Bacterial quantitative trait-locus mapping
Inventors
Michener, Joshua K. • Jacobson, Daniel A. • Vasileva, Delyana • Streich, Jared
Assignees
Publication Number
US-12362039-B2
Publication Date
2025-07-15
Expiration Date
2040-06-15
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
This disclosure provides methods for performing quantitative-trait loci (QTL) analysis in bacteria. The methods of the instant disclosure utilize multiple rounds of protoplast fusion-induced genomic recombination to break genetic linkages in bacterial genomes. The methods of the instant disclosure allow determining which genetic elements (QTL) are associated with phenotypic al features.
Core Innovation
The invention provides methods for performing quantitative-trait loci (QTL) analysis in bacteria by utilizing multiple rounds of protoplast fusion-induced genomic recombination to break genetic linkages in bacterial genomes. This approach enables determination of which genetic elements are associated with phenotypic features in bacteria, overcoming the lack of sexual recombination that hinders standard QTL mapping techniques in bacterial systems.
The background section identifies the problem that bacteria do not naturally perform sexual recombination at the scale necessary to break genetic linkages, making it difficult to apply established eukaryotic QTL mapping methods to bacteria. Previous methods for bacterial recombination have been limited in scope, scale, or genomic coverage, and direct measurements of recombination parameters are challenging due to mechanistic constraints.
The disclosed methods address this problem by inducing multiple rounds of protoplast fusion-induced homologous recombination between bacterial strains containing compatible selectable genetic markers. This iterative genomic recombination coupled with selective growth media allows for expansion of recombinant bacterial populations with mixed parental genomes. Sequencing and phenotyping of these populations enable population-wide analyses to identify genetic variations that statistically associate with specific phenotypes, facilitating bacterial QTL mapping.
Claims Coverage
The patent includes one independent claim focusing on a method for identifying quantitative trait-loci in bacteria and several dependent claims elaborating on specific features of the method.
Method for bacterial QTL identification via iterative genomic recombination and selection
Providing two starting bacterial strains with compatible selectable markers, inducing at least two rounds of genomic recombination between populations of these strains or their progeny, selecting recombinant populations using selective media that kill starting strains, obtaining final generation progeny populations, determining their genome sequences and genetic variations, phenotyping these populations, and performing population-wide analysis to identify genetic variations associated with phenotypes to identify quantitative-trait loci.
Use of antibiotic resistance genes disrupting essential bacterial genes as selectable markers
Selectable markers comprise antibiotic resistance genes inserted into bacterial genomes to disrupt genes essential for bacterial survival, allowing selection based on resistance and auxotrophy.
Phenotypic traits for QTL mapping
The phenotype to be determined includes bacterial growth rate, chemical resistance, target biochemical production, environmental niche transfer and persistence, modulation of host phenotype, inhibition or promotion of target organism growth, and growth under restrictive conditions.
Genomic recombination achieved by protoplast fusion-induced homologous recombination
Each round of genomic recombination is performed by protoplast fusion-induced homologous recombination.
Application to Gram-negative bacteria and specific genera
Methods apply to Gram-negative bacteria including genera Pseudomonas, Novosphingobium, Sphingobium, Sphingomonas, Escherichia, Zymomonas, and Cupriavidus.
Gram-negative genomic recombination by antibiotic treatment and protoplast fusion
For Gram-negative strains, genomic recombination involves treating bacteria with an antibiotic inhibiting peptidoglycan biosynthesis (fosfomycin), followed by protoplast fusion in a high osmolarity medium containing 0.5 M to 1.2 M sucrose.
Protoplast fusion via chemofusion or electrofusion
Protoplast fusion is achieved by chemofusion or electrofusion, with chemofusion optionally using polyethylene glycol.
Application to Gram-positive bacteria and specific genera
Methods apply to Gram-positive bacteria including genera Bacillus, Corynebacterium, Streptomyces, Propionibacterium, Clostridium, and Lactobacillus.
Gram-positive genomic recombination by lysozyme treatment and protoplast fusion
For Gram-positive strains, genomic recombination involves treating bacteria with lysozyme, followed by protoplast fusion in a high osmolarity medium comprising 0.5 M to 1.2 M sucrose.
Population-wide analysis by sequence mapping and variant detection
Population-wide analysis comprises mapping genome sequences of each final progeny strain to starting bacterial strains and determining presence of single nucleotide polymorphisms and insertions.
Structural variant detection by de novo genome assembly
Method further includes de novo assembly of genomes of final generation progeny strains and determining structural variants by comparison to starting strain genomes.
Variant calling by genotype mapping to starting strains
Includes variant calling step mapping genotypes of final progeny bacterial strains to genotypes of starting bacterial strains.
Pruning of variants with indistinguishable phenotype association
Population-wide analysis includes pruning variants that are in close genomic proximity and have indistinguishable association to a given phenotype using a parameter sweep function.
Haplotype calling step to identify recombination patterns
Population-wide analysis comprises a haplotype calling step to identify continuous sets of variants and regions prone to not recombine.
Use of Continuous Wavelet Transform analysis
Population-wide analysis includes performing Continuous Wavelet Transform analysis to identify genomic regions correlating with recombination and phenotype associations.
The claims collectively cover a comprehensive method for bacterial QTL mapping via iterative protoplast fusion-induced genomic recombination using selectable markers, selective growth, sequencing, and detailed population genetic analyses, applicable to both Gram-positive and Gram-negative bacteria with specific protocols and analytical steps to identify phenotype-associated genetic loci.
Stated Advantages
The methods allow routine generation and analysis of highly recombined bacterial strains from diverse clades.
The process efficiently breaks genetic linkages in bacterial genomes enabling QTL mapping, which was previously difficult due to lack of sexual recombination in bacteria.
The approach allows fine mapping of genetic loci associated with phenotypic traits at sub-gene resolution.
Protoplast fusion-induced homologous recombination enables large-scale genome-wide recombination resulting in mosaic chromosomes with unique phenotypic potential.
Documented Applications
Identifying genetic loci associated with bacterial growth rate.
Mapping genetic elements conferring resistance to chemical compounds.
Identifying genes influencing production of target biochemicals such as siderophores.
Analyzing bacterial abilities related to transfer into and persistence within new environmental niches.
Determining bacterial influence on host phenotypes when established within host microbiomes.
Identifying genetic determinants for ability to inhibit or promote growth of target organisms.
Studying bacterial growth under restrictive culture conditions.
Interested in licensing this patent?