Plasmid instability and molecular cloning in Bacillus subtilis

Bron, Sierd; Meijer, Wilfried J. J.; Holsappel, Siger; Haima, Peter

doi:10.1016/0923-2508(91)90068-l

Cited by 47 publications

(44 citation statements)

References 27 publications

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“…This fact likely refers to the described plasmid instability (15,16). However, the ability to produce nonribosomal peptides and the well established genetic and fermentation methods for B. subtilis make it an attractive target for such studies.…”

Section: Discussionmentioning

confidence: 99%

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Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

Eppelmann

Doekel

Marahiel

2001

Journal of Biological Chemistry

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Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.

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Section: Discussionmentioning

confidence: 99%

“…Due to the instability of plasmids containing large insertions of foreign DNA in B. subtilis (15,16), expression of giant recombinant gene clusters like those coding for NRPSs can only be achieved by a stable chromosomal integration.…”

Section: Construction Of the B Subtilis Atcc 21332 Srfa Deletionmentioning

confidence: 99%

Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

Eppelmann

Doekel

Marahiel

2001

Journal of Biological Chemistry

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“…The positions of the copy number control genes per and aes of pGA1, and of the collagen-like protein gene of pTX14-2 are also indicated. doi:10.1128/microbiolspec.PLAS- 0035-2014.f2 for the construction of vectors for gene cloning and expression, provided a functional sso is present to minimize the generation of the recombinogenic ssDNA intermediates, which can lead to structural and segregational plasmid instability (33)(34)(35)(36)(37). Nevertheless, it has been reported that cloning of heterologous DNA in RCR plasmid vectors can result in the generation of linear highmolecular-weight (HMW) plasmid multimers in relative amounts that correlate positively with the size of the DNA insert (38,39).…”

Section: General Aspects Of Plasmid Rolling-circle Replicationmentioning

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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“…These plasmids are stable in B. subtilis as they are, however, instability often occurred with these vectors due to rearrangement or deletion of the inserted foreign gene (Bron et al, 1991;Gurakan et al, 1997). Rolling circle replication (RCR) plasmids based on an endogenous plasmid of B. subtilis were proved to be superior to those nonnative-based plasmids (Bron et al, 1991;Meijer et al, 1995). In addition, the theta-replication plasmids were also used to construct the expression vectors with improved structural stability (Nguyen et al, 2005).…”

mentioning

confidence: 99%

“…Simultaneously, various expression vectors were also constructed, mainly based on foreign plasmids, such as pUB110 (Gryczan and Dubnau, 1978), pE194 (Ehrlich, 1977), pC194 (Gryczan and Dubnau, 1978), pAMβ1 (Jannière et al, 1990), pTB19 (Imanaka et al, 1981) and pSB6 (Bron et al, 1988;Gurakan et al, 1997;Titok et al, 2003). These plasmids are stable in B. subtilis as they are, however, instability often occurred with these vectors due to rearrangement or deletion of the inserted foreign gene (Bron et al, 1991;Gurakan et al, 1997). Rolling circle replication (RCR) plasmids based on an endogenous plasmid of B. subtilis were proved to be superior to those nonnative-based plasmids (Bron et al, 1991;Meijer et al, 1995).…”

mentioning

confidence: 99%

Construction of novel shuttle expression vectors for gene expression in <i>Bacillus subtilis</i> and <i>Bacillus pumilus</i>

Shao

Cao

Zhao

et al. 2015

J. Gen. Appl. Microbiol.

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A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

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Plasmid instability and molecular cloning in Bacillus subtilis

Cited by 47 publications

References 27 publications

Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis

Plasmid Rolling-Circle Replication

Construction of novel shuttle expression vectors for gene expression in <i>Bacillus subtilis</i> and <i>Bacillus pumilus</i>

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