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Journal of Bacteriology, August 1998, p. 4093-4101, Vol. 180, No. 16
Department of Microbiology, La Trobe
University, Bundoora 3083, Australia
Received 9 February 1998/Accepted 2 June 1998
The mechanisms by which gene products inhibit the conjugal transfer
of IncP plasmids (e.g., RP1) have been little studied. We have isolated
and characterized one such gene, fipA (624 nucleotides), from the SmaI (14.8 kb)-AatII (15.6 kb) region
of pKM101(IncN). This gene, which is also conserved in other IncN
plasmids, is transcribed in an anticlockwise direction, probably as
part of a transfer operon that includes traHI. The FipA
protein (24 kDa) appears to be cytoplasmic and, when expressed from a
multicopy plasmid, retards the growth of Escherichia coli
WP2. The mode of action of fipA was compared with that of
the apparently unrelated pifC gene from F(IncFI). Both
genes inhibit the transfer of IncP Conjugative transfer by prototypes
of the IncP The conjugal transfer functions control mating pair formation (the Mpf
or PIL system) and DNA transfer and replication (the Dtr or MOB system)
(9, 50). In the case of RP1(IncP Homologs of TraG occur in various conjugal systems (30, 41)
and are essential both for transfer of the conjugative plasmid and for
mobilization of nonconjugative plasmids (23). However, the
frequency of mobilization can vary widely depending on the conjugal
system being used, even when transfer of the conjugative plasmid is
highly efficient. The finding that RP1 TraG can replace the TrwB
homolog of R388 for the purposes of mobilization but not self-transfer
raises the possibility that different domains of these homologs are
involved in interactions with the relaxosome and the DNA transport
complex (8). The degree to which these homologs can be
interchanged has been the subject of a recent study (9).
In this report, we describe genes that inhibit the conjugal transfer of
RP1 and which may prove useful in elucidating the roles of the transfer
genes. Inhibition genes are common in plasmids (27), and
those in pKM101(IncN) (76) and F(IncFI) (68)
inhibit RP1 transfer without apparently affecting pilus function. The gene in F, pifC, has roles in the regulation of the F
pif operon (11, 31, 44) and the initiation of F
DNA replication (33, 44, 69), but neither pifC
nor the gene in pKM101 (75) is an essential tra
gene. We characterize the gene in pKM101, here named fipA,
and show that both fipA and pifC exert their
inhibition effects via traG. We also present evidence that
the interaction between pifC and traG occurs at
the protein level and not, as previously suggested (43), by
binding to RP1 DNA.
(A preliminary report of this work was presented at Plasmid Biology 96:
International Symposium on Plasmid Molecular Biology [59].)
Nomenclature.
Restriction cleavage sites and their locations
are presented in the format KpnI (24.1 kb). The designations
MOB and PIL are used as phenotypic descriptors of the groups of genes
from different plasmid type required, respectively, for conjugal DNA
metabolism and for pilus synthesis and function (MOBP,
PILN, etc.).
Strains, plasmids, and media.
The Escherichia
coli K-12 derivatives used were HB101 (recA13
Strr) (7), a rifampin-resistant mutant of HB101
(LT101 [48]), UB1301 (Rifr
[21]), and UB5201 (recA56 Nalr
[58]). WP2 (25), a tryptophan auxotroph of
E. coli B/r, and Pseudomonas aeruginosa
PAO2637(RP1) (18) were also used.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Both the fipA Gene of pKM101 and the
pifC Gene of F Inhibit Conjugal Transfer of RP1 by an Effect
on traG
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
and IncP
plasmids but to
different degrees. They also inhibit the mobilization of RSF1010 (which
requires the RP1 pilus genes and traG) but not of CloDF13
(which encodes a traG homolog). Evidence that
traG was the specific target of inhibition was obtained in an artificial system in which cloned traG was used to
enhance RSF1010 mobilization via the N pilus system. Such enhancement did not occur in the presence of fipA or pifC.
