Download The spread of plasmids as a function of bacterial adaptability

FEMS Microbiology Ecology 15 (1994) 23-32
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
23
FEMSEC 00557
MiniReview
The spread of plasmids as a function of bacterial
adaptability
H. Tsch[ipe *
Robert Koch Institut, Bereich Wernigerode, Burgstr. 37, D-38885 Wernigerode, FRG
(Received 25 November 1993; revision received 29 March 1994; accepted 18 April 1994)
Abstract: The horizontal spread of plasmids among natural bacterial populations serves as an evolutionary function for adaptation
to the ups and downs in nature. Recent evolutionary challenges are the introduction of antibiotics and the creation of new
environmental conditions in agriculture and medical care. As a consequence, surviving bacterial populations have acquired new
genetic determinants which enable the colonisation and maintenance in distinct ecological niches. The acquisition of new genetic
determinants can take place rather rapidly because of the plasmids' biology: their self-transferability and their ability to pick up
genes. As an example of horizontal gene transfer, from an ecological and evolutionary viewpoint, the emergence of resistance to
streptothricins (sat genes) is described.
Key words: Plasmid; Self-transferability; Streptothricin; sat Gene
Introduction
It becomes more and more obvious how common plasmids are among bacteria. The reason for
the ubiquity of plasmids is discussed from aspects
of the adaptability and phylogenetic flexibility of
bacteria, which is a pronounced feature of most
members of the bacterial kingdom. Since bacteria
have been found to lack sexual reproduction and
meiotic exchange, plasmids are today understood
as an essential evolutionary 'compensation' for
recombination and genetic acquisition of new
properties and entities [1-4]. However, plasmids
* Corresponding author.
SSDI 0 1 6 8 - 6 4 9 6 ( 9 4 ) 0 0 0 4 0 - 9
themselves are symbionts, from a phylogenetical
point of view, and might exhaust, as selfish genetic elements (replicons), cellular host functions.
They will serve only accidentally as carriers or
vehicles for horizontal gene transfer, at least when
it is requested through specific selection pressure.
Therefore, plasmid functions necessary for horizontal gene transfer among the various bacterial
populations and bacterial species are the capability for self-transmission and for picking-up foreign DNA (exchange of genes between the respective host chromosomes and the various plasmid replicons). By these mechanisms plasmids
endow their host bacteria with genetic variability
and flexibility in response to environmental
stresses.
24
Although human, agricultural, and industrial
activities most likely accelerated evolution in bacteria rather than changing its nature, it has become apparent that plasmids certainly increased
in number and evolved rapidly within the last
years, but they preceeded man's effects on the
environment. This is why the prevalence of plasmids and horizontal gene transfer due to their
action, must be regarded as a natural phenomenon for bacterial adaptation and for successful colonization of ecological niches.
The genetic background of horizontal gene transfer in nature
The exchange of relevant DNA sequences between strains and species within an ecological
niche and between various ecological niches has
been operatively designated horizontal gene
transfer. The background of horizontal gene
transfer is tightly connected to the biology of
bacterial plasmids.
Plasmids have to fulfill the following three
tasks for their role in genetically determining
bacterial adaptability.
(1) Ability to acquire chromosomal genes
This occurs either by integrative-excisive recombination (as far as studies with Hfr and F'
formation are concerned) or by 'insertion' of gene
cassettes. The integrative-excisive recombination
takes place by the action of insertion elements
which leads finally to integration of 'somatic
Hfr 13 map position between 7 and 13 min.:
...IS3-1ac-lS2-1S3-RelPIS3-Tre-orN-IS2-1S3-proC-purE-IS3,..
F-Prime13:
lac-lS3-purE-proC-lS3-1S2
(chromosomalsequence)
IS2-1S3-Rep-IS3-Tra-oriV
(plasmidsequence)
Fig. 1. Sequence of chromosomal and plasmid encoded genes
before and after excisionof the F plasmid [4].
