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Journal of Bacteriology, November 1998, p. 6039-6042, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Carboxyl Terminus of Protein TraD Adds
Specificity and Efficiency to F-Plasmid Conjugative Transfer
J. Ignacio
Sastre,
Elena
Cabezón,
and
Fernando
de la Cruz*
Department of Molecular Biology, University
of Cantabria, 39011 Santander, Spain
Received 20 May 1998/Accepted 5 September 1998
 |
ABSTRACT |
We isolated and characterized traD mutants with an
altered specificity of interaction with relaxosomes of various
conjugative (F and R388) and mobilizable (RSF1010 and ColE1) plasmids.
The change in specificity was due to a loss of some amino acids in the
carboxyl terminus of TraD that resulted in a broadening of the range of
mobilizable relaxosomes at the expense of a decrease in the efficiency
of F-plasmid transfer.
| |
TEXT |
Bacterial conjugation is a process
of DNA transfer that is widespread among bacteria. Essential functions
for conjugation include mating-pair formation (Mpf) and conjugative DNA
processing. In every conjugative system there is a protein that is
essential for conjugation but is not required for any of these
processes; this protein presumably connects the relaxosome (a
nucleoprotein complex for conjugative DNA processing that forms at
oriT) and the membrane-spanning protein complex for DNA
translocation (encoded by Mpf genes), and so it has been called the
"coupling protein" (5). Mobilizable plasmids (such as
RSF1010 and ColE1) bear functions required for relaxosome formation but
encode neither Mpf proteins nor the coupling protein, which have to be
provided by a conjugative plasmid (3-5, 12). Conjugative
plasmids bear all functions required to promote their own transfer,
including a gene for a coupling protein, which has been found in
different conjugative plasmids from gram-negative bacteria
(traG in RP4, trwB in R388, and traD
in F) and also in the Agrobacterium tumefaciens T-DNA
transfer system (virD4). Proteins encoded by these genes, which are termed the TraG protein family, show significant similarities among their amino acid sequences and share transmembrane domains and
sequence signatures for nucleoside triphosphate binding (1, 13). Interactions of the TraD coupling protein with different components of the F-plasmid relaxosome have already been suggested, both in vivo (TraD with TraI) (6) and in vitro (TraD with
TraM) (8).
E. coli strains and plasmids.
Escherichia
coli strains and plasmids used in this study are listed in Tables
1 and 2,
respectively.
Effects on conjugation of a deletion in the carboxyl terminus of
protein TraD.
Because of the homology among coupling proteins,
some attempts have been made to interchange them for conjugative
transfer and mobilization. Cabezón et al. (4), working
with R388, demonstrated that efficient transfer of mobilizable plasmid
RSF1010 could be achieved when R388 trwB was replaced by RP4
traG. However, F traD only very inefficiently
complemented R388 trwB for RSF1010 mobilization (5). Amino acid sequence alignment of TrwB, TraG, and TraD indicated that TraD displays a carboxyl terminus longer than those of
TrwB or TraG (Fig. 1). We suspected that
differences in RSF1010 mobilization could be due to this feature,
so the TraD carboxyl terminus was deleted by introducing a stop
codon in traD by site-directed mutagenesis. The stop
codon was introduced just after amino acid 576, which corresponds to
the point in the TraD sequence where homology with TrwB ends. The
resulting protein (TraD576 [Fig. 1]) lacks the last 140 amino acids
of TraD (nearly 20% of the whole protein).
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FIG. 1.
Comparison of TrwB with TraD and a series of TraD
mutants in the carboxyl-terminal region. The upper part of the figure
shows a schematic alignment of TrwB and TraD. Solid boxes represent
predicted transmembrane segments, and shaded segments represent
theoretical nucleoside triphosphate-binding motifs. When the amino acid
sequences of TrwB and TraD were aligned, C-terminal residue 507 of TrwB
aligned with residue 576 of TraD. The lower part of the figure shows
the amino acid sequences of the carboxyl-terminal regions of TraD and
its derivatives. The plasmids that encode each of the proteins are
shown in parentheses. Superscript numbers correspond to the positions
of the corresponding amino acids in the sequence of TraD. Amino acids
that differ from those of TraD are underlined. Asterisks represent the
ends of the proteins.
|
|
Plasmid pSU4327, containing traD576, was used to
complement pSU1456, a trwB mutant of R388, for
self-transfer and for mobilization of either ColE1 or RSF1010.
