Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria

Domínguez, Wilfredo; O’Sullivan, Daniel J.

doi:10.1099/mic.0.061408-0

Cited by 35 publications

(22 citation statements)

References 56 publications

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“…oriT simultaneously. In addition, RK2 has a capability of transferring DNA from E. coli to various organisms, including Gram-positive bacteria (Trieu-Cuot et al 1991, Mazodier et al 1989, Dominguez and O'Sullivan 2013), yeast (Nishikawa et al 1990, and CHO cells (Waters 2001), as well as to gram-negative bacteria without a specific receptor on the recipient.…”

Section: Rk2 a Multidrug Resistant Broad Host Range R Plasmid Isolatmentioning

confidence: 99%

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Yokoi

Itaya

Mori

et al. 2019

J. Gen. Appl. Microbiol.

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The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double

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Section: Rk2 a Multidrug Resistant Broad Host Range R Plasmid Isolatmentioning

confidence: 99%

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Yokoi

Itaya

Mori

et al. 2019

J. Gen. Appl. Microbiol.

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“…In the complementation test, a conjugation system using Escherichia coli SM10 pir (45) with the RP4 plasmid (46) was used to transfer the plasmid with the wild-type malK sequence into M2, according to the methodology described previously (47). However, we failed to produce a successful clone under the conditions tested.…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

Keung

Sham

et al. 2017

Appl Environ Microbiol

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Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1 H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1¡3,1¡6)-␤-glucans and (1¡4,1¡6)-␣-glucans. Although the YCWG were composed of 78.3% ␤-glucans and 21.7% ␣-glucans, only ␣-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of ␣-glucancatabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast ␣-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.

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“…Unlike electroporation, conjugation is not limited by the size of vectors that can be transformed, and has been used successfully to transfer entire genomes in E. coli (54). Additionally, conjugation uses the transfer of DNA as a single strand that is relatively insensitive to the majority of the restriction systems of the cells(55). Thus conjugation may have advantages over electroporation.…”

Section: Discussionmentioning

confidence: 99%

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Gao

Murugesan

Hoßbach

et al. 2018

Preprint

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Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from E. coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The presence of homologous TG1 and R4 attB sites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.ImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species, Amycolatopsis marina, as tools for genome integration. The high level of conservation between the attB sites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

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Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria

Cited by 35 publications

References 56 publications

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

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