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Previous Article | Next Article 
Journal of Bacteriology, May 2002, p. 2447-2454, Vol. 184, No. 9
0021-9193/02/$04.00+0 DOI: 10.1128/JB.184.9.2447-2454.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
and Stuart Austin*
National Cancer Institute-Frederick, Frederick, Maryland 21702-1201
Received 6 November 2001/ Accepted 16 February 2002
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ABSTRACT |
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INTRODUCTION |
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The ParA protein is an ATPase (8), which interacts specifically with the parS-ParB assembly (28) but only forms a stable complex with the other par components in vitro when continually charged with ATP (3, 5). Its ATPase activity, which is essential for partition, is stimulated by interaction with ParB and nonspecific DNA in vitro (7). ParA does not appear to contact parS directly (7). It was suggested that ParA is not a permanent part of a partition complex but interacts transiently with ParB during partition to promote some essential aspect of the process (8).
A mutant ParA protein, ParAM314I, acts to block plasmid maintenance (28). Unlike typical Par mutations, parAM314I completely prevents propagation of the plasmid under nonpermissive conditions. The mutant protein presumably acts by blocking replication or by preventing segregation of the plasmid even by random diffusion such that one daughter cell retains all the plasmid copies (28). It was suggested that this mutation stabilizes the ParA interaction with the ParB-parS complex such that ParAM314I becomes locked into the complex, preventing some essential dissociation of the plasmid copies from each other, or from some host attachment site during partition (28).
The partition sites of the P1 and F plasmids have been associated with a silencing activity (17, 24). Silencing inhibits the expression of genes linked to, but often some distance from, the partition site. It occurs when P1 ParB, or the equivalent F SopB protein, is supplied to its cognate partition site. Silencing did not require the ParA component. It may reflect the formation of an extended coating of the DNA by ParB that is nucleated by binding to the partition site (24). Alternatively, it may be due to the nonspecific binding of ParB to a region of DNA surrounding the partition site promoted by binding of parS to some host structure containing multiple ParB molecules (17). In either case, the ParB-parS interaction promotes spreading of ParB binding to an extended region adjacent to parS.
Plasmid partition sites can be regarded as analogs of centromeres of eukaryotic chromosomes. For budding yeast, Doheny et al. (10) followed the formation of a protein complex at the centromere by placing it between the promoter and the open reading frame for a reporter gene. The formation of a complex was inferred by a block to the transcription of the gene, and mutations which relieved this block identified genes contributing to the formation of the complex (10). Here, we employ a similar approach to probe the formation of a complex at P1 parS and to select mutants that impose or relieve a transcription block.
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MATERIALS AND METHODS |
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Escherichia coli strains.
Strain BR6903, described as "construct I" by Rodionov et al. (24), has a parS site integrated in the chromosome approximately 1 kb upstream of the test locus consisting of a constitutive plasmid P1 repA promoter followed by the lacZ open reading frame (24). Approximately 1 kb downstream from lacZ is the cat (chloramphenicol acetyltransferase) gene with its own promoter. This strain was a gift from Michael Yarmolinsky. We transduced BR6903 to recA+ by using a linked marker, cysC3152::Tn10kan, to create CC4253 (Fig. 1). CC4248 contains the "transcription block" construct, and its construction is detailed below. CC4250 is CC4248 with the cat gene and its promoter inserted 
200 bp downstream of lacZ in the 
phage sequences (Fig. 1). This strain was made using Red-mediated linear recombination by a modification to the protocols described by Yu et al. (30) to be described elsewhere. WJW26 and WJW45, gifts from Helen Wilson, are W3110 with 
(lacI-Z)M445 and (lacIZYA-argF)U169, respectively. ZH1142, a gift from Jian-guang Zhou, is W3110 (lacIZYA-argF)U169 gal490 [N::lacZ imm (cro-bio)] rnc14. CC4249 is CC4248 with ihfA82::Tn10. LE30 is mutD5 strR aziR gal. CC2056 was used in the colony color partition assay as previously described (22).
