Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

Bartosik, Aneta Agnieszka; Markowska, Aleksandra; Szarlak, Jolanta; Kulinska, Anna; Jagura-Burdzy, Grażyna

doi:10.1016/j.mimet.2011.10.011

Cited by 14 publications

(16 citation statements)

References 56 publications

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“…The conjugative transfer module of RA3 (coordinates 9437-22925 nt) was previously cloned into the high-copy-number plasmid pBGS18 [41] together with korC gene (coordinates 3391-3705 nt) encoding RA3 global transcriptional regulator [42,51]. Such construct pJSB1.24 was capable of self-transmission [42] at the frequency comparable with parental RA3 (approximately one transconjugant per donor cell).…”

Section: Resultsmentioning

confidence: 99%

“…Such construct pJSB1.24 was capable of self-transmission [42] at the frequency comparable with parental RA3 (approximately one transconjugant per donor cell).…”

Section: Resultsmentioning

confidence: 99%

“…In the mobilization experiments the donor strain DH5α carried the RA3 or pJSB1.24 (pBGS18 [41] derivative with Tra RA3 module and korC gene; [42] as the helper plasmid and the mobilizable pPT01 derivatives with the variants of oriT - mobCp region inserted. In conjugation experiments strains: DH5α(pUC18), DH5α(pJSB2.57), DH5α(pJSB2.58)(pBBR1MCS-1), DH5α(pJSB2.58)(pJSB4.1), DH5α(pJSB2.58)(pJSB4.2) were used as donors.…”

Section: Methodsmentioning

confidence: 99%

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MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

2014

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BackgroundThe IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group.ResultsMobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower.ConclusionThe MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0235-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

“…Such construct pJSB1.24 was capable of self-transmission [42] at the frequency comparable with parental RA3 (approximately one transconjugant per donor cell).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

2014

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“…The pCCATCH plasmid (12,455 bp, a derivative of pABB20 vector; Bartosik et al 2012): The transformation vector pCCATCH for stable transformation of chloroplast, containing 5′ UTR and 3′ UTR of C. merolae psb A (CMV047C), separated by cat CH cassette. The expression of the cat CH cassette was driven by dna K (CMV163C) promoter.…”

Section: Methodsmentioning

confidence: 99%

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments

et al. 2016

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Key messageWe have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism.AbstractHere we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae—an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi:10.1007/s00709-015-0936-9). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae—a new model organism.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-016-0554-8) contains supplementary material, which is available to authorized users.

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“…We therefore checked the effect of KorB excess on the self-transmission of plasmid pJSB1.24, a pBGS18 derivative carrying a functional RA3 conjugative transfer module (52). E coli DH5␣(pJSB1.24) was transformed with pAMB9.37 (pBBR-MCS1 lacI q , tacp) as a control or its derivative pAKB5.50 tacp-korB.…”

Section: Partitioning Protein Korb (Ra3) Is a Transcriptional Repressormentioning

confidence: 99%

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

Kulinska

Godziszewska

Wojciechowska

et al. 2016

Appl Environ Microbiol

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The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parS RA3 , forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nic operon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding Large, low-copy-number plasmids displaying a broad host range (BHR) carry an extended backbone of operons involved in replication, copy number control, maintenance, and in the case of self-transmissible plasmids, also conjugative transfer. Expression of these genetic units is driven by high-activity promoters and therefore imposes a substantial metabolic cost on the bacterial cell. To minimize the metabolic burden to the host, regulatory networks have evolved to diminish the backbone gene expression to a low basal level while still allowing for rapid upregulation of the transcription when needed. The regulation is achieved by autogenous repressors that produce negative-feedback loops, mediumrange repressors that control particular modules, and globally acting regulators that bind operators scattered along the plasmid molecule to coordinate expression of all modules. Fine-tuning of gene expression is postulated to be achieved by the action of corepressors and their cooperative binding in the promoter regions, which makes the plasmid highly responsive and facilitates its adaptation (1-4).BHR plasmids from the incompatibility group IncU are widespread and ubiquitous in various aquatic environments, freshwater, fish farms, and clinical isolates (5-8). The modular-mosaic backbone of the IncU plasmids is extremely well conserved not only in its overall genetic organization but also at the nucleotide sequence level (8-10). The mosaic character of genomes from this group is demonstrated by homology of the functional blocks involved in replication, stable maintenance, and conjugative transfer to the respective functional modules of plasmids from different incompatibility groups. Thus, the stability region of RA3 encodes seven homologs of IncP-1 proteins (11), whereas the RA3 conjugative transfer region clusters with similarly organized modules from the PromA group of plasmids (12-16). The RA3 plasmid (Fig. 1), the group archetype isolated from the aquatic bacterium Aeromonas hydrophila (17), is the best-studied IncU representative. Its DNA sequence has been established (GenBank accession no. DQ401103) (10), and individual functional modules have been analyzed ...

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Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

Cited by 14 publications

References 56 publications

MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

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