The availability of an in vivo assay of PifC enabled us to show that F
pif operon expression increased in cells carrying
F'lac and traG, but only if the
traG coding sequence was intact. This finding suggested that conjugal inhibition of RP1 was most likely due to a PifC-TraG protein interaction. On phenotypic grounds inhibition of
traG by fipA is also likely to occur
posttranscriptionally. Whether or not the selection of traG
as the inhibition target is an evolutionary tactic to limit the spread
of P plasmids, we anticipate that fipA and pifC
will prove useful in further investigation of the conjugal roles of
traG and its homologs.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
and IncP plasmid subgroups has been the subject
of considerable study (23, 38, 50, 71). This has
included comparisons with the F(IncFI) paradigm system (16,
19) and those determined by IncW (6, 42) and IncN
plasmids (26, 52, 53, 75), as well as studies of the
interplay between P conjugal activity and the replication and
promiscuous transfer of these plasmids (27, 28). More recently, sequence comparisons have suggested a common ancestry for
conjugal DNA transfer and that of oncogenic T-DNA transfer from
Agrobacterium tumefaciens to plant cells and the
Bordetella pertussis toxin export system (30,
41).
), the Mpf genes are
located in the Tra2 region except for traF, located in Tra1
(18, 24, 39, 40). The Mpf system is required for P-pilus
formation and, in the form of a membrane-associated Mpf complex,
promotes DNA export and the adsorption of donor-specific phages
(13, 35). The Dtr genes are located in the Tra1 region, and
their products, TraI, TraJ, and TraK, interact at oriT to form the relaxosome and initiate transfer of the single DNA strand (38, 49, 51). An additional Tra1 gene, traG, that
is not part of the Mpf or Dtr systems is essential for DNA transfer, possibly serving to link the relaxosome to the DNA transport apparatus (38, 70).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
plasmids pJP4, R772, and R751 (64). Additional plasmids used
were mutants of pVS659 and pVS520 (described in Tables 4 and 6,
respectively) and plasmids that carried parts of the RP1 region (from
position 48.869 kb [PtraG] through
traG [48.495 to 46.588 kb] to position 46.573 kb) cloned
directly from a 2.3-kb PCR product (see below) into pUC19 by utilizing
internal SphI or terminal EcoRI sites. These
plasmids, and the regions cloned in the sense orientation with respect
to the Plac of the vector, were pVS1160 (48.869 to 48.707), pVS1161 (48.707 to 47.826), pVS1162 (47.826 to 47.561),
pVS1163 (47.561 to 46.798), and pVS1164 (46.798 to 46.573).
TABLE 1.
Bacterial plasmids used
Conjugation procedures and growth experiments. Late-exponential-phase cultures in NB were used for conjugation experiments conducted by the quantitative filter method described previously (48). Experiments were performed three times, and the average transfer frequency (transconjugants per donor cell) was calculated.
For growth experiments, WP2 and its plasmid-carrying derivatives were each grown overnight in 10 ml of NB, and then the cells from a 2-ml aliquot were harvested, washed twice in minimal E medium, and suspended in 200 ml of the same medium. The 500-ml culture flasks were incubated, with shaking, at 37°C, and the absorbance of the culture at 560 nm was measured each half hour until stationary phase was reached. The set of growth experiments were performed in parallel on three occasions, and the results were subjected to multiple regression analysis (46).Methods using phages.
The virulent phage T7 (obtained from
R. A. Skurray via H. Dean, Monash University, Melbourne,
Australia) and the donor-specific phage PR4 (66) were
propagated on bacteria [HB101 and PAO2637(RP1), respectively] grown
in soft-agar overlays on NA (1). The efficiency of plating
of T7 was determined by spot phage assay (66) except that
flood lawns were prepared with diluted (ca. 5 × 107
cells/ml) exponential-phase cultures. Sensitivity (Dps+) or
resistance (Dps
) of bacteria to PR4 was determined by the
same method but using a single drop of phage (ca. 109 PFU).
DNA techniques. Standard molecular cloning techniques were performed as described previously (57). The fipA-containing SmaI (14.8 kb)-AatII (15.6 kb) region of pKM101 was sequenced on both strands by using overlapping subclones prepared from pVS1127. The sequencing reactions were performed with T7 DNA polymerase, universal or reverse primers, and protocols supplied with the Autoread sequencing kit (Pharmacia). The DNA sequence was determined with a Pharmacia LKB A.L.F. DNA sequencer and was analyzed by using software provided by the Australian National Genome Information Service (University of Sydney).
