INTRODUCTION

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IncP plasmids transfer between and replicate in most gram-negative bacterial species (36). They are very stably maintained under a range of environmental conditions. A key part of their genetic organization is what we have termed the "central control operon" which coordinates the expression of genes for replication, stable inheritance, and transfer (Fig. 1; see also review in reference 28). Two of the genes in this operon, incC and korB, encode proteins which are related to the products of the parA (incC) and parB (korB) gene families (25), respectively, found on many other plasmids and near the replication origins of most bacterial chromosomes sequenced to date. These proteins play a vital role in the active partitioning of bacterial plasmids (40), and recent studies of their chromosomal homologues in Bacillus subtilis (8, 20, 30) and Caulobacter crescentus (22) suggest that they are important for a process which ensures proper genome segregation during both vegetative growth and differentiation.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Summary of the organization of the RK2 genome, the central control region, and the global circuits that control expression of the replication and transfer genes. Gene designations are as follows: kle, genes that are coregulated by the central control region and are implicated in stable inheritance; oriV, vegetative replication origin; trfA, gene encoding the activator of oriV; tra and trb, blocks of transfer genes; oriT, origin of transfer replication. korA, korB, and incC1/2 are as defined in the text. Replication functions are shown as solid black, transfer functions as diagonal hatching, partitioning and control regions as horizontal and vertical hatching, auxilliary maintenance functions as light gray, and phenotypic markers and transposable elements as dark gray. korA, korB, and incC are shown in the central control region, with incC1 and korA overlapping. General transcriptional organization of the replication, transfer, and stable inheritance functions are shown by arrows above and below the lines. The location of the KorB binding sites are shown as black filled circles, which are labelled in order starting with OB1 associated with the korA promoter. OB class refers to location very close to a promoter (I), up to 190 bp away from a promoter (II) or more than 1 kb from a promoter. The global regulatory circuits provided by KorB and KorA are indicated by the lines and arrowheads above the diagram.

The central control regions of RK2 and R751, archetypes of the IncP and IncP subfamilies, express an active partitioning phenotype, showing better than random segregation when joined to an unstable low-copy-number plasmid (21, 25). This phenotype depends on both incC and korB. KorB (358 amino acids [aa] [18, 32]) is a global regulator that binds to 12 operators, O_B1 to O_B12, on the plasmid genome (Fig. 1) (3, 41). For RK2 we have shown that the cis-acting sequence necessary for the active partitioning phenotype involves O_B3, one of the three korB operators in or close to the central control region (42). IncC (364 aa) belongs to the ParA family of proteins, which share an ATP binding motif (25). ParA of P1 and SopA of F have been shown to have ATPase activity (4, 39) and are DNA binding proteins with an autoregulatory function (1, 7, 24). The ATPase activity is necessary for DNA binding and autoregulation (4). However, by replacing the normal parA promoter in the P1 system with a weak constitutive promoter it was possible to demonstrate that the ATPase activity is also necessary for partitioning (5). Stimulation of this ATPase activity by ParB indicates an interaction between ParA and ParB. However, the relationship between the members of the ParA-ParB pairs does not appear to be consistent. Studies of the chromosomal homologues suggest that they are not equally necessary for genome inheritance (22), and there is no reported direct evidence of interactions between them.