DNA' into the plasmid DNA (Fig. 1). These
pieces of 'somatic' or foreign D N A can be recognized by the fact that they are flanked by insertion elements [see 4,5]. Also, transposon structures are understood to be processed by this kind
of additive recombination. In contrast to the action of insertion elements, the integration of gene
cassettes takes place by the action of the 'enzyme'
integrase which recognizes and recombines at
specific small recombination sites (e.g. GTGAGGC), which give rise to integron structures
(Fig. 2). This type of recombination can build up
transposon structures. In summary, there is a
sophisticated system to ensure the recombinational 'pick up' of additional sequences [5]. It is
not suprising therefore that under natural conditions a large number of bacterial functions have
been identified as plasmid-borne, such as drug
resistance, virulence or metabolic properties [4].
(2) Stable maintenance and autonomous replication in the host population
In order to prevent rapid plasmid segregation
in the bacterial host cell, two phylogenetical
strategies have evolved. Plasmids will either replicate to multicopies (between 15 and 50) or they
will kill plasmid-free segregants. The strategy for
preventing the 'out-segregation' of plasmids by
replicating to multicopies is restricted to plasmids
of smaller size (1-20 kb) because of the genetic
load. However, plasmids of larger size (over 30
kb) occur always in low copies which might allow
a relatively easy loss. However, low copy number
plasmids remain rather stable in the cell and in
the population because of their sophisticated
killing systems for any plasmid-free segregants
which occur by chance at cell division. The killing
action is carried out by a kind of cytotoxic protein
(normally inhibited by an antagonistic protein;
alternatively, an RNA for the transfer replication,
see hok:sok system) [7,8]. In Fig. 3 the killing and
antikilling principle, occurring within the vegetative as well as within the transfer replication
systems is briefly summarized.
When a plasmid is already established in a cell
or within a population it will be hard for the cells
to get rid of the plasmid DNA. If new plasmids
encoding functions of selective value are to be
25
introduced into the bacterial population, the new
plasmid should not possess the same replication
machinery if the plasmid is to become established. Thus, only a genetically different replicon
will be in the position to become established with
the resident plasmid. Therefore, selection pressures must exist to favour the horizontal spread
of genetically different replicons. Indeed, a large
scale of various genetically different replicons
occurring in wild-type strains have been detected
and operationally typed by determination of their
size, and transfer and incompatibility properties
[4,9-12] (Table 1). 'Incompatibility' implies a
phenotype of plasmids which is a result of their
sophisticated replication control mechanisms.
These control mechanisms will only recognise related replicons (incompatible) and neglect unrelated replicons (compatible) [10]. Therefore, the
typing of plasmids with respect to both establishment and segregational incompatibility reflects
the various replication machineries [10]. However, from an evolutionary point of view, it is of
interest that distinct replicons (RepV) are corre-
/
/
lated with distinct transfer machineries (transfer
replication, RepT). This RepV/RepT correlation
is the basis of the 'plasmid species theory' [4,12]
and allows the classification of plasmids (see
Table 1).
(3) Horizontal gene transfer beyond borders of
species, genus or family, and populations
Horizontal gene transfer between various bacterial species, genera or families and between
various bacterial populations show that some
plasmids are able to cross taxonomic borders in
various ecological niches [13,14]. Since plasmids
serve only as vehicles for horizontal gene transfer, respective genes need to be integrated into
the plasmid replicons (gee above) and the plasmid
transfer and replication will give rise to amplification as well as to environmental spread of the
respective determinants.
The transfer process of self- transmissible plasmids is replicative. The donor DNA is replicated
at or through a 'transfer pore' that originates
from the sex pilus attached to the recipient cell
Ri
determinants for transposition
~
promotor
assettes
~
P
5r~~~'m~i~lai~~itite
dhfrl (~i) sat(cryptic) (~1) aadA1~~~ GAGGc
Tn7
"
"°"
..,,
Fig. 2. Integronand cassette structureson Tn7-1iketransposons[6].