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Phage BDC531 (29) was used to pick up the transcription block construct from pALA2302 by using bla and lacZ homology. This phage stock was used to lysogenize strain WJW45 to make CC4243. P1 lysates were made on CC4243 and used to transduce the transcription block construct into ZH1142 by using the technique described by Yu and Court (29), selecting ApR (10 µg/ml) at 37°C to generate CC4245. The transcription block construct was then transduced into WJW26 (selecting ApR) to generate CC4248. The construct present at the phage lambda attachment site of this strain is shown in Fig. 1. Note that this construct contains only about 200 bp of phage DNA near attL and attR. The DNA sequence of the construct in CC4248, including the PlacUV5 promoter, parS and the start of lacZ, was determined and was found to be correct.
Vectors. Plasmids pBR322 and pGB2 were as previously described (2). Plasmid pALA1858 consists of the PstI fragment of pSP102 (20), containing the Cm resistance gene, inserted into the PstI site of pBR322, thus inactivating the Ap resistance gene.
Plasmids. The plasmids used in this study that contained the P1 par locus or modified versions of it are shown in Table 1. All par genes in this study were expressed using the constitutive promoter found in pALA1570 (8). In pALA1570, the constitutively expressed par operon is present on an EcoRI-BamHI fragment inserted between the same sites of pBR322. Likewise, mutant versions of par operons were inserted into pBR322 as HindIII-BamHI fragments. Cm resistance versions of these plasmids were made by cloning the PstI fragment of pSP102 into the PstI site of each plasmid.
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Plasmids that contained a large in-frame deletion of parA were made by cutting with XhoI and SacII and inserting an oligonucleotide consisting of the annealed single-stranded sequences, 5'TCGAGCTGCCGC and 5'GGCAGC. This deletion of parA maintains the parA stop codon and leaves the parB gene intact. To construct a large deletion of parB, the relevant plasmid was cut with BglII and XbaI and an oligonucleotide consisting of the annealed single-stranded sequences 5'GATCTAACTGATAT and 5'CTAGATATCAGTTA was inserted. This restored the 3' end and stop codon of the parA gene, produced a complete deletion of the parB open reading frame, and introduced an EcoRV site.
Plasmids -P1:5RCm and -P1:5RKm are P1- hybrid constructs that can be grown lytically as phage or they can lysogenize E. coli as a low-copy-number plasmid under P1 replication control (26). They confer Cm and Km resistance, respectively. Unless noted otherwise, the plasmids were stably maintained, as they contained the par region.
The parS test plasmid was -P1:5R1005::pALA1952, which contains the parS site but no par genes and can be monitored for segregation by the colony color assay (22). This plasmid also contains the cat gene.
Isolation of mutations. Mutations were isolated as follows. Strain LE30 (mutD) was transformed with either pALA2306 or pALA2308. Plasmid DNA was extracted from the transformants by using Wizard Plus midi-preps (Promega, Madison, Wis.). These mutagenized plasmid preparations were used to transform CC4248 to Sp resistance on mac-lac-Sp plates.
Plasmid pALA2306 was mutagenized to isolate mutants that have a tighter transcription block to lacZ expression (white colonies on mac-lac plates). Cells transformed by mutagenized pALA2306 produced white colonies at a frequency of 2 x 10-4. Plasmid pALA2308 was mutagenized to generate mutations that relieve the parAM314I-dependent transcription block to lacZ expression (yielding red colonies on mac-lac plates). Red colonies from cells containing mutagenized pALA2308 were isolated at a frequency of 10-3. Candidate mutants were first struck on mac-lac-Sp plates to confirm their color. Mini-preps were then made and used to retransform CC4248 to confirm that the new phenotype was linked to the plasmid.
Estimating levels of par operon expression. The levels of par operon expression from plasmids producing wild-type Par proteins were estimated by measuring the steady-state levels of ParA protein in cells grown in L broth.
Strain WJW26 was transformed with plasmid pALA2306, pALA1855, pALA2319, pALA1570, or pALA2310. A 50-ml culture was grown to an optical density at 600 nm (OD600) of 0.4 at 37°C in L broth supplemented with the appropriate antibiotic. A -P1:5RKm lysogen of WJW26, grown to an OD600 of 0.4 at 30°C in 50 ml of L broth supplemented with 20 µg of kanamycin/ml, was used as a reference sample. The cells were harvested by centrifugation at 5,000 x g for 15 min at 4°C. After the cells were washed with 50 ml of 25 mM Tris-HCl (pH 8.0), 1% (wt/vol) glucose, 0.1 mM EDTA, and 20 mM dithiothreitol (DTT), the pellets were resuspended in 2.5 ml of B-PER Bacterial Protein Extraction Reagent (Pierce, Rockford, Ill.) supplemented with 20 mM DTT.