There are a number of points which justify the study of the incC-korB gene pair in addition to the well-characterized P1 and F systems. First, examination of the aligned polypeptide sequences shows that of all the known plasmid-encoded ParA and ParB homologues, those of the IncP families (IncC and KorB) have the highest sequence similarity to the chromosomal members of these families. Second, KorB is clearly not just a partitioning protein since it plays a key role in global regulation on the IncP genome (26, 34, 37). A third interesting feature of these genes is that incC contains two translational start codons (35). The first start codon yields IncC1, which is 364 aa in length, with an approximately 100-aa N-terminal region like ParA of P1 and SopA of F. The second start codon gives IncC2, which is 259 aa and starts just before the first ATP binding motif as found for some plasmid- and all chromosomally encoded homologues studied to date. Significantly, we found that IncC2 is sufficient for the active partitioning phenotype of the central control region in both RK2 (42) and the related IncP-1 plasmid R751 (21). However, before the involvement of incC in partitioning was discovered we had made various observations concerning its activities. Initially, we implicated incC in the temperature-sensitive phenotype of a mini-IncP-1 plasmid with a defect in korA which resulted in derepression of the korA-incC operon (33); the temperature-sensitive phenotype could be suppressed by a deletion in incC. korB was not present in this plasmid. We subsequently found that the effect of the cloned wild-type korA-incC region in displacing a resident IncP-1 plasmid in an incC-dependent manner was dependent on the presence of korB, suggesting that incC normally works through korB or affects a process that is regulated by korB (34). However, these conclusions were complicated by the overlap of korA and incC: the 101-codon open reading frame (ORF) for korA starts 11 bp downstream of the incC1 start codon and is thus included entirely within the incC1 ORF (34). The korA stop codon overlaps the start codon for incC2. Therefore, the fragment containing incC always also produced the transcriptional repressor KorA, which acts on its own promoter (korAp) as well as on the promoter for the replication operon, trfAp.

To remove this complication, we have generated a site-directed mutant which no longer produces KorA but which still expresses IncC normally. We have used this to examine the effects of incC on the stable inheritance of RK2 and on transcriptional repression by korB. In addition, we have used purified IncC1 to study KorB binding to DNA. The results show that incC1 modulates the action of korB both in vivo and in vitro, providing evidence for direct interaction between these proteins and indicating that IncC adds another layer to the global regulation circuits that control and coordinate the majority of the genes of this plasmid system.

	MATERIALS AND METHODS

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Bacterial strains and growth. The Escherichia coli strains used were the K-12 strains C600K (thr-1 leu-6 thi-1 lacY1 supE44 ton21 galK) and BL21 [F ompT hsdS_B(r_B m_B) gal dcm] (phage DE3) (Novagen, Inc.). Bacteria were generally grown in L broth (17) at 37°C or on L agar (L broth with 1.5% [wt/vol] agar) supplemented with the following antibiotics as appropriate: benzyl penicillin, sodium salt (100 µg ml¹ in liquid medium and 300 µg ml¹ in agar plates) or ampicillin (100 µg ml¹) for penicillin resistance, kanamycin sulfate (50 µg ml¹) for kanamycin resistance, tetracycline hydrochloride (20 µg ml¹) for tetracycline resistance, and streptomycin sulfate (50 µg ml¹) for streptomycin resistance.

Plasmids. Descriptions of the standard vectors can be found in Sambrook et al. (29). RK2 (60 kb; Pn^r Km^r Tc^r) and pUB307 (53 kb; Km^r Tc^r) are natural isolates of IncP-1 plasmids which have been described previously (10, 28). The constructed plasmids more specific to this work are listed in Table 1. All tacp expression plasmids also carry the lacI^q gene to allow regulated expression controlled by the addition of IPTG (isopropyl--D-thiogalactopyranoside). Details of the construction of the plasmids not previously described are given below.

Plasmid DNA isolation, analysis, cloning, and manipulation of DNA. Plasmid DNA was isolated by standard procedures (29). Large-scale plasmid purification was carried out by using alkaline sodium dodecyl sulfate (SDS), followed by CsCl-ethidium bromide density gradient centrifugation. Digestion of plasmid DNA with restriction enzymes was carried out under the conditions recommended by the suppliers on 0.8 to 2.0% (wt/vol) agarose gels. These and all other methods were carried out by standard protocols (29). DNA sequencing was performed by AltaBioscience by the dye-terminator method with an ABI 373 automated DNA sequencer. Sequences were aligned and analyzed with programs of the Wisconsin package (6). Standard PCRs (27) were performed as described previously (12). Fragments were amplified on a pCT690 (14) template for RK2. All PCR-derived clones were analyzed by DNA sequencing for verification. DNA PCR-amplified fragments were labelled with terminal transferase and [-³²P]ddATP. Restriction fragments were isolated from the agarose gel, purified by using GeneClean extraction (Bio 101), and radioactively labelled with [-³²P]ATP and T4 polynucleotide kinase.

