TEXT

Top Abstract Text References

Proteic plasmid stabilization systems have been discovered on a number of plasmids and include the ccd system of plasmid F (7), the identical parD/pem and kis/kid systems of plasmids R1 and R100 (1, 16, 19), the parDE system of plasmids RP4/ RK2 (14) and the phd/doc system of phage P1 (10). These systems consist of a long-lived toxin which is expressed at low levels and a short-lived, highly expressed antidote (8). On cell division, if a progeny cell fails to inherit the plasmid, it loses the ability to make the shorter-lived and more abundantly produced antidote and is unable to counter the toxic effects of the poison. As a result, plasmid-free cells are killed or their cell division is inhibited, depending on the type of poison-antidote system.

The 12.2-kb mobilizable, broad-host-range plasmid pTF-FC2 (GenBank accession nos. M64981 and M35249 [13]) was originally isolated from Thiobacillus ferrooxidans. This natural hybrid plasmid has a replicon clearly related to those of the IncQ plasmids (e.g., R1162 and RSF1010) (4) and a mobilization region with low but clear similarity to those of the IncP plasmids (e.g., R751 and RK2/R68/RP4) (15). Situated within the IncQ-like replicon and between the repB and repA genes (Fig. 1) is a proteic poison-antidote system named pas (for plasmid addiction system). This system is unusual in that it consists of three genes; pasA encodes an antidote, pasB encodes a toxin (which is bacteriocidal rather than bacteriostatic), and pasC encodes a protein that appears to enhance the neutralizing effect of the antidote (17).

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Layout of the pTF-FC2 pas showing its location within the plasmid replicon. The positions of the genes are relative to the ClaI site of pTF-FC2 (5).

Efficiency of the pas stability system in Escherichia coli is strain dependent. The ability of the pTF-FC2 pasABC system to stabilize a heterologous plasmid in E. coli JM105 had previously been shown (17) by cloning the pasABC genes into the unstable, low-copy-number, test plasmid pOU82 (6). We repeated the stability assays in E. coli CSH50 to compare the efficiency of the pTF-FC2 pas to those of other poison-antidote systems in a host strain background identical to that used by other workers (9). It was observed that pas varied in its ability to act as a plasmid stabilization system depending upon which strain (Table 1) was used as host. Plasmid stability was determined by growing plasmid-containing E. coli cells in batch culture for 100 generations without selection in TB (24 g of yeast extract, 12 g of tryptone, and 4 ml of glycerol per 900 ml with 100 ml of sterile 0.17 M KH₂PO₄-0.72 M K₂HPO₄ added immediately before use). Aliquots were taken at 20-generation intervals and grown at 37°C overnight in the absence of selection. One hundred colonies were transferred to Luria agar (LA) plates with plasmid selection (ampicillin [100 µg ml¹], chloramphenicol [30 µg ml¹], or kanamycin [50 µg ml¹], as required), and the percentage survival was used to calculate plasmid loss. The stability assay for pOU82 and derivatives was performed as described above, except that aliquots were plated on LA containing 40 µg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) per ml. Plasmid-containing cells form blue colonies, whereas plasmid-free cells are white. At least three stability tests were performed for each strain, and the loss frequency was calculated by the method of Gerdes et al. (6). After 100 generations, a test plasmid containing the pasABC genes (pOU-pasABC [Table 1]) was stabilized approximately 2.5-fold in an E. coli JM105 host (Table 2). However, in an E. coli CSH50 host, the pas enhanced plasmid stability about 100-fold. In contrast, the pas was ineffective in enhancing the stability of the test plasmid in an E. coli JM107 host strain or its recA derivative, E. coli JM109. Surprisingly, the base level of pOU82 stability in E. coli JM109 was 10-fold higher than in strain JM107 (Table 2). The fact that E. coli strains JM107 and JM109 are isogenic except for the recA gene implies that the recA system has an effect on the stability of the pOU82 test plasmid. The reason for this increased stability is unknown, but the finding is similar to the finding that mini-RK2 plasmids were threefold more stable in E. coli JM109 than in JM107 (14).