Tn1826
Tn 1825
26
[see 4]. The replication of donor D N A through
the pore into the recipient cell is termed 'transfer
replication' and it is somewhat similar to vegetative replication. However, the origin of transfer
replication (oriT) and of vegetative replication
(oriV) are distinct. The transfer replication is a
rather complicated and sophisticated molecular
process, which remains to be unraveled. Moreover, the number and size of genes needed for
transfer replication and the pilus architecture
a)
cell ~
wall
Killing
~ hok-mRNA
sok mRNA
t
stable
unstable(Repression)
(5
b)
CcdB
cell ~
wall /
CcdA"
TT->
CcdB
Stopcell division
and chromosomal
partitioning,filament
formation, interaction
with/~,raseA subunit
Fig. 3. T h e killing and anti-killing principles of TncF plasmids
[7,8]. (a) Connected with transfer replication. T h e hok m R N A
is a very stable molecule. It is translated into a 52 amino acid
residue containing protein with cytotoxic effects. The expression of hok m R N A is repressed by the antisense sok m R N A
which is an unstable molecule. As long as the F plasmid is
present, sufficient sok m R N A is provided to prevent killing of
the host. If the F plasmid is eliminated the stable hok m R N A
is left and is then translated into the killing principle. (b)
Connected with vegetative replication. The stable cytotoxic
protein CcdB is inhibited by the antagonistic protein CcdA.
This protein has a short turn-over time. If the F plasmid is
lost from the cell, the antagonistic CcdA protein cannot be
further synthesized and killing of plasmid-free segregants by
CcdB will take place. CcdB interacts with the GyrA subunit
which will lead to D N A cleavage.
Table 1
Incompatibility grouping in relation to size and pilus phage
specificity of plasmids from Gram-negative bacteria (for further information see [4])
Group
R a n g e of size (in kb) Pilus phage specificity
IncA (see IncC)
IncB
95-120
unknown
IncC i
135-170
~cl
IncD
65-80
M13
IncE (see IncFIX)
IncFI
75-110
M13, R17
IncFII
75-110
M13, R17
IncFIII
75-90
M13, R17
IncFIV
80-100
M13, R17
IncFVI
100-150
M13, R17
IncFIX
50-60
M13, R I 7
IncG (cancelled)
IncH1
150-200
4~H1
IncH2
200-240
q~H1
IncH3
220-240
~bH1
IncH4
210-220
&H1
IncI1
90-110
ifm
IncI2
75-90
ifm, ike
IncJ
0 (plasmid D N A always integrated)
IncK
70-80
unknown
IncL (see IncM)
IncM 1
70-90
&M
IncN ~
40-55
ike, PR4
Inc0 (see IncB)
IncOF
65-75
M13
IncP1 1
55-70
PR4, PRR1
IncP2 2
300-400
unknown
lncP3 (see IncC)
IncP4 (see IncQ) '
IncP5 2
100-200
unknown
lncP6 2
50-60
unknown
IncP7 2
100-160
unknown
IncP8 2
120
unknown
IncP9 2
90
unknown
IncPlO 2
70
unknown
IncP11 2
80
unknown
IncP14 1,3
18
no pilus
IncQ 1,3
9-12
no pilus
IncS (see IncH2)
IncT
120 (single type)
~T
IncU
35-55
unknown
IncV
100 (single type)
unknown
lncW1 i
30-40
PR4
IncW2 1
30-40
PR4
IncW3 1
30 single type
PR4
IncX1
45-55
~hxl
IncX2
50-70
&x2
IncY 3
95 (phage P1 family) no pilus
IncZ
110-140
unknown
27
Notes to Table 1:
1 Broad host range plasmids.
2 Plasmids restricted to Pseudomonas.
3 Non-selftransmissible plasmids; the classification of further
non-selftransmissible plasmids has not been performed.
However, replicons belonging to ColE1 are described in
more detail.
All other plasmids are restricted to enterics.
among the various plasmid groups is different
(e.g. tra genes of IncW plasmids comprise about
15 kb, tra genes of IncH1 plasmids about 130 kb).