Determination of XylE activity. Catechol 2,3-oxygenase (the product of xylE) activity was assayed in logarithmically growing cells (optical density at 600 nm of 0.6) as described by Zukowski et al. (43). The protein concentration was determined by the biuret method (9).

Purification of His₆-tailed polypeptide. Exponentially growing C600K(pCT800) (ptac-his₆incC1) was induced with 0.5 mM IPTG at a cell density of approximately 4 × 10⁷ CFU/ml, grown for 4 h with shaking at 37°C, harvested by centrifugation, and then purified as recommended by Qiagen for soluble native proteins on Ni-agarose columns with an imidazole gradient in phosphate buffer at pH 6. IncC1 eluted between 0.15 and 0.20 M imidazole. Polypeptides during purification were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) by using the Pharmacia PHAST gel system.

Analysis of protein-DNA interactions by gel retardation and DNase I footprinting. For gel retardation experiments, radioactive fragments were incubated either with purified RK2 KorB (41) or with wild-type His₆IncC1 and then separated by PAGE under conditions described previously (14). DNase I footprinting was performed as outlined earlier (14). After a standard binding reaction at 37°C for 30 min, an equal volume of 2× DNase I buffer plus DNase I was added, and the mixture was incubated at room temperature for 1 min before the addition of stop buffer, precipitation, washing, and loading onto 6% urea-6% PAGE gels.

	RESULTS

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incC alone can efficiently displace a resident IncP plasmid. We previously showed that incC is necessary for the central control region of IncP plasmids to be able to displace a resident IncP plasmid when introduced in trans on a compatible replicon. To determine whether incC alone is sufficient to express incompatibility, we amplified the incC gene by using a primer which changed the korA start codon (internal to incC) from ATG to ACG (see Materials and Methods) and inserted the fragment downstream of tacp (pGBT3; Table 1). The sequence change in incC (CAT to CAC) caused no alteration in the amino acid sequence since both triplets code for histidine. We determined that the induction of transcription from tacp did not result in the expression of any repressor active directly against a korA-regulated promoter by using xylE reporter fusions with trfAp and trbAp (data not shown). This result confirmed that we had inactivated korA. Induction with IPTG gave the expected products of incC: an IncC1 of ca. 39,000 M_r and an IncC2 of ca. 28,000 M_r. We also subcloned this incC gene into pDM1.1, which has the same tacp expression system controlled by lacI^q but is based on a lower-copy-number Sm^r IncQ replicon, resulting in pOLE1 (Table 1). This allowed us to select for the incC expression plasmid in the presence of RK2, which confers resistance to kanamycin, penicillin, and tetracycline. After the introduction of both pDM1.1 (vector alone) and pOLE1 into a strain with RK2, we induced transcription from tacp by the addition of different levels of IPTG and monitored the loss of RK2 over a period of 20 generations in the absence of selection for RK2. Table 2 shows that while pDM1.1 had no effect on RK2, a major loss of RK2 occurred when pOLE1 was present after the addition of 0.1 and 1.0 mM IPTG. Since pOLE1 should produce both IncC1 and IncC2, it was pertinent to ask whether incC2 alone was sufficient to mediate the displacement. Plasmid pGBT302 has incC2 under the control of tacp. Production of IncC2 was confirmed by SDS-PAGE analysis of the bacterial extracts after standard induction by IPTG (1 mM). The production of active IncC2 was also confirmed by the ability to complement an incC mutant derivative of an active partitioning test plasmid that is unstable due to its loss of the incC gene (data not shown). To increase the chances of seeing even a weak incompatibility, we used pGBT302 directly rather than subcloning into a lower-copy-number PolA-independent IncQ vector. However, this meant that we had to use pUB307 as the test Ap^s plasmid to be displaced. Comparison of the effects of the high-copy-number plasmids pGBT30 (no RK2 DNA), pGBT36 (incC1/2), and pGBT302 (incC2) showed that pGBT36 caused dramatic displacement of pUB307 (<0.01% survival after 20 generations), while no effect was found for pGBT30 or pGB302. We concluded that the incompatibility towards effectively complete IncP-1 plasmids is associated with incC1.

