INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Enteroinvasive strains of shigella species and Escherichia coli contain related large plasmids which encode many of the factors required for virulence (11). The large virulence plasmid pMYSH6000 of Shigella flexneri contains a stability element (Stb) which can promote stable inheritance of plasmids carrying the replication region of pMYSH6000, or the P1 replicon in E. coli (11, 17). A 1.1-kb fragment of the plasmid was sufficient to promote plasmid stability in the absence of other pMYSH6000 sequences. It did so without any apparent increase in the plasmid copy number (12). The fragment contains three open reading frames (17). The 240-codon trbH open reading frame is a homolog of the trbH gene of the F plasmid of E. coli, a gene of unknown function that resides in the conjugal transfer region (12). The product of the pMYSH6000 trbH open reading frame could be interrupted by a nonsense mutation without impairing function, suggesting that a TrbH product is not needed for plasmid stability (17). The two other open reading frames, STBORF1 (75 codons) and STBORF2 (133 codons) lie in the opposite orientation and completely overlap trbH. Homologous open reading frame pairs are found in F trbH, in the chromosomes of certain pathogenic bacteria (17), and in plasmids of the pathogenic organisms Salmonella dublin and Dichelobacter nodosus (4, 15, 17). The STBORF1 and STBORF2 open reading frames show little similarity to any known plasmid stability element, but their general organization resembles that of postsegregational killing systems. Such systems encode a toxin and an unstable antidote. When the plasmid is lost from the cell, the antidote decays but the toxin persists, eventually killing most of the progeny of the plasmid-free cell. This promotes the maintenance of the plasmid in the growing population (5, 20). Here, we show evidence that the Stb element does encode a postsegregational killing (PSK) system and that the STBORF1 and STBORF2 open reading frames produce an antidote and toxin protein, respectively. We propose to name the genes mvpA and mvpT (for maintenance of virulence plasmid) and the protein products MvpA and MvpT.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Media, chemicals, and DNA manipulations. Media, reagents, enzymes, buffers, and chemicals used were as previously described (1). Enzymes were obtained from New England Biolabs (Beverly, Mass.) and Boehringer Mannheim (Indianapolis, Ind.), and used under conditions recommended by the manufacturers. Concentrations of antibiotics were as follows: ampicillin, 100 µg/ml; chloramphenicol, 10 µg/ml; and spectinomycin, 10 µg/ml. Cloning methods were as previously described (19), with strain BR2846 used for plasmid growth and transformation for DNA manipulations.

Map coordinates. All map coordinates are given in base pairs and refer to the 1,127-bp sequence of the mvp (originally Stb) region (17).

Bacterial strains and plasmids. The bacterial strains BR825 trp polA::Tn10 (13), BR2846 supE44 hsdR17 recA1 endA1 gyrA96 thi relA lacU169, a derivative of DH5 (8), and W3110 (2) were derived from E. coli K-12.

292-309

294-396

Measurement of plasmid maintenance stability. The ability of various inserts to stabilize the plasmid pALA136 when it is replicating as a mini-P1 plasmid at low copy number in strain BR825 polA was measured as previously described (17).

Measurement of plasmid copy number. The copy numbers of pALA136 and its mvp derivatives were measured in Luria-Bertani (LB) broth at 30°C as previously described (17). This method compares the yield of plasmid DNA to that of the mini-P1 plasmid -P1:5RCm in a fixed quantity of cells that is mixed with the test sample prior to cell lysis and DNA extraction.

Measurement of transformation and survival frequencies. Plasmid DNA of pALA2546 mvpA mvpT⁺ was isolated from strain CC4451 which produces MvpA from the bacterial chromosome (see Results). Competent W3110 cells carrying the relevant resident pHGB2-based plasmid were prepared after growth at 30°C in the presence of spectinomycin (10 µg/ml). The cells were then transformed by the incoming plasmid to chloramphenicol resistance and the transformation frequency was determined by plating on agar with chloramphenicol and spectinomycin at 30°C. Pure lines of the transformants were grown in the presence of chloramphenicol and spectinomycin to an optical density at 600 nm (OD₆₀₀) of 0.1, and dilutions were plated on prewarmed LB agar at 30 and 42°C to determine survival under conditions where the resident plasmid is lost.

