enzyme is
being investigated in more detail. It appears that the amount of the amiR gene
product is also controlled (see below) and that this complex regulatory system
may account for the very large variations observed in the rates of amidase
synthesis under various growth conditions.
THE A M I D A S E R E G U L A T O R Y GENE
Since amidase expression was low, and the amiE gene was located very close
to one end of the cloned fragment, it was thought initially that amiR might be
missing from these constructs. However, deletion of D N A sequences downstream
from amiE was found to reduce amidase expression to a very low level in E. coli
and further experiments showed that this had the same effect in P. aeruginosa.
This indicated that amiR was probably located some distance downstream from
amiE (Fig. 7). For the P. aeruginosa experiments, plasmid pCL28, with the
presumed amiR sequence deleted, was joined to a broad host-range plasmid of
the IncQ group to give pCL34. Plasmid pCL34 was mobilized into PAC mutant
strains and tested under inducing and non-inducing growth conditions. The amiE
mutations of the PAC mutants were complemented by plasmid pCL34 (presumed
to the amiE § amiR-) and the regulatory phenotypes were those of the amiR gene
of the host chromosome. These data confirmed the positive control model (40). A
more detailed study was made by Diane Cousens using a series of deletion
constructs of pJB950.
In E. coli, amidase activity was reduced to a basal level for plasmids carrying
deletions extendings between the Clal target at 3.502 and the X h o l target at
4.452. This located amiR in a 950 bp D N A fragment about 2 kbp downstream
H
emi
E
X
b)
O
1
2
3kb
Fig. 7. Restriction maps of the Pseudomonas aeruginosa DNA insert in plasmids pJB950 and pCL28.
(a) pJB950 showing the direction of transcription of amiE. (b) pCL28 showing the location of the
deletion which removed arniR. Restriction enzyme sites: H, HindlII: X, Xhol; S, Sail (40, 41).
118
Clarke and Drew
I
o
I
,~ml E
I
I
I
II
2
3
I
I
aml R
I
4
I
pDC 3 5
)
(H)
( H)
(Bin)
I
5 kb
(
pDC 53
(
pDC 20
,(
pDC 55
(
. pDC 44
,(
pDC 45
(
,- p D C 4 8
Fig. 8. Restriction map of the Pseudomonas aeruginosa DNA insert in plasmid pJB950 showing the
fragments subcloned into the broad-host-range plasmid pKT231, pDC35 contained the amiR gene in
the correct orientation. Restriction enzyme sites: H, HindlII; Sin, Smal; B, Ball; P, PvulI; X, Xhol;
K, Kpnl; C, Clal; S, Sall (41).
from amiE (Fig. 8). In E. coli, plasmid pDC35, carrying the 1.5 kbp X h o l / X h o l
fragment, complemented a compatible plasmid carrying amiE (pDC5), giving
high constitutive expression of amidase. Plasmid pDC35 also complemented arniR
mutations of P. aeruginosa. Studies with plasmids containing the 1.5 kb fragment,
inserted in opposite orientations, showed that the amiR gene was transcribed in
the same direction as amiE (41). The amiR sequence has now been determined
(R. E. Drew, manuscript in preparation).
One surprising result was that deletion of some of the DNA sequences
between arniE and amiR resulted in higher levels of amidase expression. This was
first observed in E. coli, where expression is normally very low, but was also seen
in a P. aeruginosa mutant in which both arniE and amiR had been deleted (Table
Table 3. Amidase activities in E. coli JA221 and P, aeruginosa strains
carrying recombinant plasmids with sequences deleted between amiE and amiR
Strain
Plasmid
Succinate
Succinate/
lactamide
E. coli
JA221
none
pJB950
pDCll
pDC17
0.0
4.1
17.2
15.2
0.0
4.1
17.2
14.7
none
pGRll
pGR17
none
0.1
375
411
57.2
0.1
225
317
51.1
P. aeruginosa
PAC452
(amiER z~)
PAC433
The plasmid vector for E. coli JA221 was pBR322. The plasmid vector for P.
aeruginosa PACA52 was pKT231. PAC433 was the strain from which the cloned
ami genes had been derived (41). Amidase activities were measured in intact
cells by the transferase method (8) and are given as /~mol aeethydroxamate
formed rain- ~mg bacteria- 2.
Enzyme Evolution
119
3). The possibility that the intervening sequence codes for a repressor would
appear to be ruled out by the absence of any effect when the entire sequence was
cloned and tested in trans in E. coli. Amidase activities were higher when both
arniE and amiR were carried on a multicopy plasmid than when multiple copies of
amiE were present on a plasmid with a single copy of the amiR gene on the
chromosome. This suggests some form of regulation of expression of amiR. Since,
with the deletion derivatives of pJB950, there was an indication that amiR could
be transcribed from more than one promoter it is possible that the deletions
enhancing amidase activity bring amiR nearer to a strong promoter. Other
possibilities are that the deletions remove terminator sequences, binding sites for
other regulator proteins, or otherwise alter the secondary structure of the D N A
(41).
REFLECTIONS
Some of our original questions have been answered, at least in part. Unlike
the fl-galactosidase of E. coli, the amidase of P. aeruginosa is under positive
control as are certain E. coli enzymes and many catabolic enzymes of pseudomonads. This is far from the whole story. For amidase, there is some evidence
that catabolite repression involves cyclic AMP (25), but this needs to be
re-investigated with an in vitro preparation. It is also possible that the expression
of amiR, and therefore amiE, is affected by regulatory genes controlling nitrogen
metabolism. Further studies on amidase regulation are in progress.
In recent years there have been several conferences on the biodegradation of
xenobiotics in the natural environment. The general conclusions are that there
are several ways in which new metabolic activities may arise and more than one
process may be involved. These include mutations resulting in derepression of
pre-existing genes, mutations resulting in altered enzyme activity, rearrangement
of genetic material by plasmid transfer and genetic recombination. These
phenomena have all been observed in the laboratory and it is reasonable to
assume they can occur in the natural environment under the appropriate selection
pressure. The robust nature of many of the amidase mutants obtained by strong
selection pressure suggests that laboratory strains of this type may be useful for
large scale chemical conversions.
The amidase of P. aeruginosa was very amenable to experimental evolution
in the laboratory and novel growth phenotypes were shown to relate to mutations
in structural and regulatory genes. There was no reason to suppose that
experimental evolution of amidase had been exhausted, but some years ago it was
decided to investigate the known strains in depth rather than to search for further
mutants. It was evident that single mutations in the structural gene altered
substrate specificity and we concluded that in most, if not all cases, they affected
substrate binding by altering the protein conformation rather than by changing
active site residues. More information is needed but, unfortunately, the enzymes
have not yet been crystallized although many attempts have been made. Among
other techniques now available for further studies on the structure and functions
of amidase and its activator protein is that of site-directed mutagenesis using
cloned fragments and this is now possible.
120
Clarke and Drew
ACKNOWLEDGEMENTS
The experimental evolution of amidase was actively encouraged by Ph.D.
students, postdoctoral research workers and technicians who were most ingenious
in devising new ways of approaching the problems. It is a pleasure to
acknowledge their work, the support of the SERC and other bodies and, not
least, the invaluable cooperation of P. aeruginosa.
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