PCR amplification was carried out with the primers listed in Table 1 and reaction mixtures that contained 10% (vol/vol) dimethyl sulfoxide. The mixtures, overlaid with sterile mineral oil, were placed in a thermal cycler for 36 cycles under the following conditions: 92°C, 3 min (first cycle only); 92°C, 1 min; 60°C, 1 min; 72°C, 2 min; and 72°C, 5 min (final cycle only). The PCR product obtained (2.3, 1.9, or 1.1 kb) was cloned into pBluescriptII SK+ to create pVS1141, pVS1140, or pVS1150, respectively (Table 1). DNA of various IncN-group plasmids was isolated by cesium chloride-ethidium bromide density gradient centrifugation and digested with a combination of SmaI and AatII. The DNA fragments, separated by electrophoresis, were transferred to a Hybond(N+) nylon filter (Amersham) and hybridized at 60°C to a probe prepared from the 0.8-kb fipA-containing AatII-SmaI fragment from pVS1127. Labeling of the probe and detection of hybridization were performed with DIG-DNA Labeling and DIG-Luminescent Detection kits (Boehringer Mannheim), respectively.Expression of FipA protein.
Detection and molecular weight
determination of FipA and truncated FipA (FipA
) were performed by
the T7 RNA polymerase-promoter system (67) as previously
described (3). The bacteria (HB101 carrying pGP1-2 and
either a vector or recombinant) were exposed to 10 µCi of
[35S]methionine-cysteine mix (New England Nuclear). In
some cases, the labeled cells were fractionated (57) first
by treatment with lysozyme (1 mg/ml) to release periplasmic proteins
and then by four cycles of freezing (1.5 min at 
70°C) and thawing
(1.5 min at 37°C) to release cytoplasmic proteins. The remaining cell debris, resuspended, constituted the crude membrane fraction. Cell
samples or fractions were denatured in sample buffer (3% sodium
dodecyl sulfate [SDS], 5% -mercaptoethanol) at 100°C for 3 min
(3) prior to electrophoresis on denaturing polyacrylamide gels (12%) containing 0.1% SDS. The reference proteins used were Sigma Dalton Mark VII-L molecular weight standards. After
polyacrylamide gel electrophoresis (PAGE), proteins were stained in
0.25% Coomassie brilliant blue (Sigma)-50% (vol/vol) methanol-9%
(vol/vol) acetic acid; after destaining, autoradiographs of the dried
gels were prepared.
Nucleotide sequence accession number. The sequence reported here has been entered in GenBank under accession no. U42978.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning and characterization of fipA.
The
fip region of pKM101 was localized between the
endonuclease (nuc) and transfer (traHI) genes, in
the vicinity of map position 15.0 kb (Fig.
1) (73, 76). To isolate the
fip gene(s), parts of this region were cloned into pUC18 or
pBluescriptII SK+, and the resulting plasmids were tested for the
ability to inhibit the conjugal transfer of pUB307, a derivative of
RP1. Plasmids pVS1107 [with the pKM101 SmaI (14.8 kb)-KpnI (17.0 kb) fragment] and the pair pVS1126 and
pVS1127 [both with the SmaI (14.8 kb)-AatII (15.6 kb) fragment] were Fip+ and inhibited pUB307 even
more strongly than did pKM101 (Table 2),
probably reflecting the higher copy number of the vector replicons.
Plasmids pVS1128 and pVS1129 [both with the BglII (15.0 kb)-AatII (15.6 kb) fragment] were Fip. These
data suggest that the fip gene(s) occurs in the pKM101 SmaI (14.8 kb)-AatII (15.6 kb) fragment and that
the BglII (15.0 kb) site is necessary for its expression
(Fig. 1).
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) were expressed in vivo from the T7 
10 promoter of
the vector (67) and analyzed by SDS-PAGE (Fig.
