Of these 13 mutants, eight showed a significantly increased exponential growth rate in single culture and five were indistinguishable from the parent strain. Some chromosomes have fragile spots where breaks occur which result in the . McCutcheon JP, Moran NA. Cells were grown overnight and diluted 1000-fold before added to a bioscreen plate in quadruplicates. (S6)(S8) in Text S1 with N=107 and s=0.005 and 0.05, respectively. Moran NA, Mira A. In the middle, the structure and gene content of the Tn10 construct and the resulting phenotypes of loss of the cat, moaA and lacZ genes, respectively, are shown. Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. Before The quantity of adapter ligated fragments was determined by qPCR using the KAPA SYBR FAST library quantification kit for Illumina GA (KAPA Biosystems). The remaining seven deletion mutants showed no increase in competitive ability, even though they had a faster exponential growth rate in single cultures in LB- or M9-medium or both. Another possibility is that an adaptive process drives gene loss because superfluous genes confer a fitness cost to the bacterium, providing positive selection for gene loss. The GPL locus gene mps2 has been previously implicated in M. abscessus isolates with rough morphology [48, 49]. https://doi.org/10.1371/journal.pgen.1002787.s003. Genome evolution and adaptation in a long-term experiment with Escherichia coli. Cells were grown overnight and 100 ml of the overnight culture was mixed with 100 ml 1.5% low melting point agarose (Sigma) and lysozyme (1 mg/L final concentration) (Fluka). The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. The bacterial strains used in this study were derived from Salmonella enterica var typhimurium LT2 (designated S. typhimurium throughout this paper) and are listed in Table S4. The origins of modern proteomes. Numbers above the deletometer indicates nucleotide position from left to right within the deletometer (11,777 bp long) and numbers above solid line indicates nucleotide position in the chromosome. It's controlled by a process called quorum sensing, a form of bacterial communication. Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. and enteroinvasive Escherichia coli. Since most of the deletions remove several genes (many of which have no assigned function) it is difficult to explain why a specific deletion will have a beneficial, neutral or deleterious effect on fitness. It is notable that the calculated expected cost of running flagella is 4.5% of the cells total energy expenditure (Text S1), which corresponds well with the 3.2% fitness increase observed in the non-motile fliG deletion mutant. Every day 1.5 ul culture (approximately 106 cells) was transferred to 1.5 ml fresh LB medium and grown over night at 37C, 200 rpm. When the cell repairs the cut, it can either join the cut ends back together, resulting in a non-functional gene, or introduce a mutation that disrupts the gene's function. The engineered transposon was allowed to transpose into the chromosome of a strain where the chromosomal copy of the native moaA gene had been deleted, and 11 transposon insertions distributed over the Salmonella chromosome were chosen to measure the local deletion rates. Lindroos H, Vinnere O, Mira A, Repsilber D, Naslund K, et al. Modeling the rate of fixation for the parameter values determined in this study (Text S1), showed that take-over by deletion mutants could occur in less than 1,000 generations (Figure S4). Logarithmic ratios of YFP vs. CFP were plotted against number of generations and the growth advantage/disadvantage (s) of the mutant population was obtained from the slope of the curve. Homologous recombination, a key DNA repair mechanism in bacteria, enables the insertion of genetic material acquired through horizontal transfer of genes and transformation into DNA. These stem cells now lacking the gene could be used in vivo, for instance in mice, by inserting them into early embryos. For the remaining deletions (11/30), 4 bp or no homology was found at the endpoints. Federal government websites often end in .gov or .mil. We thank Diarmaid Hughes for comments on the manuscript and David Blair for help in calculating the energetic cost of running flagella. Six independent lineages of wild type bacteria were grown by repeated serial passage for 1000 generations in rich LB medium (Materials and Methods)_ENREF_20. From these results it is difficult to assess the relative contributions of drift and selection to gene loss in natural settings but it is likely that both processes contribute with different rates and dependence on population structure. The rate of selection-driven gene loss will be determined by two main parameters: the deletion rate at different chromosomal regions and the resulting fitness effects of these deletions. The deletometer is indicated as a rectangle where differences in shading indicate left (light) and right (dark) ends of the element. The engineered transposon was allowed to transpose into the chromosome of a