Transposon-mediated directed mutation in bacteria and eukaryotes

Saier, Milton H.; Kukita, Chika; Zhang, Zhongge

doi:10.2741/4553

Cited by 13 publications

(15 citation statements)

References 55 publications

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“…In the presence of phosphotransferase system (PTS) sugars such as glucose, IIAGlc will instead bind and inhibit GlpK. In the absence of PTS sugars, IIAGlc will become phosphorylated, bind, and activate AC's ability to produce cAMP, ultimately reducing the effect of CCR [36].…”

Section: Key Mutations Increase Through the Bottom-up Convergence Of mentioning

confidence: 99%

Causal mutations from adaptive laboratory evolution are outlined by multiple scales of genome annotations and condition-specificity

et al. 2020

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Background Adaptive Laboratory Evolution (ALE) has emerged as an experimental approach to discover mutations that confer phenotypic functions of interest. However, the task of finding and understanding all beneficial mutations of an ALE experiment remains an open challenge for the field. To provide for better results than traditional methods of ALE mutation analysis, this work applied enrichment methods to mutations described by a multiscale annotation framework and a consolidated set of ALE experiment conditions. A total of 25,321 unique genome annotations from various sources were leveraged to describe multiple scales of mutated features in a set of 35 Escherichia coli based ALE experiments. These experiments totalled 208 independent evolutions and 2641 mutations. Additionally, mutated features were statistically associated across a total of 43 unique experimental conditions to aid in deconvoluting mutation selection pressures. Results Identifying potentially beneficial, or key, mutations was enhanced by seeking coding and non-coding genome features significantly enriched by mutations across multiple ALE replicates and scales of genome annotations. The median proportion of ALE experiment key mutations increased from 62%, with only small coding and non-coding features, to 71% with larger aggregate features. Understanding key mutations was enhanced by considering the functions of broader annotation types and the significantly associated conditions for key mutated features. The approaches developed here were used to find and characterize novel key mutations in two ALE experiments: one previously unpublished with Escherichia coli grown on glycerol as a carbon source and one previously published with Escherichia coli tolerized to high concentrations of L-serine. Conclusions The emergent adaptive strategies represented by sets of ALE mutations became more clear upon observing the aggregation of mutated features across small to large scale genome annotations. The clarification of mutation selection pressures among the many experimental conditions also helped bring these strategies to light. This work demonstrates how multiscale genome annotation frameworks and data-driven methods can help better characterize ALE mutations, and thus help elucidate the genotype-to-phenotype relationship of the studied organism.

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Section: Key Mutations Increase Through the Bottom-up Convergence Of mentioning

confidence: 99%

Causal mutations from adaptive laboratory evolution are outlined by multiple scales of genome annotations and condition-specificity

et al. 2020

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“…Diverse ecological triggers for NGE activity are presented in Section 8 and Table 16 , and posted online ( ). There is also extensive documentation of targeting mechanisms at work directing mobile DNA element insertions to specific genome sites or regions in organisms that range from bacteria to plants and animals [ 696 , 697 , 698 , 699 ], ( ; ; ). Targeting and activation are particularly relevant to the larger question of positive bias in evolution when they operate via a global genome control process like epigenetic genome modification as, for example, in mouse embryonic stem cells [ 696 ].…”

Section: Further Reflections On Genome Rewriting By Nge As a Core mentioning

confidence: 99%

Living Organisms Author Their Read-Write Genomes in Evolution

Shapiro

2017

Biology

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Evolutionary variations generating phenotypic adaptations and novel taxa resulted from complex cellular activities altering genome content and expression: (i) Symbiogenetic cell mergers producing the mitochondrion-bearing ancestor of eukaryotes and chloroplast-bearing ancestors of photosynthetic eukaryotes; (ii) interspecific hybridizations and genome doublings generating new species and adaptive radiations of higher plants and animals; and, (iii) interspecific horizontal DNA transfer encoding virtually all of the cellular functions between organisms and their viruses in all domains of life. Consequently, assuming that evolutionary processes occur in isolated genomes of individual species has become an unrealistic abstraction. Adaptive variations also involved natural genetic engineering of mobile DNA elements to rewire regulatory networks. In the most highly evolved organisms, biological complexity scales with “non-coding” DNA content more closely than with protein-coding capacity. Coincidentally, we have learned how so-called “non-coding” RNAs that are rich in repetitive mobile DNA sequences are key regulators of complex phenotypes. Both biotic and abiotic ecological challenges serve as triggers for episodes of elevated genome change. The intersections of cell activities, biosphere interactions, horizontal DNA transfers, and non-random Read-Write genome modifications by natural genetic engineering provide a rich molecular and biological foundation for understanding how ecological disruptions can stimulate productive, often abrupt, evolutionary transformations.

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“…Considering that the fraction of noncoding sequences in the B. longum genome is much smaller than the fraction of coding regions, bias toward noncoding regions suggests that insertion is not entirely random. This characteristic is disadvantageous for random transposon mutagenesis but is widely known (28). For example, insertions of IS2 and IS3, which are both IS3 elements, were also reported to be biased toward noncoding regions in E. coli (29).…”

Section: Discussionmentioning

confidence: 99%

“…For example, insertions of IS2 and IS3, which are both IS3 elements, were also reported to be biased toward noncoding regions in E. coli (29). DNA secondary structure was recently proposed as the basis of biased insertion (28,30), although this has not been verified for ISBlo11.…”

Section: Discussionmentioning

confidence: 99%

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

Sakanaka

Nakakawaji

Nakajima

et al. 2018

Appl Environ Microbiol

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Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for subsp. 105-A (JCM 31944) based on IS, a native IS family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the IS transposase. An artificial transposon plasmid, pBFS12, in which IS terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >10 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of subsp. Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for subsp. , a representative health-promoting species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.

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Transposon-mediated directed mutation in bacteria and eukaryotes

Cited by 13 publications

References 55 publications

Causal mutations from adaptive laboratory evolution are outlined by multiple scales of genome annotations and condition-specificity

Causal mutations from adaptive laboratory evolution are outlined by multiple scales of genome annotations and condition-specificity

Living Organisms Author Their Read-Write Genomes in Evolution

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

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