2). A single protein was expressed from
pVS1127 (24 kDa; lane 1) and pVS1129 (22 kDa; lane 5) but not from the
corresponding plasmids with the insert in the opposite orientation
(pVS1126 and pVS1128) (data not shown) or from the plasmid vector (lane
9). These findings suggest that the pKM101 SmaI (14.8 kb)-AatII (15.6 kb) fragment encodes a single intact gene,
here named fipA, that is transcribed in an anticlockwise (AatII
SmaI) direction (Fig. 1), yielding a
protein of 24 kDa. The BglII site near the 3' end of
fipA accounts for the expression of a truncated FipA from
pVS1129. Cells expressing FipA and FipA were also fractionated
prior to SDS-PAGE, and the distribution of the proteins was
compared with that of chloramphenicol acetyltransferase (Cat; a
cytoplasmic protein [62]) and -lactamase (Bla; a
periplasmic protein [15]) expressed in a parallel
experiment from cells carrying pVS915 (Fig. 2). Although the expression
of the two FipA proteins was relatively poor (lanes 1 and 5), each
occurred predominantly in the cytoplasmic fraction (lanes 3 and 7), as
did Cat (lane 12).
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10 and 35
E. coli-like promoter sequences (45, 65)
were detected in the upstream sequence. Hydropathy analysis
(37) of the deduced 23.8-kDa FipA protein (208 amino acid
residues) revealed a hydrophobic region at the amino terminus (residues 32 to 48) predicted by the ALOM program (32) to be a
transmembrane domain. No probable signal sequence was detected. Despite
these predictions that FipA may be membrane associated, the
experimental findings are more suggestive that it is cytoplasmic. No
significant similarities were detected between fipA and
sequences in the GenBank/EMBL databases when the nucleotide or
translated sequences were compared.
Conservation of fipA-like sequences and the effect of fipA on the growth of E. coli WP2. Members of the IncN plasmid group share a range of phenotypic characteristics, only one of which is the conjugal inhibition of IncP plasmids (10, 26, 27, 74). When seven IncN plasmids from diverse sources and with different resistance profiles were tested, all inhibited the conjugal transfer of pUB307 (103- to 105-fold [data not shown]). These plasmids had distinguishable SmaI/AatII restriction profiles, but six (pCU1, pVS101, pVS144, pVS151, R199, and R309) carried a 0.8-kb SmaI-AatII fragment that hybridized with the corresponding fipA-carrying fragment from pKM101 (data not shown). The seventh plasmid (N3) lacked this fragment, but hybridization occurred with a fragment of >9 kb. These data suggest that fipA-like sequences are conserved in IncN plasmids.
The ability of fipA to retard the growth rate of E. coli WP2 was also tested, as such an effect has been associated with the pKM101 BalI (14.6 kb)-KpnI (17.0 kb) region (25), shown here to include fipA. The doubling time of WP2 (54 min) was not significantly affected by the carriage of pKM101 (52 min), pBluescriptII SK+ (59 min), or pVS1129 (Fip) (60 min) but
was increased by the carriage of pVS1127 (Fip+) (69 min; significant at P < 0.05). These data
demonstrate that fipA can retard the growth of WP2, but in
our hands, this occurred only when the gene was cloned (i.e.,
multicopy) and not, as observed by Hall (25), when present
in the native pKM101.
Characteristics of fipA and pifC and the
ability of these genes to inhibit RP1-mediated mobilization of
nonconjugative plasmids.
The pifC gene of plasmid F,
like fipA (76), inhibits the conjugal transfer of
RP1 without affecting surface exclusion (Sfx+) or
sensitivity to donor-specific phages (Dps+) (43,
68). Inhibition by pifC is, however, at least 100-fold weaker than that by fipA. This is the case whether the genes
occur on the native plasmid (F'lac versus pKM101) or have
been cloned (pVS1150 versus pVS1127) and whether the target is an
IncP plasmid (the RP1 derivative pUB307) or an IncP plasmid
(e.g., R751) (Table 2).
4), as has been observed previously
(8). Evidence supporting traG as the inhibition
target, and also demonstrating that pilus function is normal in the
presence of fipA or pifC, was obtained in
experiments using CloDF13a, a nonconjugative plasmid that encodes a traG homolog (9). In this case, the
presence of fipA or pifC inhibited conjugal
transfer of RP1 but had no effect on the ability of RP1 to mobilize
CloDF13a (Table 3).