From a taxonomic point of view it is important
that some plasmids perform conjugative D N A
transfer and vegetative replication within a wide
range of host bacteria. Indeed, some plasmid
groups have a very broad host range like IncQ,
IncP and IncW plasmids, however others such as
IncI 1 are not able to cross the taxonomic border
from Escherichia coli to Proteus or even to Pseudomonas. A border for natural horizontal gene
transfer is the Gram barrier. Transfer between
Gram-positives and Gram-negatives is limited,
possibly due to the different cell walls, which
obviously prevents genetic exchange under natural conditions. However, some rare plasmids can
be transferred under laboratory conditions (e.g.
IncQ; E194). The molecular reason for the
broadly varying host ranges is unknown. It is very
likely that each phylogenetic group of bacteria
selected for and adapted their own plasmid
species.
From an ecological point of view it is interesting to ask where transfer of plasmids between the
various bacterial populations take place [13-15].
From laboratory experiments, it is deduced that
plasmids require optimal temperatures, sufficient
oxygen pressure and nutrients for conjugative
transfer. Concerning 'optimal conjugation' temperature, it is of interest that many plasmids
exhibit transfer only at lower temperatures (below 30°C). For example, the IncM plasmids stop
their transfer at temperatures over 30°C, since
temperature controls induction or stopping of
transfer replication as well as pilus production
[16]. Most of the conditions for conjugative transfer are sometimes fulfilled in the open environ-
ment [13,14], and often even to a greater extent
than in the gut of human or animals.
Resistance d e v e l o p m e n t to streptothricins - a
m o d e l for a s s e s s i n g horizontal gene transfer as a
function of adaptation to e n v i r o n m e n t a l conditions
The introduction of streptothricins in animal
husbandry at the beginning of the 1980's and the
observation of streptothricin resistance development before, during, and after application, proved
to be a good model for assessing horizontal gene
transfer. The streptothricins, belonging to one
distinct family of antibiotics, consist of the
residues fl-lysine, gulosamin and streptolidine
[17]. In spite of their excellent antimicrobial
properties, particularly for Gram-negative bacteria, they remained excluded from therapeutic application and have been used only rarely for plant
protection and nutritive purposes in animal husbandry. Between 1980 and 1990, nourseothricin, a
mixture consisting of streptothricin D and F, was
used in Eastern Germany for nutritive purposes
in the pig breeding industry. The application of
nourseothricin was carried out countrywide, replacing the tetracyclines, and therefore placing a
severe selective stress on bacterial populations.
Ecological and genetic investigations before the
application of nourseothricin did not reveal any
plasmid-borne streptothricin resistance among
animal and human strains. However, 1 to 2 years
later, plasmid-borne resistance was detected
among strains isolated from pigs, and subsequently in isolates from manure, river water, food,
and human beings [18-20]. These plasmids code
for the enzyme streptothricin acetyltransferase
(sat) which can detoxify the antimicrobial agent
by acetylating the fl-lysin residue [18,21]. Up to
now, 6 different determinants have been analysed, two (sat-l, sat-2) on a Tn7-1ike transposon,
one (sat-3) linked to IncQ plasmids, two (nat, sta)
from Streptomyces and one (sat-4) from Campylobacter coli. From the sequence specificities of
the sat determinants, respective gene probes and
oligonucleotide primers for PCR were designed
and, by means of these molecular tools, environ-
28
(1) There is a constant rise in the incidence of
Table 2
First o c c u r r e n c e of sat d e t e r m i n a n t s on various p l a s m i d
species i n d e p e n d e n t of the t i m e p e r i o d of s e l e c t i o n p r e s s u r e
Y e a r of isolation
sat-1
(Yn 1825)
1982 a (begin of
application)
1983
1984
1985
1986
1987-1993
-
sat-2
(Tn 1826)
streptothricin resistance which coincides with
the selection pressure due to streptothricin
application. The development of resistance is
mediated by the spread of plasmids carrying
resistance determinants (sat genes) mostly organised in transposons. It was found that
Tn7-1ike transposons acquired respective gene
cassettes (Fig. 2) inserted into various plasmid
species (Table 2).
(2) The plasmid pool used by the streptothricin
resistance determinants was not the drug resistance plasmids common among enterics
from animals and human beings, since only in
very rare cases sat genes have been found to
be associated with multiple drug resistance.