strain where the chromosomal copy of the native moaA gene had been deleted, and 11 transposon insertions distributed over the Salmonella chromosome were chosen to measure the local deletion rates. The mixture was transferred into small plastic wells and allowed to solidify on ice for 30 min. Yes Compare with Figure S4 for a different normalization method (Text S1). Once this occurs, the cell's repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ), which involves directly ligating the two cut ends together. oryzae (Xoo) is a causative agent of rice bacterial blight (BB). Performed the experiments: SK SS OGB. Using published data on protein levels from S. enterica growing in LB medium [36], we calculated the nL values for the genes that were included in the different deletions (Table S3). Yes Affiliation To examine the importance of selection as a driver of gene loss, we determined how frequently and to what extent gene loss could confer an increase in fitness under controlled experimental conditions. Furthermore, it has been shown that inactivation/loss of the cadA gene, encoding lysine decarboxylase, can increase virulence of Shigella, providing a potential driving force for gene loss [6][8]. Primers were designed to map the deleted regions at 10 kb specificity. Growth rates were measured in exponential phase in both LB and M9-media supplemented with 0.2% glycerol. The biased mutation spectrum and increased mutation rate possibly result from the deletion of DNA repair genes, a gene class that is reduced or absent in many bacteria with reduced genomes [16], [19], [22][26]. An early instance of the use of this technique in Escherichia coli was published in 1989 by Hamilton, et al. (A) Relative exponential growth rates (parental strain set to 1.0) in single cultures for mutants with deletion of different chromosomal regions during growth in rich LB medium. This results in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. (C) Selection coefficients of deletion mutants obtained from competition experiments in rich LB medium with an isogenic parental control (defined as s=0). Fitness was initially measured as growth rate during exponential growth in rich LB media and M9-media supplemented with glycerol for 5 different deletions from each of the 11 different chromosomal regions, representing a total of 55 mutants (Materials and Methods)_ENREF_20. Here, we established a time-effective variant of consecutive single-crossovers. The sizes of the deletions in the respective region were estimated using pulsed field gel electrophoresis (PFGE). Moran NA, Plague GR. Table 5-2. Single-nucleotide polymorphisms (SNPs) have been used to track strains of clinical relevance or, when they are stably linked to other parts of the bacterial haplotype, to predict phenotypic. Ehrenberg M, Kurland CG. Here we have tested the latter idea, and our results show that a surprisingly high fraction of random deletions introduced into the Salmonella chromosome do in fact increase fitness as measured by exponential growth rate. All plasmids were prepared using the E.Z.N.A plasmid mini kit (Omega Bio-Tek) and transferred between strains using transformation or transduction. Calculated take-over times to reach 50% of the population as a function of the deletion rate. One deletion removed 5.2 kbp of DNA, including the uvrC, uvrY, yecF, sdiA, yecC, yecS genes, and the second removed 54 bp in the fliG gene. The mechanisms and driving forces involved in reductive evolution and loss of non-used gene functions are still largely unknown. Fitness was initially measured as growth rate during exponential growth in rich LB media and M9-media supplemented with glycerol for 5 different deletions from each of the 11 different chromosomal regions, representing a total of 55 mutants (Materials and Methods)_ENREF_20. HHS Vulnerability Disclosure, Help [5] If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.[6]. Our results suggest that selection as well as non-adaptive processes might drive genome reduction and that for certain chromosomal regions gene loss may occur by a rapid adaptive process. For 15/55 deletion mutants (varying in size from 18 to 44 kbp and located at 0.35, 2.07 Mbp and 3.80 Mbp) an increased growth rates were observed in either rich or poor growth media, and for 3/55 mutants growth rates were increased in both media. For gene knockout investigations, RNA interference (RNAi), a more recent method, also known as gene silencing, has gained popularity. However, this is an inefficient process, as homologous recombination accounts for only 102 to 10-3 of DNA integrations. Another possibility is that an adaptive process drives gene loss because superfluous genes confer a fitness cost to the bacterium, providing positive selection for gene loss. Yes All PCRs were run with Taq gold enzyme (Applied biosystems) according to the following protocol in a Geneamp 9700 (Applied biosystems); 94C 2 min, then 31 rounds of 94C 30 s, annealing (5565C) 30 s, elongation 72C (30 s-2 min) and a final elongation at 72C