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Development of a conjugal system to assay RP1 traG activity. To further investigate the possibility that the RP1 traG gene is the target of inhibition by fipA and pifC, we developed an artificial conjugal assay of traG activity based on the finding that the weak mobilization of RSF1010 by IncN plasmids (9, 81) is greatly enhanced in the presence of the RP1 Tra1 genes (Table 4). In this assay, the donors carried three compatible plasmids: RSF1010t(MOBQ), a fipA mutant of pKM101(pVS793; MOBNPILN), and a clone of the entire RP1 Tra1 region (pVS659; MOBP traF+G+ oriT+). By substituting pVS659 with mutants of the Tra1 genes, the roles of the N and P transfer genes in the pKM101fipA-mediated mobilization of RSF1010t and pVS659 could be assessed.
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3). This showed that the MOBP and
PILN systems can function together in conjugation in the
same way as has been found with MOBW and PILN
(6). As expected, mutations in the MOBP genes of
Tra1 (traI and traJ) or in traG
abolished mobilization of the pVS659 mutant. This also confirmed that
pKM101 genes cannot complement mutations in these Tra1 genes
(18). In contrast, a mutation in the PILP gene,
traF (70), had no effect on mobilization
presumably because the need for this gene was obviated by the intact
PILN system.
The second observation related to RSF1010t (Table 4), which
was efficiently mobilized by pKM101fipA in the
presence of pVS659 (1.1 × 102) but not in its
absence (2.6 × 106). Such mobilization did not
require traJ, traI, or traF, as
RSF1010t encodes the requisite MOBQ system
(14) and PILN, as described above, provides the
contact system. The traG gene, however, was essential for mobilization, as is also the case when RSF1010 is mobilized by RP1 alone (40, 70). The significant feature of the conjugal assay was that efficient mobilization of RSF1010 by
pKM101fipA occurred when traG was provided by
pVS659 (1.1 × 102) but not when it was provided by
RP1 (using pVS766) (Table 4). This finding suggested that the high
copy number of pVS659, and hence hyperexpression of traG
(and other Tra1 genes), was the basis for the unexpectedly
efficient mobilization of RSF1010t by pKM101fipA. Such
mobilization did not occur when pKM101fipA was
replaced by pKM101 (fipA+) (Table 4, line 4),
confirming that a Tra1 gene(s), namely, traG, is
the target of fipA-mediated inhibition. Finally, a
prediction from these findings was that hyperexpression of
traG should also permit mobilization of an RP1pil
mutant (pVS766). This was found to be the case but, as mentioned above,
is an example not of complementation but of successful combination of
the MOBP and PILN systems.
Hyperexpression of traG enhances pKM101-mediated
mobilization of nonconjugative plasmids, but this effect is abolished
in the presence of fipA or pifC.
To confirm that
traG is the target of inhibition by fipA and
pifC, traG was isolated alone and its effect was
tested in the conjugal assay described above. This was done by using a
2.3-kb fragment containing traG and its promoter
(PtraG), which was PCR amplified from pVS729 and
then cloned into pBluescriptII SK+ (Table 1). The transformants
recovered all carried the insert in the antisense orientation with
respect to the Plac of the vector. One such
derivative, pVS1141, when used in the conjugal assay promoted efficient
mobilization of RSF1010t by pKM101fipA (5.1 × 102 [Table 5]). This
showed that traG is the sole RP1 gene required for such
mobilization. When fipA was also present in the donor, mobilization of RSF1010t was reduced to the control level (5.0 × 106). This confirmed that traG is the target
of inhibition by fipA.