The cells were lysed by incubating the resuspended pellets for 30 min at room temperature with 250 µg of lysozyme/ml and 10 U of Omnicleave Endonuclease (Epicentre Technologies, Madison, Wis.) per 1 OD600 of cell culture. The resulting lysate was cleared by centrifugation at 27,000 x g for 30 min at 4°C. The supernatant was desalted by chromatography on an 8.5-ml Sephadex G-25 column equilibrated with 200 mM ammonium sulfate, 40 mM HEPES (pH 7.5), 0.1 mM EDTA, 10 mM DTT, and 15% (vol/vol) glycerol. The lysates were stored at -70°C for further use.
The proteins were concentrated by precipitation with trichloroacetic acid and separated on a 12% (wt/vol) polyacrylamide mini gel. Western blottings were done as previously described (4). The blots were probed with rabbit polyclonal anti-ParA antibody and developed using the ECL Western blotting Analysis System according to the supplied protocol (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.). ParA protein of known concentration was used as a quantitative standard. By comparison of the ParA band intensity with that of a series of dilutions of the ParA protein standard, the weight of ParA protein per milliliter of culture was determined. Knowing the number of cells in the culture, the number of ParA proteins per cell was calculated.
Cat assays. Chloramphenicol acetyltransferase assays were carried out using the Fast Cat Yellow kit from Molecular Probes (Eugene, Oreg.) by following the manufacturer's protocols. After thin-layer chromatography, the spots were quantitated using a Molecular Dynamics Typhoon 8600 analyzer by using its accompanying software.
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RESULTS |
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attachment site. In addition, the P1 parS site was inserted between the promoter, PlacUV5, and the lacZ gene (Fig. 1). The strain formed red colonies on lactose indicator plates. Thus, the parS site itself does not block transcription from proceeding from the lac promoter into the lacZ gene. Assays of ß-galactosidase (Table 2) showed that this modified lacZ operon was repressed by the LacIq repressor and was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). The fully induced level (ca. 300 units) was below that normally achieved by the wild-type lacZ gene in its normal context (ca. 1,500 Miller units, data not shown). This may reflect the altered context of the lac promoter or the properties of the mutant LacIq repressor. However, part of the reduction appears to be due to IHF protein binding to the parS site, because introduction of an ihfA mutation into strain CC4248 caused lacZ expression to increase by some 50% (Table 2).
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parS site in CC4248 appears to be functional. As supplying the wild-type Par proteins had little effect on lacZ transcription, the possibility existed that the parS site was nonfunctional or somehow inaccessible to binding proteins in this construct. To test this, we determined whether the parS site in the construct would exert incompatibility against an incoming plasmid containing the P1 Par system. Partition-mediated incompatibility prevents the stable establishment of replicon utilizing a par system when the cell already contains a copy of that system (1). This is due to the ability of the resident parS site to compete with the incoming replicon for binding of the par proteins or for binding to some cellular component required for partition (19).
Strain CC4248 was first lysogenized with the P1 mini-plasmid -P1:5RCm (Par+). The mini-P1 lysogens were then grown nonselectively for approximately 25 generations and retention of the plasmid was scored. In CC4248, 44% of the cells contained the -P1:5R plasmid after nonselective growth. In an isogenic control strain without the parS construct in the chromosome, 97% of the cells maintained the plasmid. We conclude the parS site in CC4248 is functional, at least as defined by this competition assay.