Therefore, the source of plasmid species (see
Table 2) must be a pool of cryptic plasmids.
However, the search for such cryptic transfer
plasmids among enterics revealed only a limited number of known respective plasmid
species such as IncFI, IncFII, IncIa, IncH1,
but no IncM or IncC plasmids (unpublished
observations).
sat-3
I2
W3
Q
I1, FII, X
N, X
H2
FII, FI
OF, D, M, U, K
H1
a d d i t i o n a l p l a s m i d species carrying
r e s p e c t i v e sat g e n e s have not
been detected
I A t the b e g i n n i n g of s t r e p t o t h r i c i n a p p l i c a t i o n in a n i m a l
h u s b a n d r y , t r a n s f e r a b l e or t r a n s p o s a b l e sat d e t e r m i n a n t s
c o u l d not b e d e t e c t e d a m o n g gut b a c t e r i a of h u m a n b e i n g s
and animals.
mental studies on bacterial populations with and
without selection pressure have been performed
[22].
The ecological and evolutionary data achieved
from the streptothricin model may be summarized as followed:
Table 3
S p r e a d of p l a s m i d s p i E 6 3 6 (IncI2, 80 kb, Tn1826), p i E 6 3 8 (IncW3, 38 kb, Tn1825) and p i E 6 6 3 (IncX, 52 kb, Tn1826) e n c o d i n g
s t r e p t o t h r i c i n r e s i s t a n c e (sat), in v a r i o u s b a c t e r i a l p o p u l a t i o n s in the e a s t e r n part of G e r m a n y
Origin
1982
initial
application
1983
1984
1985
1986
1987
1989-1993
end of
application
gut flora of pigs
pig m a n u r e
gut flora of
personal 1
gut flora of
family m e m b e r s 2
gut flora of
healthy adults 3
food 4
sick a n i m a l s
u r i n a r y tract
infections
salmonellosis
shigellosis
-
12, W 3
12, W 3
-
12, W3, X
I2, W3, X
I2, X
X, W3
-
-
12, W3, X
12, W3, X
I2, W3, X
-
-
12, X
-
-
-
I2, W3, X
-
-
-
X, W3, I2
-
-
12, W3, X
.
-
X, 12
X, W3, I2
X, 12, W3
-
X
-
12, W3, X
I2, W3, X
X
12, X
X
I2
none
- -
.
.
.
.
.
.
.
.
.
.
.
x E m p l o y e e s in the a n i m a l farm.
2 R e l a t i v e s a n d family m e m b e r s of the e m p l o y e e s .
3 P e o p l e not r e l a t e d to a n i m a l h u s b a n d r y , u r b a n p o p u l a t i o n .
4 M a i n l y m i l k a n d raw m e a t have b e e n investigated.
Tn1826 (sat-2, a a d A , TnT-like).
Tn1825 (sat-l, a a d A , Tn7-1ike).
.
29
(3) Under conditions of local selection pressure
the horizontal spread of genes is 'activated' in
such a way that it will take place also to
ecological niches and bacterial populations
which are not under selection pressure and
where apparent selective advantage does not
exist. In following three distinct streptothricin
resistance plasmids, plE636(IncI2), pi E
638(IncW3), and plE663(IncX) (Table 3) these
were detected first among strains obtained
from pigs and later among bacteria which
were not in direct contact with streptothricins
or associated with animal husbandry. Due to
the 'activated' plasmids, the respective genes
will even move into bacteria in niches which
do not gain an apparent advantage by acquiring them (see Shigella in Table 3).
(4) In spite of their absence in human and animal
strains, sat determinants occurred and persisted among environmental bacterial populations without selective pressure [22]. They
were found to be identically organized as in
bacteria under selection pressure (sat-l, sat-2
on Tn7 like transposons, sat-3 on an IncQ
replicon). Since the sat determinants have
(5)
been found similarly in bacteria under selection pressure, environmental bacteria appear
to serve as a reservoir for sat determinants.
However, the question remains why streptothricin resistance determinants exist naturally in environmental populations.