Measurement of cell killing on loss of plasmids carrying the mvp genes. Strain W3110 carrying the relevant pHGB2-based plasmid was grown overnight at 30°C in LB broth with spectinomycin. The culture was diluted 100-fold in the same medium and grown to an OD₆₀₀ of 0.1. An aliquot was withdrawn, and the number of viable cells was determined by plating on LB agar with and without spectinomycin. The culture was diluted 50-fold into LB broth at 35 or 42°C, and exponential growth was continued for approximately 400 min. Periodically, the culture was diluted with prewarmed LB broth in order to keep the OD₆₀₀ between 0.01 and 0.1. At the time points indicated, aliquots were withdrawn and the number of viable cells was determined by plating on LB agar with and without spectinomycin at 30°C. In some experiments, a second plasmid based on pALA136 was present. In these cases, the overnight cultures contained chloramphenicol in addition to spectinomycin. As these second plasmids contain the origin of plasmid pBR322, they are maintained as multiple copies in strain W3110 and are not lost from any of the cells during the course of the experiment (data not shown).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Deletion analysis of the 1,127-bp mvp region. A series of deletions were constructed in the 1,127-bp mvp fragment present in the mini-P1 plasmid pALA2515 (Fig. 1). This plasmid relies on the function of the mvp region for its proper maintenance when present in a polA strain where the plasmid replicates at a low copy number under P1 control (17). Deletion analysis shows that a 714-bp region is necessary and sufficient to promote stable plasmid maintenance (pALA2529) (Fig. 1). This sequence includes the two short open reading frames, mvpA and mvpT, and a putative promoter region immediately upstream of them.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Physical map of the mvp region showing phenotypes of deletion derivatives. Shaded bars indicate regions present in the numbered plasmids. Arrows indicate the trbH and mvp open reading frames. Asterisks mark the positions of nonsense mutations in pALA1589 (TAG in mvpT) and pALA1585 (TGA in mvpA). Two brackets under the mvpA open reading frame () mark the extents of the deleted bases in the 6- and 61-codon in-frame deletions in pALA2534 and pALA2546, respectively. In stability tests (STAB.), a plus denotes >80% retention and a minus denotes <5% retention of plasmid in 25 generations of unselected growth. In incompatibility tests (INC.), a plus denotes >90% loss and a minus denotes <5% loss of resident plasmid pCS1367 (mvp+) in ca. 25 generations after the introduction of the incoming plasmids listed on the left.

The 714-bp mvp fragment is very effective in promoting stable plasmid maintenance. The stability conferred by the minimal 714-bp mvp fragment was consistently better than that conferred by its 1,127-bp progenitor (Table 1). Loss of the mini-P1 plasmid with the smaller fragment was undetectable, even after 100 generations of unselected growth, showing that the mvp system is highly efficient in promoting the maintenance of this construct. The mvp system proved more effective in stabilizing the mini-P1 plasmid construct in this assay than did the P1 par system (Table 1). P1 par promotes the active partition of daughter plasmids to daughter cells and is the system primarily responsible for the accurate maintenance of the P1 plasmid and its stable miniplasmid derivatives (14). As was the case with its 1,127-bp progenitor (12), the 714-bp mvp fragment confers stability without any apparent increase in plasmid copy number. The average copy numbers of the 714-bp derivative (pALA2529) and the pALA136 vector were not significantly different at 7.8 and 6.4 per cell, respectively, grown in LB broth at 30°C (see Materials and Methods).

The phenotypes of mutations in mvpA and mvpT. A nonsense mutation (amber) at base 465 in mvpT abolished the stability phenotype (pALA1589) (Table 1). The amber mutation was designed such that it does not alter the coding of the trbH gene as it substitutes one leucine codon (CTT) for another (CTA). We conclude that the mvpT gene and likely its protein product are essential for the stability phenotype. The fact that truncation of a large carboxy-terminal segment of mvpT (pALA2527) (Fig. 1) also abolishes stability is consistent with this conclusion.

