Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle

Angelov, Angel; Li, Haijuan; Geißler, Andreas; Leis, Benedikt; Liebl, Wolfgang

doi:10.1016/j.syapm.2013.06.001

Cited by 21 publications

(18 citation statements)

References 31 publications

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“…This allows the generation of multiple gene deletions/mutations in the parental strain to produce a final strain unmarked by an antibiotic resistance gene. (ii) The previously reported deletion systems based on pyrE (6), bgl (18), or upp need a pyrE-, bgl-, or uppdeficient host strain. In contrast, no codA ortholog exists in T. thermophilus HB27, and the wild type can be used directly as a parental strain in the reported deletion system.…”

Section: Discussionmentioning

confidence: 99%

“…(iii) Furthermore, the codA sequence used in the integrative plasmid should not cause unexpected homologous recombination with the genome sequence. However, pheS (5), rpsL1 (17), or bgl-lacZ (18), as well as the two promoters P slpA and P treha that are also part of the integrative vector, originate from T. thermophlilus and could potentially recombine with the native genomic genes, leading to false-positive clones after the first recombination step. Using pheS as a counterselectable marker, a spontaneous large-scale deletion, including the carotenoid synthesis genes and the ␤-glycosidase gene in the megaplasmid, was observed, apparently mediated by insertion sequence (IS) elements (5).…”

Section: Discussionmentioning

confidence: 99%

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Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Wang

Hoffmann

Watzlawick

et al. 2016

Appl Environ Microbiol

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We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a ␤-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Wang

Hoffmann

Watzlawick

et al. 2016

Appl Environ Microbiol

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“…While several other counterselections for T. thermophilus have been reported, the system described here has the advantage that it does not require genetically marked strains, as in the case of pyrE (11) and bgl (23). There are several aspects to this system that could be expanded upon in future versions.…”

Section: Discussionmentioning

confidence: 99%

Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

et al. 2015

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Thermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase ␣-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog pchlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on ␤-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well. IMPORTANCEThermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into the T. thermophilus genome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

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“…Although this method reduces the mutant selection process, it also necessitates the creation of a pyrF − pyrR − double-knockout parent strain and results in a final auxotrophic mutant; it would therefore be unsuitable for commercial strain production unless the mutations were repaired at the final stage. An alternative counter-selection system was described that utilized β-glucosidase (Bgl) and the synthetic substrate X-Glu (5-bromo-4-chloro-3-indolyl-β- d -glucopyranoside) [16]. This study demonstrated that an increase in the concentration of X-Glu led to a considerable reduction in the size of colonies; this was confirmed to be the result of toxic Bgl cleavage products of X-Glu by creation of a bgl deletion strain that showed no sensitivity to X-Glu.…”

Section: Introductionmentioning

confidence: 99%

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

et al. 2017

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Background Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.ResultsOur technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.ConclusionThe selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

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Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle

Cited by 21 publications

References 31 publications

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

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