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2 (Table
5). This suggested that if traG is also the target of inhibition by pifC, then the effect of pifC is
almost negated by traG hyperexpression in pVS1141, a finding
that accords with the weaker inhibition by pifC than
fipA (Tables 2 and 3). Implicit in this explanation is the
notion that pifC-mediated inhibition of traG may
be a posttranscriptonal event. Data consistent with this view
were obtained when plasmids with reduced traG
expression were used. These plasmids carried a promoterless
traG gene cloned in the sense (pVS1142) or antisense
(pVS1140) orientation relative to the Plac of
the vector (Table 1). Compared to pVS1141, the reduced expression of
traG was clearly reflected in the intermediate level of
RSF1010t mobilization effected by pVS1142 (7.4 × 103) and the very poor level of mobilization effected by
pVS1140 (4.3 × 105). When the two comparable donors
also carried pifC, the inhibitory effects of this gene were
now discernible by the reduction in RSF1010t mobilization to the
control level. This confirmed that traG is the target of
inhibition by pifC. The behavior of fipA was
different from that of pifC in that it abolished RSF1010t mobilization in combinations with all three traG clones.
Whether this reflects differences in the mechanism of action of these genes is not known, but it seems likely that inhibition by
pifC is exerted at the protein level.
Levels of transfer similar to those shown in Table 5 were also obtained
in corresponding experiments in which pSC101c replaced RSF1010t (data
not shown). This was not surprising, as the mobilization of these
plasmids is dependent on traG (4, 18, 40).
Use of F'lac Pif activity to study pifC-mediated inhibition of traG. The findings described above show that traG is the target of inhibition by fipA and pifC, also raising the possibility that pifC may mediate its effect at the protein level. To investigate this further, we took advantage of the role of PifC to negatively regulate the F pif operon, the expression of which inhibits the propagation of phage T7 (denoted Pif+) (31, 56). Miller et al. (43) showed that F'lac Pif activity increased in cells that also carried RP4 (=RP1), reflecting the removal of PifC with consequent reduced plating and altered plaque morphology (denoted Pif++). The Pif phenotype thus provides an in vivo assay of the levels of PifC, and we used this phenotype to further study pifC-mediated inhibition of traG.
The results obtained (Table 6) showed that Pif activity of F'lac (Pif+) was indeed enhanced by the presence of the RP1 derivative pVS520 (Pif++). We have also shown that an equivalent effect was produced if the only RP1 gene present was traG, with (pVS1141) or without (pVS1140) its native promoter. This suggested that traG may be the sole RP1 gene required to enhance F'lac Pif activity. Significantly, enhanced Pif activity did not occur if traG was disrupted at different sites by a Tn5 insertion mutation (e.g., pVS580) or if only parts of the traG gene were present (e.g., pVS1161). The fact that the coding sequence per se was important and that this must be present intact implied that the removal of PifC is more likely to occur by an interaction with the TraG protein than by the binding of PifC to traG DNA. This conclusion is contrary to that drawn by Miller et al. (43), who favored PifC binding to RP4 DNA. Indeed, a possible PifC-binding sequence has been identified by computer analysis within the overlapping traI and traH genes of RP1 (50). When a plasmid which carries this sequence but not traG (i.e., pVS658 [Table 6]) was tested, it failed to enhance F'lac Pif activity. These combined data suggest that the conjugal inhibition of RP1 by pifC probably arises by a PifC-TraG protein interaction and that the same interaction may be responsible for the enhanced Pif activity of cells carrying both F'lac and RP1.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study concerned the mode of action of two genes that can inhibit the conjugal transfer of the P-group plasmid RP1. One gene, fipA [from pKM101(IncN)], was isolated and characterized in this work. The other, pifC [from plasmid F(IncFI)], has been the subject of considerable study, including two reports concerning transfer inhibition (43, 68). We confirmed that the sole inhibitory effect of pKM101 and F was to depress transfer of RP1 (68, 76) and showed, in parallel experiments, that inhibition by pifC is weaker than by fipA even when their effects are enhanced by cloning (Table 2). These studies also showed that fipA is the sole gene in the pKM101 fip region (76) required for inhibition. The fipA gene, of 624 nt (pifC is 1,086 nt [11]), occurs in the pKM101 SmaI (14.8 kb)-AatII (15.6 kb) fragment and is transcribed anticlockwise, yielding a protein of 24 kDa (predicted mass, 23.8 kDa) (Fig. 2). Deletion of the SmaI (14.8 kb)-BglII (15.0 kb) portion resulted both in truncation of the protein (to 22 kDa) and abolition of its inhibition effect, showing that the 23 C-terminal amino acid residues are essential for FipA activity. Removal of the two C-terminal residues from the PifC protein (40 kDa [12]) significantly reduced its inhibition effect (from 4,000- to 70-fold) (43).