Under conditions of selective advantage, the
pool of plasmids in respective bacterial populations can be maximal. Since 1990, there has
been no more application of streptothricin,
but the prevalence of streptothricin resistance
plasmids is still high. This implies that determinants introduced once by selective advantage can persist for a long time, even if they
are not longer needed.
A protocol of plasmid transfer under natural
conditions
Horizontal gene transfer under natural conditions has been observed in a small sewage plant
of 10 m 3 with a total turnover of 3 weeks (unpublished data). Since this plant is localized in a
suburban area and belongs to a 12-member fam-
107 [ %
,o°1\
..--e-----
,o,
/\
,ol !
\
,o l!
,o,]/
_
\
..---1-'--"
.
lr
'I"
'i"
"I"
9"
0
1
2
4
5
i
i
...--.-.-41
-r
r
T
T
10
12
14
18
days
[~BIHE oR+BIHE -e-R+donor ]
Fig. 4. In vivo transfer of a naturally occurring plasmid in a coliform sewage population to E. coli K12 CV601. CV601: E. coli
CV601 counts. R + CV601: E. coli CV601 containing 96 kb multiple drug resistance plasmid of IncFII group encoding resistance to
chloramphenicol, streptomycin, sulphonamide, tetracycline, ampiciilin, kanamycin, and mercury. R + donor: indigenous population
harbouring the respective plasmid. All counts were carried out on M9 minimal medium containing the respective drugs and amino
acids for E. coli K12 CV601. The points in the figure represent the average of three counts.
30
ily house, the bacterial flora observed within the
sewage plant originated from the family members
only. No family member had been treated by
antibiotics or was hospitalised in the last 10
months. Over a 6-month period, a distinct 96 kb
large, IncFII multiple drug resistance plasmid
could be detected among the coliform population
of this sewage. However, we failed to find this
plasmid in bacteria from stool samples taken from
the family members on several occasions. In order to analyse the nature of this plasmid persistence, we introduced E. coli K12 CV601 in the
sewage and screened for respective exconjugant
cells. The donor bacterial count was always between 7.5 x 105 ml-1 and 3 X 10 4 ml 1, the temperature in the sewage plant was about 12°C
during the experiment. Twenty 1 of overnight
culture of the recipient bacterial strain CV601
were introduced via the toilet bowl. Immediately
after introduction and on each day, viable counts
of CV601 and of exconjugants of CV601 were
determined. As shown in Fig. 4 there was a rapid
loss of CV601 from the sewage plant population
within a few days whereas the donor count remained around 105 cfu m l However, after 12 days of absence of E. coli
K12 CV601, it reappeared, now carrying the 96
kb IncFII plasmid with the multiple drug resistance function. Since selection pressure favouring
this particular drug resistance function could not
be detected, it remains an open question whether
the plasmid-encoded functions were of selective
advantage under these environmental conditions,
or if plasmid transfer should be characterized as
a selfish event. Nevertheless, this observation
demonstrates the in vivo transfer of plasmids
under natural conditions in a sewage plant even
when the temperature and the concentration of
donor and recipient bacteria are not optimal.
Conclusions
(1) The environmental spread and persistence of
plasmids and plasmid-borne genes among
bacterial populations is mainly dependent on
the biological properties of the plasmids, such
as replication and transfer. Irrespective of the
selective advantage supporting the incidence
of respective genes in bacterial populations
under environmental stress, 'selfish' plasmid
transfer may enable the spread of genes
among the population.
(2) The biology of plasmids is different as far as
the various phylogenetic systems (plasmid
species) are concerned. They can respond to
various environmental signals.
(3) The recruitment of foreign DNA, the broad
host range, the stable maintenance and the
transferability under environmental conditions enable the plasmids to represent a 'task
force' for the bacteria in order to overcome
environmental stress situations.
(4) Plasmid biology and the various plasmids
species existing in nature are only beginning
to be understood. Proceeding with studies on
environmental bacterial populations we will
detect more plasmid species and molecular
mechanisms involved in horizontal gene
transfer.
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