Vector Systems

Basically, three different vector systems have been developed (Bron, 1990): autonomously replicating plasmid vectors, integrative vectors, and bacteri-ophage vectors. Based on their mode of replication, plasmid vectors can be divided into two groups. The first group replicates according to the rolling circle mechanism (RCM) and the second uses the theta mechanism. Most small plasmids (smaller than ^12 kb) from Gram-positive bacteria replicate via the RCM, while larger plasmids use the theta mechanism (Bron et al., 1991; Ehrlich et al., 1991). A major distinction between the two modes of replication is the generation of single-stranded (ss) DNA intermediates by RCM plasmids (see below). Due to sometimes low transformation frequencies using B. subtilis as primary host, it is often more convenient to carry out the initial cloning steps with a shuttle plasmid in E. coli and then transfer the recombinant plasmids to B. subtilis. Shuttle plasmids contain two different origins of replication; one able to drive replication in E. coli and the second in another host, here B. subtilis. Integrative vectors allow insertion of the gene of interest into the B. subtilis chromosome, and most bacteriophage vectors are based on the temperate phage f 105 (Errington, 1993).

A. Rolling circle-type replication vectors 1. Natural rolling circle-type plasmids

A number of small plasmids have been observed to accumulate ssDNA during replication (Gruss and Ehrlich, 1989). Several of these plasmids were studied in detail and shown to replicate by a RCM (Sozhamannan et al., 1990; te Riele et al., 1986). The RCM requires two replication origins, one called ''plus'' and is used for the synthesis of the ss replication intermediates, while the other, termed ''minus,'' is used for the conversion of the ss intermediates into a mature double-stranded molecule. Minus origins are noncoding, highly palindromic sequences, usually about 200-300 bp, which function only in one orientation. In their absence, plasmid ssDNA accumulates. Initiation of replication at the two origins does not occur simultaneously and therefore results in the accumulation of the ssDNA replication intermediate (Novick, 1989).

The four plasmids pUB110, pC194, pE194, and pT181 were initially identified in Staphylococcus aureus. While pUB110 specifies resistance to kanamycin and neomycin and has a copy number of 30-50 per chromosome (Lacey and Chopra, 1974), pC194 codes for chloramphenicol resistance and is maintained at a copy number of about 15 (Iordanescu et al., 1978). The third plasmid, pE194, confers resistance to macrolide-lincosa-mide-streptogramin B (MLS) antibiotics and is present in —10 copies per chromosome (Iordanescu, 1976). Most importantly, pE194 is naturally temperature-sensitive for replication (Repts phenotype) and does not replicate above 45 °C in B. subtilis. When cells containing pE194 were grown on erythromycin-containing media at 50 °C, erythromycin-resistant cells were selected in which pE194 was found to be integrated into the bacterial chromosome at a variety of sites (Hofemeister et al., 1983). The fourth plasmid, pT181, is similar to many other tetracycline-resistant plasmids, for example pT127 and pSN1 (Iordanescu, 1976), with a copy number of around 20 copies per chromosome. Plasmid pTA1015 belongs to the group of cryptic plasmids isolated from B. subtilis strains (Uozumi et al., 1980).

Two types of plasmid instability have been described, segregational instability involving loss of the entire plasmid population from a cell and structural instability involving the loss, rearrangement, or acquisition of DNA sequences by the plasmid. Loss of the whole plasmid is the consequence of unequal segregation during cell division: all copies segregate into one of the two daughter cells. While low-copy number plasmids are stably maintained by partitioning functions ensuring their accurate segregation at cell division, high-copy number plasmids are segregated randomly at cell division. One important reason for segregational instability of high-copy number plasmids is their tendency to form multimeric forms (in particular of RCM plasmids). The mechanism that controls the copy number of a plasmid ensures, on the average, a fixed number of plasmid origins per chromosome. Therefore, cells containing multimeric plasmids have the same number of plasmid origins but fewer plasmid molecules, leading to a greater instability. Structural instability often leads to deletion formation caused by erroneous replication termination (Michel and Ehrlich, 1986) and aberrant nicking-closing events (Ballester et al., 1989) mediated by the replication proteins of the plasmids. Topo-isomerase-like activities were also implicated in illegitimate recombination (Lopez et al., 1984). Yet another important source for deletion formation is based on recombination between short direct repeats stimulated by the RCM (Bron et al., 1991; Janniere and Ehrlich, 1987). Here, 9-bp direct repeats (and sometimes direct repeats as short as 4 bp) are sufficient for deletion formation and create a major source for structural instability in recombinant plasmids. While the vector plasmids by themselves are normally stable, cloning of a DNA fragment can introduce direct repeats, where one repeat is located within the vector and the other within the insert.

2. Cloning vectors derived from rolling circle-type plasmids

In many cloning experiments, the natural plasmids shown in Table 6.1 have been directly used as vectors. Since these plasmids do not replicate in E. coli, several shuttle vectors have been constructed and important ones are listed in Table 6.2. Besides cloning vectors, several expression vectors allowing intra- and extracellular production of recombinant proteins are available. They contain both constitutive and inducible promoters and the coding regions of signal sequences derived from different genes coding for extracellular proteins.

B. Theta-type replication vectors

1. Natural theta-type replication plasmids

Plasmids which do not create ssDNA intermediates belong to the other class and replicate in the host through a theta-type intermediate, and these are present in low copy numbers and are structurally stable. The known prokaryotic theta plasmids can be classified into at least five groups A-E (Bruand et al., 1993; Meijer et al., 1995), where two of these groups incorporate plasmids from Gram-positive bacteria (Table 6.3). One class concerns the broad host range plasmid pAM^l of Enterococcus faecalis (Bruand et al., 1991) and the highly related streptococcal plasmids pIP501 of Streptococcus agalactiae (Brantl and Behnke, 1992) and pSM19035 of Streptococcus pyogenes (Tanaka and Koshikawa, 1977; Tanaka and Ogura, 1998). The replication region of pLS32 can support replication of a DNA fragment as large as 310 kb without gross DNA rearrangement (Itaya and Tanaka, 1997), and even of the entire chromosome of B. subtilis via bidirectional replication (Hassan et al., 1997). pBS72 has been isolated as a naturally occurring plasmid in an undomesticated strain of B. subtilis of the territory of Belarus with a copy number of 6 per chromosome (Titok et al., 2003).

2. Cloning vectors derived from theta-type replication plasmids

Several cloning vectors have been constructed based on theta-type replication vectors. All these vectors are shuttle vectors which allow the cloning and verification steps in E. coli, and the final recombinant plasmid is then shuttled into B. subtilis (Table 6.4). Cloning vectors are based on the replication functions of pAM;S1 and pTB19 active in B. subtilis and the

TABLE 6.1 Important rolling circle-type replication plasmids



Size (bp)

Original host




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