Mutant Par protein imposes efficient transcription block. The transcription block tests were repeated using a mutant version of the constitutive par operon present on plasmid pALA2312. Plasmid pALA2312 has a point mutation in parA and produces the mutant ParA protein ParAM314I. With this plasmid, the reporter strain formed white colonies on lactose indicator medium, and the induced levels of ß-galactosidase were reduced some 12-fold relative to the level achieved with the wild-type Par proteins (Table 2). The observed effect was dependent both on the parAM314I mutation and on the presence of the wild-type ParB protein: a derivative of pALA2312, pALA2316, which has a large in-frame deletion in parB did not impose the block (Table 2), and plasmid pALA1895, which contains an in-frame deletion of the parA gene removing the region containing the parAM314I mutation, largely relieved the transcription block, confirming that the mutant parA gene is required (Table 2). Thus, the mutant ParAM314I protein, in conjunction with wild-type ParB, can form a protein complex capable of blocking transcription from passing from the lac promoter into lacZ. The block was not due to overproduction of the proteins from the mutant plasmid, as all plasmids tested utilize the same constitutive promoter (28).
Block is dependent on level of Par protein synthesis.
Cells containing -P1:5RKm, a mini-P1 plasmid with a wild-type, autoregulated par operon, contained about 1,000 molecules of ParA per cell. We defined this as the 1x level of par operon expression (see Materials and Methods). Cells containing pALA2310, the pBR322-based plasmid with a constitutively expressed wild-type par operon, showed a 10x level of expression. Alternative constructs with the same constitutive promoter expressed the parA protein at 20x (pALA2306) and 0.5x (pALA1855) (see Materials and Methods). Table 3 shows that the 10x and 20x levels of par expression were sufficient to impose the transcription block with the parAM314I allele but that the 0.5x level was not. The equivalent plasmids carrying the wild-type par genes failed to exert a comparable transcription block (Table 3) even at the highest level of expression (20x).
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1 x 10-3. Samples of these were isolated and purified, and the plasmid DNA was extracted from them. Four of these plasmid isolates were analyzed by DNA sequencing. Each of the four had the original parAM314I mutation plus an additional single mutation in the par operon. Three different mutations were found in parA and one was found in parB (Fig. 2). One parA mutation, parAQ12STOP, and the parBM1I start codon change are likely to be null mutations. This is consistent with the conclusion that both the mutant ParA protein and the wild-type ParB protein are required for a transcription block.
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2 x 10-4). White colonies were purified and the plasmid DNA was extracted. The XhoI-SacII fragment, which includes the bulk of the parA gene (Fig. 2), was excised from each mutant candidate and inserted into a plasmid containing a par operon from which the XhoI-SacII fragment had been deleted (see Materials and Methods). The resulting plasmids from five of the candidate mutants were introduced into strain CC4248, and the colonies were white on mac-lac plates. Thus, the block to ß-galactosidase activity was due to a mutation within the XhoI-SacII region of parA. This was confirmed by DNA sequencing. Each mutant proved to have a single base change from the wild-type sequence (Fig. 2). Two isolates failed to cause a block when the XhoI-SacII region was transferred, suggesting that they had a mutation outside this interval. Sequencing of the original mutant isolates showed that one had a single base change in parA, parAA14V. The other had a single base change in parB, parBT12P (Fig. 2). Of the seven mutants that were sequenced, six different mutations were represented, five in parA and one in parB. The parA mutation, parAD209G, was isolated twice from different mutagenized plasmid preparations. The ß-galactosidase levels of CC4248 containing these mutant par alleles are shown in Table 4.
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Mutant proteins are partition defective and yield propagation-defective phenotype.