The results are shown in Table 3. While
self-transfer of pSU1456 was complemented by TraD to very low levels
(10
8 transconjugants/donor), this frequency
increased 1,000-fold with TraD576. The frequency of mobilization
of ColE1, however, remained unaffected. Interestingly, the
traD576 mutant also increased pSU1456-mediated mobilization
of RSF1010 by 1,000-fold, resulting in a frequency equivalent to
that obtained when RSF1010 mobilization was complemented by
trwB (104 transconjugants/donor). This result
suggested that the presence of the TraD carboxyl terminus was
hindering mobilization of RSF1010 by the MpfW system. It
seems likely that the TraD C terminus led to deficient interaction
either with the RSF1010 relaxosome or with the R388 MpfW
apparatus. To decide between both possibilities, we analyzed RSF1010
mobilization while employing the MpfF apparatus (provided
by strain JM109). With TraD as the complementing protein, RSF1010
mobilization was poor (5 × 106
transconjugants/donor [Table 3]). However, conjugation of F itself
occurred at high frequencies (1 transconjugant/donor [Table 3]), so
it can be assumed that TraD is able to interact properly with
MpfF but fails to interact with the RSF1010 relaxosome. If, as we supposed, the TraD carboxyl terminus was hindering the
interaction with the RSF1010 relaxosome, TraD576 should lead to a
higher RSF1010 mobilization frequency than TraD when complementing
JM109. In fact that was the case, and the results showed that
mobilization of RSF1010 by MpfF also increased 1,000-fold
with TraD576 (Table 3). Thus, the RSF1010 mobilization frequency was
103-fold higher with TraD576 than with TraD, regardless of
the Mpf system employed, and the lack of the TraD carboxyl terminus in TraD576 allowed better interaction of TraD576 with components of the
RSF1010 and R388 relaxosomes.
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TABLE 3.
Transfer frequencies of mobilizable plasmids, or of
conjugative plasmids deficient in the coupling protein, when
complemented by TraD variantsa
|
|
To this point, our results showed only a hindering function for the
TraD carboxyl terminus, but its physiological significance in F
conjugation was suggested by an experiment in which we used TraD576 to
complement JM109 (F traD) for F transfer. In this case, the
frequency of conjugative transfer of F traD upon
complementation by TraD576 was reduced 104-fold compared
with the frequency obtained when F traD was complemented by
wild-type TraD (Table 3). The conclusion was that the TraD carboxyl
terminus is responsible for efficient coupling with the F relaxosome,
at the cost of hindering the interaction with unrelated relaxosomes.
It is worth noting here that the frequencies of ColE1 mobilization with
MpfW (pSU1456) or MpfF (JM109) did not vary
when TraD576 instead of TraD was employed (Table 3). These results
suggest that interaction of TraD with the ColE1 relaxosome probably
does not involve the carboxyl region. These data reinforce the concept that the Mpf functions of both F and R388 can interact properly with
TraD576, and so the results reported above should be a consequence of
altered interactions with the relevant relaxosomes.
The C-terminal 37 amino acids of TraD are responsible for the
change in specificity.
Using a different experimental approach, we
randomly mutagenized traD to improve its ability to
complement conjugative transfer of R388 trwB. A method that
combined in vivo mutagenesis and selection steps was used. Random
mutagenesis was carried out by introducing pSU4305, a recombinant
plasmid containing F traD together with R388
oriT, into E. coli XL1-Red (catalog no. 200129;
Stratagene). This strain is damaged in three different DNA repair
systems (mutD mutS mutT), so its mutation rate is about
5,000-fold higher than that of common E. coli laboratory
strains. After 200 generations of growth of XL1-Red(pSU4305), plasmid
DNA was isolated and introduced into strain DH5
(pSU1456).
About 104 transformant colonies were pooled, diluted
in Luria broth to 2 × 109 cells/ml, and used as
donors for mating with the recipient strain, UB1637. The presence of
R388 oriT in pSU4305 allowed conjugative DNA processing of
this plasmid by pSU1456-encoded proteins and thus its transfer to the
recipient strain. Donor cells (107) were mated with
109 recipient cells at 37°C on a solid surface
(0.22-µm-pore-size Millipore nitrocellulose filter on prewarmed
nutritive agar plates) for 1 h. Transconjugants harboring pSU4305
derivatives were isolated. In that way, a selection step that
eliminated nonconjugative mutants and enriched the population of those
traD mutants that increased complementation of R388
oriTW transfer was carried out. Resulting transconjugants were pooled, and plasmid DNA was obtained from them and
reintroduced into XL1-Red cells to carry out another mutagenesis-selection cycle. After five of these cycles, a mutant plasmid named pSU4309, which produced a frequency of transfer of R388
trwB that was 103-fold higher than that obtained
with the original plasmid, pSU4305, was isolated (Table 3).
