Structural Instability of Bifunctional Vectors in Streptomyces
The molecular mechanisms responsible for plasmid structural istability in gram-positive bacteria and the data so far published on the same problem in Streptomyces are reviewed.
The usefulness of plasmid cloning vectors depends on their long term stability, which is sometimes affected by insertion of foreign DNA. Plasmid structural instability is frequently one of the problems hampering cloning experiments in gram-positive bacteria. Several studies of deletion end--points have indicated that plasmid rearrangements and deletions in gram--positive bacteria are often the consequence of illegitimate recombination (for review see Ehrlich et al., 1986; Anderson, 1987). Plasmid rearrangements (involving deletions) occur most frequently by recombination between short homologous sequences (3–30 bp long), but also between sequences of no homology (less than 3 bp). Two models have been proposed for illegitimate recombination between short homologous sequences. The first, called slipped mispairing, involves aberrant replication of single stranded DNA in the replication fork. The second, breakage and reunion model, implies the introduction of double stranded breaks and rejoining of the molecules between the direct repeats. Topoisomerases from prokaryotes and eukaryotes may be involved in this process. According to both models, deletions, duplications and translocations may occur (Ehrlich et al., 1986). Plasmid structural instability is often pronounced in bifunctional vectors, which consist of two replicons and qualitatively different DNAs. Most often they are used to shuttle DNA between gram-positive bacteria and Escherichia coli. If the replicon belonging to a gram-positive plasmid replicates by a olling circle(RCR) mechanism, during which process single stranded molecules of plasmid DNA (ssDNA) accumulate, the structural instability is even more pronounced. The ssDNA plasmids represent an important family of replicons in gram-positive bacteria. Besides a plus origin and a replication protein (Rep), these plasmids possess a minus origin (M-0), which serves as an efficient initiation site, recognized by host factors, for the conversion of the circular plus-strand ssDNA to double-stranded DNA (dsDNA). The properties of a ssDNA plasmid in a foreign host may depend on whether the M-0 is active in the particular host. In all hosts, a plasmid lacking an active M-0 is still viable, but accumulates ssDNA. The minimal sequences of M-0 in all described cases (Gruss and Ehrlich, 1989; Deng et al., 1988) are large (at least 130–220 bp) and contain imperfect palindromic structures (Radnedge et al., 1989). The plus origin, Rep protein activity and M-0 recognition are involved in successful host adaptation (Gruss and Ehrlich, 1989). Insertion of certain DNA fragments, e.g. from E. coli, into these plasmids results in a shift in plasmid distribution from principally monomeric (for the original wild type) to principally multimeric (for hybrids) form. The formation of high molecular weight (HMW) multimers is a function of the replication of plasmids replicating via a ssDNA intermediate. It has not been observed for a plasmid which replicates as a dsDNA molecule (Gruss and Ehrlich, 1988). The HMW form could induce non-termination of plasmid replication, delayed completion of the second strand synthesis, and re-entry into the replication pool with the appearance of the deletion in the original construct (for the model see Gruss and Ehrlich, 1989).
The discovery of Streptomyces plasmid pIJ101,a multicopy plasmid with a broad host range eliciting lethal zygosis (Kieser et al., 1982), has increased the possibility of genetic engineering with respect to commercially important Streptomyces strains. This plasmid and its derivatives belong to the described group of plasmids of gram-positive bacteria, of which more than a dozen are already sequenced. They all replicate via a ssDNA intermediate. One of the most frequently used erivatives of pIJ101, either for cloning antibiotic biosynthesis genes or or construction of shuttle vectors, is pIJ702 (Katz et al., 1983).Plasmid pIJ702 is a broad host range multicopy plasmid, obtained by insertion of the mel or tyrosinase gene into pIJ350.