Cells expressing MvpT die when MvpA expression is blocked. Plasmid pALA2547 expresses MvpA from an insert in the temperature-sensitive plasmid pHGB2. Cells containing it could be transformed by pALA2546 mvpA mvpT⁺ at 30°C, albeit at a somewhat reduced efficiency (Table 2). However, when the temperature was raised to 42°C in the presence of chloramphenicol so that the pALA2547 was lost from the cells but pALA2546 was retained, most of the cells died (Table 2). This confirms that mvpT expression in the absence of mvpA is lethal.

The mvpA gene acts as an incompatibility determinant against mvp-promoted plasmid stability. The mini-pMYSH6000 plasmid pCS1367 is stably maintained at low copy number in a polA mutant strain due to the presence of the active mvp region (17). When a second plasmid containing mvp is introduced, pCS1367 becomes unstable (17). We mapped the mvp sequences responsible for exerting this incompatibility effect to the intact mvpA gene (Fig. 1). If mvpA produces an antidote, cells containing an extra copy of this gene would be immune to PSK when a plasmid carrying the mvp system is lost, thus explaining the incompatibility effect.

PSK by the mvp system. Killing or growth inhibition by MvpT in the absence of MvpA suggests that mvp is a PSK system with MvpT as the toxin and MvpA as the antidote. We introduced the 714-bp mvp region into the temperature-sensitive replicon pHGB2 and studied the fate of cells when plasmid replication was blocked at 42°C, so that the plasmid (pALA2530) could not be maintained. The block to plasmid replication appeared to shut off cell growth after a short lag. The result was a static population of viable cells, virtually all of which retain the plasmid (Fig. 2A). This result is consistent with highly efficient PSK (5). When the temperature of the culture is raised, the pHGB2 mvp⁺ plasmid (pALA2530) stops replicating and is presumably diluted from the growing cells until the copy number approaches a value of 1 per cell. Subsequent cell divisions give rise to plasmid-free cells, virtually all of which die.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Cell killing at 42°C upon loss of a temperature-sensitive plasmid with the 714-bp mvp region. Cultures were shifted from 30 to 42°C at time zero. Viable counts were normalized to a value of ca. 1 at time zero. Open symbols, unselected viable counts; solid symbols, counts on spectinomycin-selective agar. (A) Squares, pHGB2; triangles, pALA2530 (714-bp mvp region). (B) Triangles, pALA2530 in cells containing the vector pALA136; circles, pALA2530 in cells containing pALA2527 (mvpA+). The data are from one of several experiments which gave similar results.

Lack of PSK when the mvp genes are in their normal immediate context. Surprisingly, the 1,127-bp stb fragment does not work in the PSK assay. Loss of the plasmid carrying it at 42°C had no measurable effect on the growth of the cells and a viable plasmid-free population was generated (Fig. 3). This lack of activity is not due to the temperature sensitivity of the mvp system or to an inadvertent mutation of sequences within the fragment (data not shown). Rather, it appears to be an extreme manifestation of the inhibitory context of the mvp genes when surrounded by the naturally occurring sequences. Removal of either of the sequences from the ends of the fragment that bracket the mvp genes restored most or all of the PSK in this assay (Fig. 3).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Lack of cell killing by the 1,127-bp mvp fragment is caused by bases surrounding the mvp genes. Cultures were shifted from 30 to 42°C at time zero. Viable counts were normalized to a value of ca. 1 at time zero. Open symbols, unselected viable counts; solid symbols, counts on spectinomycin-selective agar. (A) Squares, pHGB2; circles, pALA2511 (1,127-bp mvp region). (B) Plasmids derived from pALA2511 with the flanking regions deleted from upstream of the mvp genes (pALA2542) (triangles) or downstream of the mvp genes (pALA2543) (diamonds) are shown. The data are from one of several experiments which gave similar results.

