From essential to persistent genes: a functional approach to constructing synthetic life

Acevedo‐Rocha, Carlos G.; Fang, Gang; Schmidt, Markus; Ussery, David W.; Danchin, Antoine

doi:10.1016/j.tig.2012.11.001

Cited by 106 publications

(94 citation statements)

References 72 publications

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“…We sequenced the entire set of 9,250 mutants obtained in this study and assembled a library of 1,110 unique mutants in which the tagged transposon had integrated into a discrete gene. Considering that L. casei is expected to encode 2,929 genes (45), with a putative set of at least 400 essential genes (46,47), one can consider that more than half of the genome was mutagenized. Because transposon insertions could have polar effect, one can safely state that nearly all the nonvital L. casei functions have been affected.…”

Section: Discussionmentioning

confidence: 99%

Functional genomics of Lactobacillus casei establishment in the gut

Licandro

Scornec

Pédron

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Although the composition of the gut microbiota and its symbiotic contribution to key host physiological functions are well established, little is known as yet about the bacterial factors that account for this symbiosis. We selected Lactobacillus casei as a model microorganism to proceed to genomewide identification of the functions required for a symbiont to establish colonization in the gut. As a result of our recent development of a transposonmutagenesis tool that overcomes the barrier that had prevented L. casei random mutagenesis, we developed a signature-tagged mutagenesis approach combining whole-genome reverse genetics using a set of tagged transposons and in vivo screening using the rabbit ligated ileal loop model. After sequencing transposon insertion sites in 9,250 random mutants, we assembled a library of 1,110 independent mutants, all disrupted in a different gene, that provides a representative view of the L. casei genome. By determining the relative quantity of each of the 1,110 mutants before and after the in vivo challenge, we identified a core of 47 L. casei genes necessary for its establishment in the gut. They are involved in housekeeping functions, metabolism (sugar, amino acids), cell wall biogenesis, and adaptation to environment. Hence we provide what is, to our knowledge, the first global functional genomics analysis of L. casei symbiosis.commensalism | Lactic acid bacteria T he pioneering studies that led to the characterization of the gut microbiota were reviewed in 2001 (1). These studies and recent investigations have revealed mutualistic functions (2), including a barrier effect against allogenic microbes (3), fermentation of complex sugars (4, 5), and maturation and homeostasis of the immune system (6). Recent metagenomic studies have revealed an extraordinary diversity of genes constituting the gut microbiome (7), opening the way to correlative studies linking microbiome diversity, homeostasis, and diseases (5,8,9).In parallel, some representative species, i.e., "model symbionts," now are being studied functionally (10). As it was done for pathogens, it is essential to develop the cellular microbiology of symbionts and particularly to identify the genes required for their establishment and persistence in the gut. Transcriptomic profiling identified up-regulated genes linked to metabolic functions, stress responses, and pili synthesis during early colonization (11-13). Comparative genomics among Lactobacilli identified strain-specific candidate genes for extended colonization: In Lactobacillus rhamnosus, persistence was attributed to an spaCBA locus encoding LPXTG-like pilins (14), and in Lactobacillus johnsonii it was attributed to specific glycosyltransferases, a phosphotransfer system, and a protease (15). Otherwise, a functional in vivo screening based on the expression of a genomic library of Bacteroides fragilis identified a locus encoding polysaccharide utilization as essential for stable colonization of murine colonic crypts (16). Alternatively, colonization of germ-free mic...

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Section: Discussionmentioning

confidence: 99%

Functional genomics of Lactobacillus casei establishment in the gut

Licandro

Scornec

Pédron

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…They are those identified in the minimal genomes constructed for SynBio approaches. We named this set the paleome (Acevedo‐Rocha et al ., 2013). It comprises the core translation, transcription and replications machineries, together with basic membrane functions involved in waste disposal, basic ion supply, in energy generation and cell wall synthesis, as well as a key set of central metabolism enzymes.…”

Section: Bacillus Subtilis In 2017mentioning

confidence: 99%

Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement

Borriss

Danchin

Harwood

et al. 2017

Microbial Biotechnology

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SummaryGenome annotation is, nowadays, performed via automatic pipelines that cannot discriminate between right and wrong annotations. Given their importance in increasing the accuracy of the genome annotations of other organisms, it is critical that the annotations of model organisms reflect the current annotation gold standard. The genome of Bacillus subtilis strain 168 was sequenced twenty years ago. Using a combination of inductive, deductive and abductive reasoning, we present a unique, manually curated annotation, essentially based on experimental data. This reveals how this bacterium lives in a plant niche, while carrying a paleome operating system common to Firmicutes and Tenericutes. Dozens of new genomic objects and an extensive literature survey have been included for the sequence available at the INSDC (AccNum AL009126.3). We also propose an extension to Demerec's nomenclature rules that will help investigators connect to this type of curated annotation via the use of common gene names.

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“…(Color figure online) researchers to address the role of chance and necessity at the molecular level? The key to resolving these prior questions is, at least in part, to assess the degree to which our system tracks or differs from experimental systems that replace genomic components with homologs obtained from other extant organisms (Acevedo-Rocha et al 2013;Agashe et al 2013;Andersson and Hughes 2009;Pena et al 2010;Urbanczyk et al 2012).…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Experimental evolution of Escherichia coli harboring an ancient translation protein

Kaçar

Tran

et al. 2016

Preprint

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The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes, and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year old inferred ancestral variant of tufB, an essential gene encoding Elongation Factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a two-fold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2,000 generations in the laboratory, and examined the adaptive response via fitness assays, whole-genome sequencing, proteomics and biochemical assays. We found that the hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by up-regulation of protein production. We expect that this ancient-modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.

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From essential to persistent genes: a functional approach to constructing synthetic life

Cited by 106 publications

References 72 publications

Functional genomics of Lactobacillus casei establishment in the gut

Functional genomics of Lactobacillus casei establishment in the gut

Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement

Experimental evolution of Escherichia coli harboring an ancient translation protein

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