The mutant par operons that impose a transcription block were excised from their original contexts and inserted into a pBR322 vector. The intact par operon under constitutive control was restored in these constructs with the relevant parA mutation in place (10x level of expression). Strains containing these plasmids were then used in the colony color partition assay (22). Cells carrying the control plasmid with a wild-type parAB operon were readily lysogenized with the parS test plasmid (-P1:5R1005::pALA1952) by selecting for chloramphenicol resistance. Once established, the test plasmid was efficiently maintained in these cells without selection (Table 5). The test plasmid was also established readily in cells containing the pBR322 vector, but the parS test plasmid was rapidly lost without selection. Cells expressing the various mutant par operons, however, behaved differently from either of these cases. With the majority of the mutants, it was not possible to introduce the parS test plasmid even with selection into cells expressing the mutant operons (Table 5). In the two exceptional cases, small colonies were obtained under selection after 48 h of incubation. The parS plasmid was extremely unstable in these cells (Table 5). The very slow growth of the test colonies on medium selective for the parS plasmid presumably reflects this rapid plasmid loss. We conclude that the six new par mutant operons resemble those of the parAM314I mutant in not only being unable to support the partition of a parS-containing plasmid but preventing even the establishment of the plasmid within the cell (28). This ParPD (propagation-defective) phenotype may indicate that a complex at parS interferes with the plasmid DNA replication or that the plasmid copies are unable to dissociate from each other and always remain in one of the daughter cells at cell division (28).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wild-type ParA-ParB-parS complex does not participate in an efficient block and effects lacZ expression only slightly when ParA and ParB are present at high levels. We have previously suggested that wild-type ParA participates in a complex at parS only transiently during the cell cycle and that the ParAM314I mutant protein is blocked at some stage in the partition process that locks it permanently into the complex (28). Our current observations are consistent with this and provide further evidence for the existence and nature of this "locked" complex. The complex clearly requires both ParA and ParB proteins to be present in a complex bound to parS. The requirement for ParA for the transcription block is most clearly shown in the case of the parBT12P mutant, where an in-frame deletion within ParA diminishes the transcription block.
In order for the ParA-ParB-parS complex to block transcription, one of the proteins needs to contain a parPD mutation. Three of the seven effective mutations lie within regions known to be involved in a ParA-ParB interaction. Two of these are in the carboxy-terminal region of ParA, and one is on the amino terminus of ParB (Fig. 2). We suggest that these changes stabilize the ParA-ParB interaction, preventing the proteins from dissociating from each other and from the parS site at a critical step in the partition process. We are presently investigating the binding properties of the mutant proteins to see if they promote formation of a stable ParA-ParB-parS complex in vitro.
Regional silencing occurs in the transcription block strain, but only under conditions similar to the transcription block itself: it requires ParA, ParB, and a parPD mutation. We suggest that the properties of the parS site can be affected by the surrounding DNA. In the Rodionov et al. configuration (Fig. 1), the parS site can act as a loading site for wild-type ParB in the absence of ParA. This presumably allows ParB to spread to the adjacent DNA to promote silencing. It is not clear whether a stable complex is formed on the parS sequence itself under these conditions. In contrast, ParB alone has very little effect on parS or the surrounding DNA in the transcription block configuration as constructed here. The parS site does not act as an efficient loading site for ParB alone, and only a modest effect is seen on lacZ or cat expression. However, in the presence of both ParA and ParB and when one of these proteins contains a parPD mutation, a stable complex that blocks transcription through the site is formed at parS. This stable complex can now act as a loading site for ParB to spread to the adjacent DNA so that partial silencing of distant genes can occur.
Two general principles are implied. First, ParA in conjunction with ParB is capable of forming a specific stable complex at parS when one of the proteins is distorted by mutation. These mutations likely enhance an interaction that wild-type ParA normally exhibits; i.e., they stabilize the ParA interaction with ParB and/or the partition site and thereby prevent transcription from traversing parS. Second, regional silencing by ParB alone or by wild-type ParA and ParB at physiological concentrations is not a universal phenomenon. Which context results in a parS site with properties that most resemble the active site in the actively partitioning plasmid? It is unclear. However, genes in the vicinity of the parS site cannot be constantly silenced in P1 or mini-P1 plasmids that are properly partitioned. Otherwise, we could not follow the markers they carry and they could not replicate due to the requirement for expression of the adjacent rep gene. Some plasmids containing parS do appear to be subject to silencing. In these cases, the site doesn't function for plasmid partition and the plasmid is rapidly lost when the Par proteins are present (16). Thus, the properties of our integrated parS site may be closer to that of the functional plasmid parS than that present in the Rodionov et al. (24) silencing strain.
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ACKNOWLEDGMENTS |
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We thank Oleg Rodionov, Helen Wilson, Michael Yarmolinsky, and Jian-guang Zhou for providing strains and details of the silencing construct found in CC4253. We thank Don Court for critically reading the manuscript.
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FOOTNOTES |
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Present address: The Institute for Genomic Research, Rockville, MD 20850.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-P1:5R and -P1:5L. J. Bacteriol. 153:800-812.
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