Stabilization of a plasmid under conditions where DNA replication is compromised. At 35°C, replication of pHGB2 is not shut off but is marginal. Some replication occurs, as shown by a significant and continuous increase in the number of plasmid-containing cells, but plasmid loss is frequent (Fig. 4). When the plasmid contained the 714-bp mvp fragment (pALA2530), the plasmid was substantially stabilized at 35°C (Fig. 4). However, not all of this added stability was due to the killing of plasmid-free cells. Otherwise, the growth curve of plasmid-containing cells (and the total cells) should mimic that of plasmid-containing cells in the pHGB2 vector control (Fig. 4). The additional stability does not appear to be due to the function of the mvp genes; a control fragment containing unrelated sequences also conferred some stability on pHGB2 at 35°C (pALA2518) (Fig. 4). This control fragment had no effect on the fate of the cell or the plasmid when replication was completely blocked at 42°C (data not shown). It appears that the insertion of nonspecific (or relatively common) sequences into pHGB2 can aid the stability of the plasmid but only when replication is marginal. The additional stability seen with the mvp fragment at 35°C is probably due to this nonspecific effect. Note that the 1,127-bp mvp fragment (pALA2511) also confers stability at 35°C (Fig. 4), despite the fact that, like the control fragment in pALA2518, it is inactive in PSK (Fig. 2). This stabilizing effect is insensitive to the incompatibility exerted by a second plasmid expressing MvpA (data not shown), again suggesting that it is a nonspecific effect. Maintenance of the pSC101 replicon employed in pHGB2 is sensitive to the degree of negative supercoiling induced by DNA gyrase (3). Perhaps the sequences that improve pHGB2 plasmid stability at 35°C contain gyrase binding sites that increase negative supercoiling.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   The fate of cells carrying a temperature-sensitive mvp plasmid at 35°C. Cultures were shifted from 30 to 35°C at time zero. Viable counts were normalized to a value of ca. 1 at time zero. Open symbols, unselected viable counts; solid symbols, counts on spectinomycin selective agar. (A) Squares, pHGB2; circles, pALA2530 (714-bp mvp region). (B) Triangles, pALA2518 (control insert); diamonds, pALA2511 (1,127-bp mvp fragment). The data are from one of several experiments which gave similar results.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The properties of mutants of the mvp genes suggest that it is a PSK system, with MvpA as the antidote and MvpT as the toxin. Confirming this conclusion, we found that, when the replication of a plasmid containing the minimal mvp region was blocked, the number of viable cells stopped increasing and only plasmid-containing cells survived.

When the mvp system is embedded in an 1,127-bp fragment that includes surrounding sequences from the original pMYSH6000 plasmid, the system fails to kill cells when plasmid replication is blocked. Thus, the natural context of the system appears to inhibit function. The inhibitory sequences also appear to attenuate the ability to stabilize a mini-P1 plasmid. Removal of either the excess upstream or downstream sequences restores full mvp activity in either assay. The fact that extensions at both ends of the mvp region are required for this inhibitory effect makes it unlikely that some specific gene product is involved. For example, it is unlikely that mvp parallels the case of the pas system of plasmid pTF-FC2, where an additional 71-codon open reading frame (pasC) downstream of the antidote toxin genes attenuates pas activity (18). Rather, it seems that the naturally occurring sequences surrounding mvp have some fortuitous effect on gene expression which increases antidote levels or decreases toxin levels, rendering the system less effective at killing. Perhaps this contextual effect occurs in E. coli but not in the natural host, S. flexneri. In addition, it appears that the effect of context extends even to the vector sequences surrounding the 1,127-bp fragment. Thus, the mvp genes in the 1,127-bp fragment function in the mini-P1 vector to stabilize it (Table 1) but show no signs of cell killing in pHGB2 (Fig. 3). Our interpretation of this is that the genes function in one vector but not in another. This suggests that it is not just the immediate sequence context that can attenuate mvp function but rather some general property of the DNA region, such as supercoiling density or subcellular localization.

The minimal mvp system confers a high degree of stability on an unstable mini-P1 plasmid. The plasmid pALA136 is normally lost at a rate of ca. 5% per generation under the assay conditions, whereas no loss was seen during unselected growth of the minimal mvp derivative during many independent experiments. The apparent efficiency and small size of this element suggest that it may be useful for stabilizing plasmid vectors in practical applications.

The mvp locus is a member of a family of similar elements in a variety of disease-causing organisms. All examples so far described are linked to virulence genes on plasmids or host chromosomes (17). Perhaps mvp and its relatives play important roles in the maintenance of virulence in these organisms by efficiently killing cells which fortuitously lose blocks of virulence genes due to their deletion from the chromosome or loss of a plasmid which carries them.