IncC modulates the effect of KorB on the inheritance of the mini-IncP-1 plasmid pKK1. Our previous results suggested that the incompatibility of incC is dependent on the presence of korB. Since further analysis of the interplay of incC and korB depended on the controlled production of IncC and KorB in the same bacterial strain, it was first necessary to determine whether it was feasible to express both genes simultaneously. We therefore studied the growth of E. coli C600K carrying only the compatible vectors (pGBT30 and pDM1.1), a combination of the vector and an overproducing plasmid for single proteins (pGBT30 and pDM1.12; pDM1.1 and pGBT36), and combinations of the two compatible plasmids overproducing KorB and IncC (pDM1.12 and pGBT36, respectively). This analysis showed that when korB was induced with IPTG at concentrations of 0.2 mM, there was growth retardation and that the additional presence of induced incC allowed this effect to be seen at 0.1 mM IPTG, although incC alone had no effect. The estimation of CFU on L agar and microscopic examination of the cultures indicated that the effect of KorB on apparent culture density was due to clumping. However, when incC was also present with korB the number of CFU per milliliter was reduced up to 10-fold when the IPTG concentration was 0.2 mM, and there appeared to be a 60 to 70% loss of both plasmids from the bacteria in the culture. Consequently, only IPTG concentrations up to 0.1 or 0.2 mM were used for routine assays.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Loss of resident mini-IncP plasmid pKK1 (Tcr) indicated as log the ratio of tetracycline-resistant bacteria to total (tetracycline-resistant plus tetracycline-sensitive) bacteria when grown in the presence of tacp-korB (pDM1.12) without or with tacp-incC (pGBT36) induced with increasing concentrations of IPTG. Controls lacking a regulatory gene were provided by pDM1.1 (Smr IncQ vector control for pDM1.12) or pGBT30 (Pnr pMB1-based vector control for pGBT36). Only in the presence of korB is any loss seen, and the effect of incC is to relieve the korB effect at 0.1 mM IPTG but to potentiate this effect at higher concentrations.

Enhancement of KorB repression by IncC1 protein. Since the apparent IncC counteraction of the KorB effect on pKK1 was unexpected, we used a second reporter system based on transcriptional fusions to the xylE gene. KorB regulates the expression of seven promoters in the RK2 genome by binding to six O_Bs (binding of KorB to O_B10 in the divergent trfAp/trbAp region simultaneously downregulates both promoters [15]). In relation to three of the KorB-repressed promoter regions (trfAp, korAp, and klaAp), O_B is located immediately upstream of the 35 sequence (proximal position), while in relation to the other four (trbAp, trbBp, kfrAp, and kcrAp) KorB acts at a distance (O_B is located 80 to 180 bp from the tsp). We chose a representative selection of transcriptional fusions between RK2 promoters and the xylE cassette to determine the effect of IncC on KorB-mediated repression. The presence of incC (induced from tacp-incC in pGBT36) in the absence of KorB had no effect on gene expression (data not shown), but it potentiated the effect of KorB repression (expressed from tacp-korB in pDM1.12) with all of the promoters tested (Table 3) and therefore is independent of the position of O_B (proximal or distal to promoter sequences). We also carried out the experiment with a range of IPTG concentrations. The results, as presented for trbBp in Fig. 3, show that the stimulation of the korB effect by incC is seen over the whole range of IPTG concentrations tested. We also determined the effect of overproducing IncC2 alone (pGBT302) on the repression by KorB (pDM1.12). The results showed no effect of IncC2 on repression by KorB, suggesting that the N-terminal 105 aa are necessary for the modulation of KorB repressor function by IncC1.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Effect of IncC on repression of trbBp by KorB. XylE was assayed as described in Materials and Methods and as presented in Table 3 except that the level of IPTG was varied as shown. The plasmid combinations were as in Table 3, with the addition that pGBT36 was included with pGBT30 so as to have a strain with IncC but without KorB. The controls with neither KorB nor IncC (circles) or just IncC (triangles) were assayed only either without IPTG or at the highest IPTG concentration. KorB alone and KorB plus IncC are indicated by diamonds and squares, respectively. The log scale is used to make the difference between the latter two lines clearer.