for 7 min before cooling down to 4C. See Figure 1B for the actual locations of the different insertion positions. However, gene knockouts also have some limitations. Thus, with larger population sizes selection-driven gene loss is a faster process whereas with population bottlenecks genetic drift increases in importance. L1=M1, L2=M2, which is the minimum possible). Tracing the evolution of gene loss in obligate bacterial symbionts. One deletion removed 5.2 kbp of DNA, including the uvrC, uvrY, yecF, sdiA, yecC, yecS genes, and the second removed 54 bp in the fliG gene. It is important to note that CRISPR-based gene knock-out, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution. Each operon contains regulatory DNA sequences, which act as binding sites for regulatory proteins that promote or inhibit transcription. A central biological question is how genomes evolve with respect to size and gene content and which factors affect and constrain this evolution. Curves from Eq. sharing sensitive information, make sure youre on a federal Deleted material is indicated with a dashed line and remaining chromosomal material with a solid line. These competitions allowed the detection of fitness differences as small as s=0.003 [33]. The pioneering studies of Zamenhof and Eichhorn [1], Dykhuizen [2] and Koch [3] showed that reduced expression of certain biosynthetic and catabolic operons could for unknown reasons result in an increased fitness. Gene knockout by mutation is commonly carried out in bacteria. (B) Deletion rates at 11 different chromosomal regions. However, these findings do not exclude the possibility that adaptive processes contribute to gene deletion as well, and it has been suggested that for certain free-living bacteria selection might drive genome reduction [28][30]. 10.3791/58267 Abstract A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Selective breeding may be required to produce homozygous knockout animals. The small genome of an abundant coastal ocean methylotroph. [3] Repair processes may introduce mutations that destroy functionality of the gene. As the cost of protein synthesis exceeds that of DNA and RNA for a given gene, expression of a non-used protein of length L amino acids by n copies assuming that the protein has no other adverse effects will reduce fitness by s=nL/r official website and that any information you provide is encrypted Biotechniques. Resistance markers were removed from strains using plasmid pCP20 carrying the FLP-recombinase under thermal induction control [42]. Our results show that for large deletions (>2 kbp), fitness can be increased by deletions at 3 out of 11 random positions on the chromosome, whereas for eight regions deletions appear neutral or deleterious. The authors have declared that no competing interests exist. Gene Deletion Gene Expression Regulation, Bacterial Gene Knockout Techniques / methods* Genetic Engineering / methods* Genetic Markers Genetic Vectors Plasmids / genetics Polymerase Chain Reaction Pseudomonas aeruginosa / genetics* Favre D, Viret J. Gene replacement in Gram-negative bacteria: the pMAKSAC vectors. It is notable that the calculated expected cost of running flagella is 4.5% of the cells total energy expenditure (Text S1), which corresponds well with the 3.2% fitness increase observed in the non-motile fliG deletion mutant. This implies that for certain chromosomal regions gene loss may occur by a rapid adaptive process whereas for other regions a slower genetic drift process probably dominates. . Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. For bacterial obligate endosymbionts and intracellular pathogens that have evolved from free-living bacterial species with large genomes, their small genomes are most likely the result of increased genetic drift associated with an intracellular lifestyle and population bottlenecks [9][18]. No, Is the Subject Area "Salmonella typhimurium" applicable to this article? The apparent deletion rate at each of 11 examined chromosomal locations varied from 0.5109 to 2.2108/cell/generation (Figure 1B). Sequencing was performed using the SNP&SEQ Technology Platform in Uppsala. Objective: Phocaeicola (P.) vulgatus (formerly Bacteroides vulgatus) is a highly abundant and ubiquitous member of the human gut microbiota, associated with human health and disease, and therefore represents an important target for further investigations. government site. Then the DNA was cut inside the agarose plug with xbaI (Fermentas) in 1RE buffer for 2 h at 37C. No, Is the Subject Area "Deletion mutation" applicable to this article? That method has since been developed for other organisms, particularly research animals, like mice. Apart from the deletions identified at the transposon insertion point, these 30 mutant strains had no other sequence changes, allowing us to determine how deletions that were isolated at the different chromosomal locations affected bacterial fitness. the contents by NLM or the National Institutes of Health. Finally, from our genome-wide determination of deletion rates and previous studies [38], [39] it appears as if the terminus region is a hotspot for recombination, implying that genes in the replication terminus region are more prone to loss. Briefly, the DNA was fragmented using nebulization with compressed air at 3235psi for 6 min. (S4) in Text S1: s=0.005 (blue) and s=0.05 (red); N=107 (solid), N=108 (dashed), and N=109 (dotted). Phenotypic characterization of multiple deletion strains. Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. Moran NA, McCutcheon JP, Nakabachi A. Genomics and evolution of heritable bacterial symbionts. This is required in place of a gene knockout if the null mutation would lead to embryonic death,[8] or a specific tissue or cell type is of specific interest. Further these rough isolates 2B-7 to 2B-11 . In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Gene knockin is similar to gene knockout, but it replaces a gene with another instead of deleting it. After 1000 generations individual colonies from 3 of the 6 lineages and whole populations of the remaining 3 lineages were chosen for whole-genome sequencing. (A) Schematic representation of the Salmonella typhimurium LT chromosome and the structure and insertion points of the Tn10 construct used for measurements of deletion rates. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. The bacteria in the large culture are induced to express the target gene through addition of a chemical signal to the culture medium. [2] The construct can be delivered to stem cells either through microinjection or electroporation. Genomic DNA from 25 independent strains carrying deletions at 10 different locations was prepared using the Qiagen genomic tip 500 G kit according to instruction from the manufacturer (Qiagen). A complete gene knockout permanently inactivates the gene, while a conditional gene knockout allows for the gene to be turned off and on at specific times or in specific tissues. All plasmids were prepared using the E.Z.N.A plasmid mini kit (Omega Bio-Tek) and transferred between strains using transformation or transduction. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Every day 1.5 ul culture (approximately 106 cells) was transferred to 1.5 ml fresh LB medium and grown over night at 37C, 200 rpm. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Then the DNA was cut inside the agarose plug with xbaI (Fermentas) in 1RE buffer for 2 h at 37C. Transcription activator-like effector nuclease, "Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases", "High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library", "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering", "TALENs: a widely applicable technology for targeted genome editing", "Efficient Gene Knockout in Goats Using CRISPR/Cas9 System", Frontiers in Bioscience Gene Knockout Database (available on archive only), List of varieties of genetically modified maize/corn, Detection of genetically modified organisms, https://en.wikipedia.org/w/index.php?title=Gene_knockout&oldid=1156175883, Creative Commons Attribution-ShareAlike License 3.0, This page was last edited on 21 May 2023, at 15:39. Strains of S. typhimurium used in this study. Curves from Eq. Sequences were analyzed with CLC genomics workbench (CLC bio, Aarhus, Denmark) with analyses of SNP, DIP and low coverage regions. Sequences were analyzed with CLC genomics workbench (CLC bio, Aarhus, Denmark) with analyses of SNP, DIP and low coverage regions. Sequencing was performed using the SNP&SEQ Technology Platform in Uppsala. Circles and squares are from the stochastic model, Eqs. The DNA fragments were end-repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase (PNK), followed by purification on a QIAquick PCR purification column (Qiagen). By plotting the change in selection coefficient (s) for each mutant grown in single cultures in LB- versus M9-medium supplemented with glycerol and for each mutant grown in single culture versus competitions in LB-medium (Figure S3AS3B) it can be seen that the fitness-increasing effect of these different deletions is conditional and depends on both growth media and assay condition. The competitors were grown separately overnight and then mixed together at 11 ratio in fresh medium. CRISPR-based gene knock-out is a powerful tool for understanding the genetic basis of disease and for developing new therapies. Bacteria were grown in standard Luria Bertani-broth (LB) or M9 minimal media supplemented with 0.2% glucose or glycerol [41]. This implies that for certain chromosomal regions gene loss may occur by a rapid adaptive process whereas for other regions a slower genetic drift process probably dominates. For each independent culture the number of chlorate-resistant, white, chloramphenicol susceptible colonies was divided by the total number of cells plated and deletion rates were calculated with either the median or P0 method [45], [46]. Finally agarose plugs were transferred to 0.5 M EDTA, pH 8.0 and stored at 4C.
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