pIJ101 has an active second strand synthesis function (sti) (Deng et al., 1988), corresponding to M-0 of Bacillus and Staphylococcus plasmids hich replicate via a ssDNA intermediate. In many pIJ101 derivatives, like pIJ350 or pIJ702, the sti function is deleted,resulting in accumulation of ssDNA. Sequence analysis of the Bc1I-E fragment of pIJ101 (Deng et al., 1988; Radnedge et al., 1989) showed that the sti region stands out from the rest of the sequence because of its high degree of symmetry, with many sequences of 5–10 nucleotides occurring as direct or inverted repeats. The stability of this region of secondary structure and the presence of the GAGCGT sequence make sti act as the main initiation signal for the conversion of ss to ds forms of the plasmid (Radnedge et al., 1989). Replication of the leading strand (5’–3’) proceeds anti-clockwise. The circular ss intermediates (Schrempf, personal communication) are converted to ds forms by a rate-limiting reaction, resulting in a copy number of c. 100.The addition of sti to the basic replicon provides an efficient signal for the initiation of the synthesis of the second (lagging) strand and leads to an increase in the number of ds copies of plasmids such as pIJ2743. The copy number of pIJ101 and its derivatives is influenced by sti and by an additional trans-acting function, cop, (Deng et al., 1988). It has been shown (Gruss and Ehrlich, 1989) that the plasmids with homologous plus origins also have corresponding homologies in their Rep proteins. A published sequence (Kendall and Cohen, 1988) of pIJ101 revealed a similar amino acid motif in the Rep protein, despite the high (72%) G + C content of the Streptomyces plasmid (as opposed to 30 to 40% G + C for Bacilli and Staphylococci). Thus far, homologies in the Rep proteins have been found to include only the ssDNA-accumulating plasmids (Gruss and Ehrlich, 1989). Classification of plasmids according to their mode of replication should prove useful in the construction of cloning vectors.
One of the first examples of intraplasmid recombination in Streptomyces has been reported by Nakano et al. (1984). The authors have observed a spontaneous tandem duplication of 900 bp in plasmid pSL1 in S. lavendulae. The spontaneous duplications in the S. lavendulae plasmid have been attributed to recombination between short (5 bp) direct repeats.
Some plasmid vectors become unstable in Streptomyces only after insertion of heterologous DNA. To study intraplasmid recombination in Streptomyces using convenient and well-defined starting material, the shuttle vector, pIJ132, containing a pair of direct repeats of the mel gene from pIJ702, was constructed. The construct was much more stable in E. coli than in S. lividans, in which homologous recombination between the repeats produced a single product, pIF138 (Chen et al., 1987).
Lee et al. (1986) used E. coli plasmid pUC12 alone or in conjunction with the hepatitis B viral surface antigen gene, and cloned them into the vector pIJ702. No S. lividans transformants stably maintaining the entire hybrid plasmids pWCL1 and pWTS2 were found. In each case deletions of the hybrid plasmids were observed. Radnedge et al. (1989) also constructed a shuttle vector based on E. coli pUC8 and Streptomyces plasmid pIJ702 to study the expression of the beta-lactamase gene in Streptomyces. Both orientations of pUC8 inserted at the BglII site of pIJ702 were unstable in S. lividans.
Chen et al. (1987) attributed particular instability to the BglII--PstI sequence in pIJ702, which obviously played an important role in the instability of the above-mentioned shuttle vectors in S. lividans. Consistent with this hypothesis was the observation of these authors that spontaneous deletions, in the mel sequence usually had one end-point between the BglII and PstI sites (Chen et al., 1987).
As against the unstable pIJ702 shuttle vectors, several examples of constructs stable in Streptomyces were reported. Neesen and Volckaert (1989) described the construction of a new shuttle vector by cloning a small artificial E. coli replicon pGV462 into pIJ702 at SphI and SstI, which resulted in excision of 430 bp from the region preceding the tyrosinase (mel) gene of pIJ702. The shuttle vector pSKNO1 was stable in Streptomyces.
Radnedge et al. (1989) described the stable replication of plasmid pQR1 constructed from PstI digests of pBR325 and pIJ702. Jensen et al. (1989) reported the formation of the stable shuttle vector pSH obtained by ligating pIJ702 to pUC119 at the PstI site. The plasmid was used for successful cloning of the S. clavuligerus isopenicillin N synthase (IPNS) gene (Jensen et al., 1989).
Shuttle vectors pZG3.1 and pZG3.2 (communicated at this Symposium), as well as their ClaI-BclI deleted derivatives, were constructed by insertion of pBR328 into the PstI site of pIJ350, in both orientations. All plasmids extracted from transformant colonies less than 15 days old were completely stable in S. lividans and S. rimosus R6. However, deletions in all constructs were observed in some older colonies and upon subculture.