IncC potentiates in vitro binding of KorB to DNA. The above in vivo experiments raised the possibility that IncC directly modulates the binding of KorB to its target DNA. To initiate studies on the effect of IncC in vitro, we produced soluble cell extracts from bacteria with pGBT30 (vector alone) or pGBT36 (vector with tacp-incC) after induction with IPTG and then determined their effect on the retardation of a radioactively labelled DNA fragment with the trfAp/trbAp region whose in vivo activity was described above. Increasing concentrations of KorB, purified as previously described (41), were titrated with a range of concentrations of these extracts. It was clear that while the extracts of bacteria carrying the expression vector without IncC had no effect on the mobility of the labelled fragment, the presence of IncC in cell extracts after the induction of bacteria with pGBT36 promoted retardation when KorB was present, even at concentrations of KorB which showed negligible retardation on their own (data not shown). These results were important since they indicate that native IncC with no modifications at either end does modulate KorB DNA binding activity.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Effect of purified IncC on the ability of KorB to retard radioactively labelled fragments with the trfAp and korAp regions. For trfAp/trbAp, the 300-bp region previously described (13) was amplified by PCR and then labelled with [-32P]ddATP and terminal transferase. For korAp PCR, primers 5'-GGGATCCTCCTGAACTGGCTTTCGG-3' (RK2 coordinates 59306 to 59325) and 5'-GGGAATTCTTGTTGGGCTGGCAGTGTCG-3' (RK2 coordinates 59578 to 59597) were used to amplify a 300-bp fragment, which was labelled with [-32P]ddATP and terminal transferase. The extra non-RK2 bases correspond to the restriction sites added to allow subcloning of the products, and the extra bases were added to allow efficient cutting by BamHI and EcoRI. The fragments were incubated under conditions described previously (13) with the combinations of IncC and KorB indicated. IncC was present at 150 nM when it was added. KorB was added at 2.5, 7.5, 25, and 75 nM (the highest concentration was applied to korAp only).

View larger version (63K): [in this window] [in a new window]   FIG. 5.   Comparison of the effect of KorB on the retardation of radioactively labelled fragments kfrAp and trfAp regions without or with IncC. For trfAp, the PCR fragment described in the legend to Fig. 4 was used, whereas for kfrAp the 550-bp PpuMI fragment from pDM300 was labelled and recut with BsmAI, which generates two fragments of approximately 300 and 250 bp. KorB was added at 250 nM. IncC was added at 150 nM.

View larger version (79K): [in this window] [in a new window]   FIG. 6.   Effect of purified IncC on KorB DNase I footprints at trfAp region. Single end-labelled fragments carrying OB10 were incubated in 20-µl volumes with increasing concentrations of KorB, either without or with IncC as described in Materials and Methods. After DNase I treatment and separation of the fragments on a urea-6% PAGE gel, the image was visualized with a phosphorimager. The concentrations of KorB used were 7.5, 25, and 75 nM. IncC was used at 150 nM in all of the samples indicated prior to the addition of DNase I.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have extended our knowledge in this study of the in vivo biological activity associated with the incC gene, and we have shown that purified IncC1 can modulate KorB binding at a representative range of its operators in vitro. This helps to explain its identification as an incompatibility determinant and suggests that it adds an additional level of global regulation to that provided by KorB, which binds to 12 sites on the RK2 genome. Other members of the ParA proteins which share sequence motifs with IncC have been shown to be ATPases (4, 39). Therefore, it is possible that IncC provides a response to the energetic or physiological state of the bacteria and that this is relayed to the KorB regulon. This could be important in controlling expression of the plasmid replication functions as its bacterial host makes the transition from exponential growth to stationary phase and back again.

