Maintaining Plasmid Stability By The Hok/sok Post-Segregational Killing System

Plasmid instability is a significant concern in the industrial utilization of recombinant microorganisms, which is of both theoretical and practical importance. Two types of plasmid instability occur frequently, both in segregational and structural instability. Segregational instability was defined as the loss of the entire plasmid from the cells due to defective partitioning, while structural instability was involved in the rearrangement, loss, or insertion of plasmid DNA sequences, associated typically with transposition or recombination.Current studies on plasmid stability have been approached genetically, focusing on segregational instability, which is concerned as a major problem.Characterized by non-antibiotic selective pressure, plasmid-encoded post-segregational killing system (PSK) is established as a more efficient way to maintain plasmid stability. The PSK system does not participate directly in distributing plasmids to dividing cells, only killing plasmid-free cells by the hok gene products after distribution, which thus keeps plasmidic genetic characteristics passing from one generation to another generation. The hok/sok killer locus of E. coli plasmid R1 has two genes: hok (host killing) encodes a highly toxic transmembrane protein,denoted hok mRNA; sok (suppression of killing) specifies a small cis-encoded antisense RNA that blocks translation of hok, denoted sok RNA. According to the principle of dote-antidote, the mechanism of hok/sok locus can be interpreted as follows: in cells with a hok/sok-carrying plasmid (which produces sok RNA), sok RNA binds to hok mRNA rapidly and represses its translation; while in plasmid-free cells, the sok RNA pool is depleted owing to rapid decay of the antisense RNA, the mature and translatable form of hok mRNA accumulates, and hok protein synthesis results in killing of the plasmid-free cells. So the survived cells all contain hok/sok locus, which as a result improves plasmid stability.In order to do further research on how hok/sok stabilizes plasmids by a phenotype of post-segregational killing, the hok/sok gene was inserted into the low copy plasmid pACYC184 with its BamHⅠsite. The recombinant plasmid pACYC184-hok/sok was constructed, and then transformed into E. coli DH5α. According to the recombinant plasmid DNA sequence of pACYC184-hok/sok, the special primers were designed to do long PCR, in order to combine hok/sok△274T and the plasmid DNA sequence of pACYC184. Then the long PCR products self-ligated to develop another recombinant plasmid pACYC184-hok/sok (M), used as a control for pACYC184-hok/sok. Among other things, hok/sok△274T was obtained with one T deletion in the sequence of 274th site, which damaged the structure of hok/sok′s ORF (open reading frame). As a result, the transcription of sok antisense RNA was depressed, leading to sok RNA′s deffective unbinding to hok mRNA.To determine the segregational instabilities of the constructed plasmids, the two recombinant E. coli DH5αstrains with plasmids of pACYC184-hok/sok and pACYC184-hok/sok (M), and the control strain with pACYC184, were respectively cultured under the non-antibiotic condition, thus testifying the relations between plasmid loss and no antibiotics addiction during continuous division of host cells. The method was dedicated as follows: the recombinant bacterial strains were cultivated continuously in Luria-Bertani (LB) medium without chloramphenicol, and at regular intervals, appropriately diluted samples were spread on LB agar plates. One hundred colonies were transferred from the LB agar plates to LB plates containing 34ug/ml chloramphenicol. Those showing chloramphenicol sensitivity were assumed to be plasmid-free cells. Simultaneously,single colonies with resistance to chloramphenicol was used to extract plasmid DNA by standard alkaline lysis, to determine the presence of the plasmids. Moreover, the recombinant plasmids extracted from both the original strains and the 135th or 15th (As the recombinant strain containing pACYC184-hok/sok (M) was very unstable, only grown for 15 generations) generation strains were digested by SphⅠ, additionally with the plasmid pACYC184 as a control. Considering whether the hok/sok PSK system effects host cell growth, we charted the growth curves of two recombinant strains as well as the genetic engineering bacteria containing pACYC184.The experimental results showed that, under the non-antibiotic condition, the recombinant E. coli DH5αwith plasmid pACYC184-hok/sok were stably maintained 135 generations; but the recombinant E. coli DH5αwith plasmid pACYC184-hok/sok (M) was very unstable, only grown for 15 generations with 98% of plasmid loss. Hence, hok/sok PSK system can enhance the plasmid stability of pACYC184 obviously. Further restriction and sequencing analysis identified the consistency and correctness of pACYC184-hok/sok DNA sequence from both the original strains and the 135th generation strains. However, the similar results also happened to the recombinant plasmid pACYC184-hok/sok (M), it had little genetic importance because of the short generations maintaining by its host strain. Compared with the genetic engineering bacteria containing pACYC184, DH5α(pACYC184-hok/sok (M)) grew slowly during all stages of growth, which suggested that its low growth rate was closely correlated with plasmid instability. Whereas DH5α(pACYC184-hok/sok)grew more rapidly, its growth curve was similar to the control bacteria's.Subsequently, we came to a conclusion that, the stability of hok/sok-carrying plasmid pACYC184-hok/sok was obviously better than hok/sok-free plasmids of pACYC184 and pACYC184-hok/sok. Within the continuous division of host cells, the recombinant plasmid pACYC184-hok/sok has been evolved with good segregational stability, whereas the recombinant plasmid pACYC184-hok/sok (M) has high segregational instability. Intriguingly, under the depressed condition without antibiotics, the genetic engineering bacteria carrying pACYC184 can grow for about 50 generations without plasmid loss. Although it posses a certain segregational instability, it still agrees with the needs of industrial production.In a word, with hok/sok PSK system introducing into the plasmid pACYC184, its plasmid stability is greatly enhanced, which thus provides a more safe and efficient strategy to improve the translation of exogenous genes and genetic engineering.