The FipA protein was detected predominantly in the cytoplasmic cell
fraction but also occurred in the crude membrane fraction, a
distribution like that of Cat (Fig. 2). On the basis of this comparison, we concluded that FipA, like Cat (62) (and also PifC, which is a repressor [31]), is a cytoplasmic
protein. Additional confirmation of this is needed, as a possible
transmembrane domain occurs in the FipA N terminus (residues 32 to 48)
suggestive of membrane association. FipA was also expressed poorly
compared to Cat and Bla (Fig. 2), and this occurred when other
vectors carrying the T7 10 promoter were used (60). As
the difference is not due to a paucity of Met or Cys residues (i.e.,
poor labeling of FipA), it suggests that efficient inhibition of RP1
(ca. 105-fold [Table 2]) is elicited even when
fipA expression is at its lowest, in pKM101.
The fip region mapped in pKM101 (76) occurs
between two transfer regions, one encoding surface exclusion
(54) and pilus genes (53) and the other
with oriT and four other tra genes (75). We found that fipA is transcribed
anticlockwise in pKM101 but detected no associated promoter, suggesting
that fipA may be part of an operon. This possibility seems
likely from an inspection of the upstream DNA sequence which encodes
the traHI ORF (73). This ORF is read in the same
direction as fipA, and its stop codon partially overlaps the
fipA start codon, indicating translational coupling of gene
expression (Fig. 1). If this overlap is correct, then the
pKM101
1246::Tn9 mutation (Tra+
Fip+) which demarked traH and fipA
(75, 76) probably occurs within traHI. This
indicates that neither the loss of C-terminal residues from TraHI nor
the reduction in FipA levels expected from polarity is sufficient to
alter the phenotypic effects of these proteins. The possibility that
fipA may be part of a tra operon is also interesting in light of the association between nuc and
traG (53, 55). These two operons are transcribed
convergently with nuc and fipA (which are
adjacent) as the last genes (Fig. 1). The linkage to tra
genes may account for our finding that fipA-like sequences
are common in IncN plasmids even though fipA is not required
for pKM101 transfer (76). Instead, fipA can
affect the growth of E. coli WP2 (25) but,
in our hands, only when hyperexpressed. The significance of this
finding is not known. The nuc gene, which encodes an
EDTA-resistant nuclease (55), may also be conserved, as all
IncN plasmids tested by Winans and Walker (74) elaborated
such an activity.
The pifC gene, like fipA, does not play a role in the conjugal transfer of its native plasmid but, rather, is involved in its replication (69). The two genes have similar phenotypic effects, namely, inhibition of RP1 transfer but not of other conjugal functions (Sfx or Dps [68, 76]). From the known distribution and roles of the RP1 tra genes (50), this phenotype suggested interference with one or more of the Tra1 components, i.e., the mob genes (traIJK), oriT, or traG. Given these possible targets and the nonidentity of fipA and pifC, it was surprising to find that inhibition by both genes was directed to traG. This was suggested first (Table 3) by the ability of fipA and pifC to block RP1-mediated mobilization of nonconjugative plasmids reliant on traG (RSF1010, ColE1, and pSC101 [18, 40, 70]) but not of CloDF13, which encodes a traG homolog (9, 38). The demonstration that traG is indeed the inhibition target was obtained in an artificial system in which the N-conjugal functions were supplemented with traG (discussed further below). The mobilization of RSF1010 (or pSC101) from such donors could be blocked in the presence of fipA or pifC, confirming traG as the inhibition target (Table 5).
Significantly, fipA and pifC differed in that
RSF1010 mobilization was totally blocked by fipA (a strong
inhibition gene), but this only occurred with pifC (a weaker
inhibition gene) when traG expression was low (Table 5). The
fact that inhibition by pifC could be negated by high
traG expression suggested titration of PifC, by binding to
either traG DNA, its transcript, or TraG protein.
