Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

Seegers, Jos F. M. L.; Zhao, Adam C.; Meijer, Wilfried J. J.; Khan, Saleem A.; Venema, Gerard; Bron, Sierd

doi:10.1007/bf00290234

Cited by 51 publications

(45 citation statements)

References 29 publications

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“…Plasmid pWV01 has been shown to be able to replicate in both gram-positive and gram-negative bacteria, including E. coli (20,30). Demonstration of functional roles of replication elements during the pGKV21 replication in gramnegative bacteria, not in gram-positive bacteria, is important to understand the replication mechanism of pGKV21 as a broadhost-range plasmid.…”

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Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

et al. 1997

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A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13⌬lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.Primer RNA synthesis for the replication of Escherichia coli single-stranded DNA (ssDNA) phages may be accomplished by one of the three different modes of priming (19). Initiation mechanisms of complementary-strand synthesis have been distinguished on the basis of the minus-origin sequences and host function requirements. In each case, an RNA primer is synthesized prior to the initiation of DNA synthesis. This RNA primer is synthesized by primase and RNA polymerase in case of G4 and M13 phages, respectively (4, 7). In X174, conversion of ssDNA to double-stranded DNA (dsDNA) requires the formation of a primosome, a multiprotein complex composed of at least seven host proteins (29). Primer RNAs are synthesized at multiple sites located throughout the X174 genome due to the mobile nature of this protein complex (1,26,28). It was found that many plasmids isolated from E. coli contained specific nucleotide sequences required for priming DNA synthesis on an ssDNA template. These sequences have been called the single-strand initiation (ssi) signals. These ssi signals were identified by their ability to complement the poor growth of an ssDNA phage in which a great part of minus origin had been deleted (17,27). The ssi signals of E. coli plasmids were also classified into four major groups with respect to their priming mode: (i) G4 type (3, 23, 24, 32), (ii) X174 type (2, 15, 23-25), (iii) ABC type (22), and (iv) plasmid-specific type (RSF1010) (12-14).Many rep...

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Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

et al. 1997

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“…The ssoW was identified in lactococcal plasmid pWVO1 (130,131). This origin is located in a 250-bp DNA fragment that contains two inverted repeats, IR I and IR II.…”

Section: The Single-strand Originmentioning

confidence: 99%

“…Thus, priming of the lagging strand from ssoW seems to occur through two different pathways: one is catalyzed by RNAP and requires the entire origin, and the other, which was suggested to involve a primosomal complex, only requires IR I. Efficient functionality of the pWVO1-ssoW seems to be confined to lactococci (131).…”

Section: The Single-strand Originmentioning

confidence: 99%

Plasmid Rolling-Circle Replication

et al. 2015

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Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

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“…This ssDNA intermediate is turned into a double-stranded molecule starting from a second origin, the single-strand origin of replication (sso). Although host-encoded RNA polymerase has been shown to participate in all replication mechanisms studied so far, the sso of the lactococcal plasmid pWV01 (26) can probably be activated by one of two routes, RNA polymerase dependent or RNA polymerase independent (42).…”

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“…Contrasting with these Rep-dso conserved associations, the sso can be divided into several types, which are not necessarily correlated to one specific family of replicons. Five different types are now recognized: the pC194 (sso a ), pUB110 (sso u ), pBAA1 (sso t ) (21), p353 (27), and pWVO1 (sso w ) (42) types.…”

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Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons

1997

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The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43°C).Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal ␦-endotoxins during sporulation. The chromosome sizes of different B. thuringiensis strains vary from 2.4 to 4.3 Mb, as described for the closely related bacterium B. cereus (10). Since most of the genes present on the small chromosome (2.4 Mb) mapped to one half of the large molecules, it is believed that the genome consists of one constant part and another less stable entity, more easily mobilized into other genetic elements, e.g., plasmids (10). Indeed, plasmid profiles of most B. thuringiensis strains are rather complex, with molecules ranging from 2 to more than 200 kb. So far, however, most of the attention has been focused on the large plasmids because of their capacity to code for the insect-specific endotoxin genes (crystal [cry] genes); very few small (Ͻ15-kb) plasmids have been characterized yet. To our knowledge, 15 cryptic plasmids, including pGI3, have been found in B. thuringiensis. For two of them (pGI1 and pTX14-2), cloning attempts have so far failed (7,13). No cloning has been reported for six others (1, 28), and only five molecules have been partially or fully sequenced.In contrast to the large plasmids that normally display a -type replication mechanism, most of the small gram-positive replicons use the rolling circle replication mode (21). In this case, the replication protein (Rep) introduces a nick at the double-strand origin (dso) from which replication starts. The plus strand is displaced, and polymerization of a new plus strand by 3Ј-OH extension from the nick occurs. Then Rep recognizes a termination sequ...

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Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

Cited by 51 publications

References 29 publications

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

Plasmid Rolling-Circle Replication

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons

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