Other examples reported by us were the shuttle vectors pZG5 and pZG6. pIJ350 was linearized at its unique PstI site and ligated to PstI-cut Bluescribe M13− or pUC18, respectively. Both vectors proved to be stable regardless of colony age or multiple subculture. Moreover, they were used for successful retransformation of three tRNA genes into S. rimosus.
However, when pBR322 was used for construction of shuttle vectors, they usually exhibited structural instability in Streptomyces (Schottel et al., 1981; Wohlleben et al., 1986). Upon transformation of S. lividans and S. rimosus with a simple shuttle vector pZG1, a high level of instability was detected (Pigac et al., 1988). The process of plasmid rearrangements and deletions occurred gradually, correlating with colony age. It was not unusual to find several plasmids with different deletions coexisting in the mycelium of a single transformant colony. Being aware that pIJ350 and pZG1 accumulate ssDNA and lack sti function, the shuttle vectors pZG4.1 and pZG4.2 were constructed (communicated at this Symposium). They were obtained by partial digestion of pIJ303 with PstI and ligation with PstI-linearized pBR322. pZG4.1 and pZG4.2 have pBR322 inserted at PstI(35) and PstI(13) of pIJ303, respectively. The presence of sti function and non-detectable amounts of ssDNA in the lysates of pIJ303 extracted from S. lividans (Schrempf and Pigac, 1986) could contribute to a reduction in the amount of ssDNA and consequently to deletions in the shuttle vectors. However, both vectors were rather unstable in Streptomyces.
On the contrary, other shuttle vectors based on pIJ101, but in combination with other E. coli replicons, proved to be stable in Streptomyces. Shareck et al. (1984) constructed pFSH102 by inserting E. coli plasmid pSAS1206, carrying a sulfonamide-resistance gene, into pIJ101 at PstI site. After initial deletions in both pSAS1206 and pIJ101 (c.4.6 kb), the remaining plasmid still possessing sti function replicated stably in Streptomyces, fully expressing sulfonamide resistance.
Stable replication in Streptomyces was also achieved with similar constructs based on pPFZ12, an in vivo deleted derivative of pIJ303 still carrying sti function, and pBR325 to create the bifunctional vector pPFZ54, which was used for construction of a streptomycete cosmid, pPFZ74 (Chambers and Hunter, 1984).
pIJ101 and its deleted derivatives are similar to the ssDNA family of plasmids in Bacilli and Staphylococci (Gruss and Ehrlich, 1989). Their mode of replication (RCR) via ssDNA, the high homology of the Rep proteins, and the sti function corresponding to M-0 stimulate homologous and illegitimate recombination, and consequently rearrangements and deletions of the parental plasmids. The reported data also indicate that the E. coli part of the vector, as well as the site of the insertion of E. coli replicon into the streptomycete plasmid may affect the structural stability of the constructed shuttle vectors in Streptomyces.
An example of host influence on stabilization was observed when the pZG1 shuttle vector, being highly unstable in S. lividans, became almost completely stable in S. rimosus R6 on persistent subsequent etransformation, indicating that some minor changes in pZG1 did occur during propagation of the recombinant plasmid in S. rimosus R6 (Pigac et al., 1988).
Yet, some Streptomyces - E. coli shuttle vectors enabled the successful expression of homologous and heterologous genes in Streptomyces. In our earlier report (Pigac et al., 1988) we concluded that plasmid structural instability in Streptomyces is a sequential process comprising the change in plasmid structure and the replacement of the parental plasmid by the newly formed one. It is obviously correlated with transformant colony age, revealing the presence of the completely intact shuttle vectors in transformant colonies up to 10 days old, which could make them useful in cloning experiments.
At the moment the data can be discussed only in terms of experience, since there has been no systematic study of plasmid structural instability nor explanation of its mechanism in Streptomyces. Therefore we hope that this mini-review, revealing the state of knowledge on this subject, could be useful in further attempts to construct new shuttle vectors or to find new approaches to explain the mechanisms of this phenomenon in these commercially important bacteria.
KeywordsShuttle Vector Structural Instability Illegitimate Recombination PstI Site Parental Plasmid
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