Interaction with the DNA seemed likely as PifC is a DNA-binding protein
(11) that negatively regulates the F pif operon
(31). Indeed, this role of PifC permits its removal to be
gauged by the enhanced inhibition of phage T7 (the Pif phenotype)
(43) (see above). In our study, Pif activity increased in
donors carrying F'lac and RP1 (or cloned traG)
but only if the traG coding sequence was intact (Table 6).
If the sequence was interrupted by various insertion mutations or only
parts of traG or PtraG were present,
Pif activity was normal. Hence, it is unlikely that PifC binds to
traG DNA or mRNA unless, in the latter case, the 5' terminus is involved. We conclude instead that PifC interacts with the TraG
protein, causing enhanced Pif activity and inhibition of TraG-dependent
RP1 conjugal functions. The mechanism of fipA-mediated inhibition of traG cannot be determined from this study, but
it is unlikely to affect transcription. FipA has no detectable
DNA-binding motif, and despite strong inhibition, there is no polar
effect on the traF pilus gene downstream of traG
(i.e., RP1 donors carrying fipA are Dps+ [Table
3]). The ability of fipA and pifC to inhibit
transfer of IncP plasmids (Table 1) was not surprising, as the Tra1
regions of RP1 and R751, and particularly the traG gene
sequences, are highly conserved (78). This may account for
the similarity of the RP1 and R751 responses compared to those of R772
and pJP4, in which sequence divergence may be greater (Table 1).
The artificial conjugation system used in this study also provided information on the interplay of P, N, and other conjugal components. The PILN system (in pKM101fipA) can mobilize a MOBP plasmid (pVS659) (Table 4) under conditions where P-relaxosome formation is normal (requiring traI and traJ) but traG is hyperexpressed (using a cloned MOBP). The dependence on hyperexpression explains why the PILN system could not mobilize an RP1pil mutant (pVS766) (Table 4). In analogous experiments, the PILN (but not PILP) system has also been shown to mobilize a MOBW plasmid (6), and this too is probably due to hyperexpression of TrwB, a TraG homolog. In both cases, the homolog presumably interacted efficiently with its relaxosome, but not with the PILN DNA transport complex unless at elevated levels. It may be that the TraG homologs, in addition to a delivery/coupling role (9, 50), may also make a contribution to nucleic acid or pilin transport in the transport complex. This may be the reason why mutations affecting TraG and F TraD can alter Dps properties (18) or pilus number (2) even though these proteins, unlike the Ti VirD4 homolog (20), are dispensable for pilus synthesis (19, 24).
The PILN system with hyperexpressed traG also
mobilized RSF1010 (IncQ) and pSC101 (Table 5). Thus, TraG can interact
with different relaxosomes and efficiently couple these to the normal PILP or TraG-supplemented PILN systems.
PILW with hyperexpressed traG can also mobilize
RSF1010 (and ColE1), but the efficiencies are lower (ca.
103/donor), comparable to those obtained with R388 itself
(using trwB function) (8). The limiting step in
this case seems to be poor association of the TraG-Q-relaxosome with
the PILW transport complex. Taken together, the data
suggest that TraG and the equivalent N-system homolog, traJ
(70a), are more similar to each other than to TrwB. Such
similarities are likely to be regional, as studies of the functional
relationships (9) and predicted topology of TraG-like
proteins (9, 19, 47) suggest that interactions with
relaxosomes and transport complexes may involve different domains. This
would be consistent with the finding that some RP1pil mutants continue to mobilize nonconjugative plasmids, albeit at reduced
levels (18).
We expect that the demonstration of genes that specifically inhibit traG, one targeting the TraG protein, will provide an additional tool for investigating the complex role of TraG and its various homologs.
| |
ACKNOWLEDGMENTS |
|---|
We thank P. R. Fisher for assistance with statistical and DNA sequence analysis and the following colleagues who generously provided strains or plasmids: F. de la Cruz, H. Dean, S. T. Fong, R. M. Hall, V. Iyer, M. E. Kovach, A. J. Pittard, and R. A. Skurray.
Financial support for this work was provided by the Australian Research Council. J.M.S. was the recipient of an Australian Postgraduate Research Award.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, La Trobe University, Bundoora 3083, Australia. Phone: (613) 9479 2317. Fax: (613) 9479 1222. E-mail: vilma{at}lumi.latrobe.edu.au.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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