The mutation present in pSU4309 that was responsible for the effect on
the frequency of transfer of R388 trwB was localized by DNA
heteroduplex analysis in mutation detection enhancement (MDE)
polyacrylamide gels (Hydrolink). Briefly, wild-type and mutant plasmid
DNAs were endonuclease digested, mixed, heat denatured, and then
renatured and run on an MDE gel. Heteroduplex DNA molecules carrying
mismatches were detected by means of their reduced electrophoretic mobility, allowing detection of single-base substitutions. In this way,
we limited mutations to a segment of 36 bp located 128 bp upstream from
the end of traD. This region and adjacent DNA were
sequenced, revealing that the only mutation detected was a deletion of
a G at position 2358 in the published DNA sequence of traD
(11). This point deletion caused a frameshift that changed the TraD amino acid sequence from position 680 onward and resulted in
the appearance of a premature stop signal at amino acid 698. The
resulting protein, named TraD680fs, had lost the last 37 amino acids of
the carboxyl terminus of TraD (Fig. 1). That small deletion produced an
effect on plasmid transfer frequencies equivalent to that observed with
TraD576: a 103-fold increase in the transfer frequencies of
both R388 and the mobilizable plasmid RSF1010 and a
104-fold reduction in F plasmid transfer frequency. A
10-fold increase in the transfer frequency of plasmid ColE1 may also be
significant (Table 3). Therefore, the determinant for efficient
interaction with the F relaxosome that hindered the interaction with
R388 and RSF1010 relaxosomes was located at the very end of TraD, its last 37 amino acids. The remaining amino acid sequence deleted in
TraD576 apparently had no additional effect, and its role could be
simply to position properly the specificity determinant in the whole protein.
We next wondered if further deletion of the TraD carboxyl terminus
beyond amino acid residue 576 would maintain the same properties. Therefore, we constructed the TraD derivative TraD518, which ends with
the substitution G518I and lacks the C-terminal 199 amino acids of TraD
(Fig. 1). When plasmid pSU4316, containing traD518, was used
to complement the transfer of either R388 trwB or F
traD, the transfer frequencies obtained were very low (Table
3), suggesting that the region between amino acids 518 and 576 contains
sequences of TraD that are essential for function.
Altogether, our results showed that the loss of even a small fragment
of the TraD carboxyl terminus causes a change in the specificity of its
interaction with different relaxosomes, resulting in a moderate
frequency of conjugative transfer for a series of them (Table 3). Thus,
the effect can be described as a widening of the range of mobilizable
relaxosomes at the expense of a reduction in the efficiency of F
relaxosome mobilization. This result could be interpreted as the
presence in F TraD of a carboxyl-terminal arm, not present in other
coupling proteins (such as TrwBR388 and
TraGRP4), that constitutes a high-affinity site for
interaction with its own relaxosome. The existence of this C-terminal
arm hinders the interaction of a second, less efficient, site with affinity for a broader range of relaxosomes. Evolutionarily, this kind
of specialization typically occurs in stable environments, where it is
advantageous to sacrifice a capacity for adaptation to different
conditions for an increase in functional efficiency (called a
K-strategy); in contrast, under unstable conditions, specialization is
a burden instead of an advantage, and an r-strategy based on a high
reproduction rate is more convenient for ecological success
(15). In nature, F exists usually as a single-copy plasmid exclusively in bacteria belonging to the Enterobacteriaceae,
which proliferate in animal digestive tracts. This ecosystem probably constitutes a stable environment, so F may have followed a K-strategy consisting of the development of a specific and efficient transfer apparatus to promote its own transfer. This is consistent with the
higher genetic complexity of the F transfer apparatus compared with
other conjugative systems (9). Meanwhile, plasmids dwelling in more unstable environments, as do promiscuous plasmids within IncN,
IncP, IncW, or IncQ, may have developed a simpler, less specialized,
and less efficient but more versatile conjugative apparatus.
| |
ACKNOWLEDGMENTS |
This work received financial support from the Spanish Ministry of
Education (DGICYT) through project PB95-1269. J.I.S. was a recipient of
grants from the Department of Education of the Basque Government and
from the Marqués de Valdecilla Foundation (in different periods),
and E.C. was a recipient of a grant from the Department of Education of
the Basque Government.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, University of Cantabria, Cardenal Herrera Oria s/n, 39011 Santander, Spain. Phone: 34-42-201942. Fax: 34-42-201945. E-mail:delacruz{at}medi.unican.es.
Present address: Laboratory of Molecular Biology of the Medical
Research Council, Cambridge, United Kingdom.
| |
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Journal of Bacteriology, November 1998, p. 6039-6042, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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