The function of some ParA and ParB protein pairs, such as those of F and P1, appear to be limited to partitioning, although the ParA protein in these systems does act in an autoregulatory capacity (2, 11, 40). On the other hand some ParB homologues are known exclusively as regulators, for example, as repressors of plasmid-encoded virulence genes (38), where they appear to work without a ParA homologue. The IncC-KorB pair clearly provides more than just active partitioning in the stable inheritance of IncP plasmids, so the IncP system may bridge these two extremes of function. Therefore, the study of IncC-KorB function in the context of the IncP plasmids is likely to provide deeper insights into the biological activities of the ParA-ParB families.

It may be significant that the modulation of korB activity by incC seems to be associated with IncC1 not IncC2. IncC1 has an N-terminal region of approximately 100 aa like ParA of P1 and SopA of F, which are associated with autoregulation (7, 23, 35). While IncC alone does not show any DNA binding activity in vitro or any regulation of gene expression on its own in our assays, it seems likely that this part of the protein is required for its role in modulating gene expression. Our studies on the partitioning activity of incC indicate that IncC2 is sufficient for partitioning activity (21, 42). Therefore this ability to modulate KorB transcriptional repressor activity is not necessary for the partitioning process. This dissection of activities may be of considerable significance, since all of the chromosomal homologues of IncC and some of the plasmid-encoded homologues lack this N-terminal region prior to the ATP binding motif.

It was also significant that in the plasmid displacement assay with low levels of induction, the expression of incC actually appeared to reduce the negative activity of korB. A possible trivial explanation for this is that incC expression reduces effective KorB levels either directly by affecting the availability and/or stability of KorB or indirectly by affecting the expression of korB. We do not think that these explanations are likely because with the same combination of expression plasmids in the promoter-xylE fusion system at similar IPTG concentrations we observed that IncC potentiated repression by KorB. This rules out the possibility that KorB levels have gone down or that KorB is sequestered into a less-active form. An alternative hypothesis is that at low levels IncC and KorB can form a complex on the DNA which counterbalances its inhibitory effect on transcription, possibly by giving better-than-random segregation due to provision of an active partitioning activity. This would be very interesting because it would suggest that O_B10 could simultaneously participate in the regulation of trfA gene expression and active partitioning, a situation which would have an attractive symmetry to it since trfA is required for replication. While recent studies with a heterologous replicon and the central control region (42) indicate that O_B3 has centromere-like activity, it is possible that a number of individual O_Bs could have this activity if present alone or in different contexts. Our current work on the sequence requirements for the cis-acting sequences which are necessary for centromere-like function should establish whether this explanation is plausible.

In studying the effect of IncC1 on binding of KorB, we made a number of novel observations. First, we found that there are significant differences in the affinity of KorB for different operators, suggesting a hierarchy of binding sites. These differences were not noticed when studies were first performed with purified KorB (3, 41), although reexamination of the data presented in those earlier studies indicates that our observations here do not contradict those studies. It will be important therefore to test all of the other operators to define their relative affinities and thus determine which ones are likely to be occupied first as the concentration of KorB rises in the cell. Second, we observed the formation of higher-order complexes of KorB-DNA at both O_B1 (korAp) and O_B10 (trfAp). Such complexes were not observed previously (3, 41), probably because of gel conditions. The absence of such complexes with O_B2 appears to correlate with its lower affinity for KorB. DNase I footprinting indicated that the complexes are not the result of coating the DNA, since the footprint remains sharply focused around O_B10. The nature of these higher-order complexes will be addressed in a separate paper. Third, we observed that IncC favored the formation of all observed KorB-DNA complexes, even those at O_B2 where it does not form higher-order complexes. Since IncC itself does not cause any retardation of the fragments studied and does not show any change in the KorB DNase I footprint, it seems unlikely that IncC is making contact with an extended segment of DNA, although we cannot rule out small yet significant contacts. Even if these contacts do occur, the data suggest very strongly that IncC acts through KorB. We propose that IncC interacts with KorB and stabilizes the primary KorB-DNA complexes. The molecular details of these interactions will be explored in future work.