2

3

Pas toxicity varies between E. coli host strains. To investigate the reason for host-strain dependent variation in stability, combinations of the pas genes were cloned behind the tac promoter of a pKK223-3 vector (Table 1). In lacI^q strains containing these constructs, tac-controlled expression of the pas genes is induced by isopropyl--D-thiogalactopyranoside (IPTG). Since E. coli CSH50 does not contain lacI^q, the F' lacI^q-containing episome was transferred from E. coli 71/18 to CSH50 by conjugation. Conjugation was carried out overnight on the surface of a LA plate followed by plating on minimal medium plus streptomycin (100 µg/ml) to select for streptomycin resistance and proline independence. The stability of the test plasmid in this E. coli CSH50-I^q strain was indistinguishable from that of the test plasmid in CSH50. The effect of IPTG-induced pas gene expression on the growth of E. coli host strains JM105, JM107, JM109, and CSH50-I^q is shown in Fig. 2. Not all of the strains were equally sensitive to the PasB toxin. Growth of strain CSH50-I^q was the most severely inhibited by induction of pTac-pasB, with JM105 less inhibited and strains JM107 and JM109 the least inhibited. Expression of the pasA gene (encoding the antidote) relieved the toxic effect of pasB, although in E. coli strain CSH50-I^q, growth inhibition was relieved only slightly. When all three pasABC genes were expressed, the growth rates of all strains increased further, although in no strain did the growth rate reach that of the vector control. IPTG-induced expression of the pasABC system was toxic to all strains, but toxicity was most severe in E. coli CSH50-I^q, which was also the strain in which the pas plasmid stabilization system was most effective.

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Growth curves of E. coli strains overexpressing the pas genes. Each graph shows pKK223-3 (control) (), pTac-pasB (), pTac-pasAB (), and pTac-pasABC (). Datum points are the means of three separate experiments. OD 600, optical density at 600 nm.

Role of Lon protease in plasmid stabilization. Plasmid proteic stabilization systems operate on the principle that the antidote is subject to a much higher rate of turnover than the toxin (8). A pOU82-pKG339-based conditional replication system (9) was used to determine which E. coli protease is involved in the selective degradation of the PasA antidote. Since one of the E. coli protease mutants was resistant to tetracycline (Table 1), the tetracycline resistance marker and pSC101 replicon of pKG339 were replaced by the chloramphenicol resistance marker and the p15a replicon of pACYC184 to produce plasmid pKGCm. When the copA gene of pKGCm is provided in trans to pOU82, replication of pOU82 can be halted by IPTG induction of the pA1/O4 promoter (Fig. 3). Therefore, addition of IPTG to E. coli (pOU-pasABC) cells will result in arrest of plasmid replication, and plasmid-free cells containing the toxin-antidote complex will result. The protease responsible for selective degradation of the antidote will digest the antidote, which will no longer inhibit the toxin, resulting in cell death. Functional antidote will persist in E. coli mutants deficient in the antidote-degrading protease, and cell death will not occur. Plasmid pOU82 and the pas-containing pOU82-based plasmid (pOU-pasABC) were transformed into E. coli SG22093 clpP1 and SG22095 lon mutants and into E. coli SG22025, a protease-proficient parental strain of these mutants, each of which contained a coresident pKGCm plasmid. The growth of all strains following the addition of 2 mM IPTG is shown in Fig. 4. Growth of the E. coli (pOU-pasABC) lon mutant strain was similar to that of the same strain containing pOU82 (Fig. 4A), suggesting that in this strain the antidote was long-lived. In contrast, growth of the E. coli (pOU-pasABC) clpP mutant and E. coli (pOU-pasABC) protease-proficient strains was reduced relative to that of the same strains containing the pOU82 control plasmid (Fig. 4B and C). The observation that growth was not reduced by forced plasmid loss in an E. coli lon mutant, whereas there was a decrease in growth following the loss of plasmids expressing the pasABC genes in lon-proficient strains, suggests that the antidote protein was stable in the lon mutant and that the Lon protease is involved in the degradation of the PasA antidote.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   pOU82-pKGCm conditional replication system based on the pOU82-pKG339 system (modified from reference 9 with permission of the publisher). The pACYC replicon and chloramphenicol resistance markers of pKGCm have replaced the pSC101 replicon and tetracycline resistance marker of pKG339. Addition of IPTG results in expression of copA from the pA1/O4 promoter, and CopA inhibits replication of the pOU82 R1 replicon. Restriction site abbreviations: B, BamHI; E, EcoRI; H3, HindIII; Sp, SphI; P1, PstI; Sa, SalI; X, XhaI; Sm, SmaI; Ss, SspI.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Growth curves of protease mutants with different plasmids. Growth curves of E. coli lon mutant SG22095 (A), E. coli clpP1 mutant SG22093 (B), and E. coli protease mutant parent strain SG22025 (C) containing pOU82 and pKGCm (control) () or pOU-pasABC and pKGCm () are shown. OD 600, optical density at 600 nm.

2

2

2