Bacterial quantitative trait-locus mapping

Patent metadata

Field	Value
Publication number	US-12362039-B2
Application number	US-202016901514-A
Country	US
Kind code	B2
Filing date	Jun 15, 2020
Priority date	Jun 14, 2019
Publication date	Jul 15, 2025
Grant date	Jul 15, 2025

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.

First claim

What is claimed is:

1. A method for identifying quantitative trait-loci in bacteria comprising:
providing two starting bacterial strains, wherein the two starting strains comprise selectable markers compatible with each other in that either at least one of the two starting bacterial strains is a wild type prototroph strain without an antibiotic resistance gene and the other starting bacterial strain is double auxotroph and comprises two different antibiotic resistance genes, or the two starting bacterial strains are each a single auxotroph strain comprising an antibiotic resistance gene and differ from each other in auxotrophy and antibiotic resistance;
inducing at least two rounds of genomic recombination, wherein the at least two rounds of genomic recombination comprises
(i) inducing a first round of genomic recombination between a population of a first starting strain and a population of a second starting strain to obtain first generation progeny bacteria,
(ii) selecting among the first generation progeny bacteria to obtain two populations of bacteria that have undergone genomic recombination using two different selective media, wherein each selective medium kills both starting bacterial strains and allows only one of the two populations of the first generation progeny bacteria to live,
(iii) inducing a second round of genomic recombination between one of the two selected populations of the first generation progeny bacteria and a population of bacteria that comprises a selectable marker compatible with the first selected population of the first generation progeny bacteria and that is selected from the group consisting of:
(a) the other of the two selected populations of the first generation progeny bacteria,
(b) a population of a strain that is otherwise genetically identical to one of the two starting strains,
(c) a population of a strain that is different from both starting strains, and
(d) a population of bacteria selected from progeny bacteria from induced genomic recombination between two different starting strains, thereby obtaining second generation progeny bacteria;

(iv) selecting among the second generation progeny bacteria for two populations of bacteria that have undergone genomic recombination using two selective media, wherein each selective medium kills both populations of bacteria used in the second round of genomic recombination, and allows only one of the two populations of the second generation progeny bacteria to live, and
(v) obtaining the selected two populations of the second generation progeny bacteria as two populations of a final generation progeny bacteria, or inducing at least one more round of genomic recombination and selecting between a first selected population of the second generation progeny bacteria and a population of bacteria that comprises a selectable marker compatible with the first selected population of the second generation progeny bacteria to obtain two populations of a final generation progeny bacteria;

determining the sequences of the genomes of the two populations of the final generation progeny bacteria, thereby determining genetic variations within the genomes of the two populations the final generation progeny bacteria;
determining at least one phenotype of the two populations of the final generation progeny bacteria; and
performing a population-wide analysis to identify genetic variations that associate with the at least one phenotype, thereby identifying quantitative-trait loci that are associated with the at least one phenotype.

2. The method of claim 1 , wherein the antibiotic resistance genes are inserted into the bacterial genomes to disrupt genes essential for bacterial survival.

3. The method of claim 1 , wherein the at least one phenotype comprises one or more of bacterial growth rate, resistance to a chemical compound, production of a target biochemical, ability to transfer into new environmental niche, ability to persist in a new environmental niche, ability to modulate a host phenotype when established in the host microbiome, ability to inhibit growth of a target organism, and ability to grow under restrictive conditions.

4. The method of claim 1 , wherein the genomic recombination at each round is achieved by protoplast fusion-induced homologous recombination.

5. The method of claim 1 , wherein the bacteria are Gram-negative.

6. The method of claim 5 , wherein the Gram-negative bacteria are selected from the group consisting of Pseudomonas, Novosphingobium, Sphingobium, Sphingomonas, Escherichia, Zymomonas , and Cupriavidus.

7. The method of claim 5 , wherein the genomic recombination at each round is achieved by protoplast fusion-induced homologous recombination comprising (a) treating the bacterial strains with an antibiotic that inhibits peptidoglycan biosynthesis; and (b) inducing the treated bacterial strains to undergo protoplast fusion in a high osmolarity medium comprising between 0.5 M and 1.2 M sucrose.

8. The method of claim 7 , wherein the antibiotic that inhibits peptidoglycan biosynthesis is Fosfomycin.

9. The method of claim 7 , wherein the protoplast fusion is achieved by chemofusion or electrofusion.

10. The method of claim 9 , wherein the chemofusion is achieved using polyethylene glycol.

11. The method of claim 1 , wherein the bacteria are Gram-positive.

12. The method of claim 11 , wherein the Gram-positive bacteria are selected from the group consisting of genera Bacillus, Corynebacterium, Streptomyces, Propionibacterium, Clostridium , and Lactobacillus.

13. The method of claim 11 , wherein the genomic recombination at each round is achieved by protoplast fusion-induced homologous recombination comprising (a) treating the bacterial strains with lysozyme, and (b) inducing the treated bacterial strains to undergo protoplast fusion in a high osmolarity medium comprising between 0.5 M and 1.2 M sucrose.

14. The method of claim 13 , wherein the protoplast fusion is achieved by chemofusion or electrofusion.

15. The method of claim 14 , wherein the chemofusion is achieved using polyethylene glycol.

16. The method of claim 1 , wherein the population-wide analysis comprises:
(a) mapping the sequences of each strain of the final generation progeny bacteria to the starting bacterial strains; and
(b) determining the presence of single nucleotide polymorphisms and insertions based on the mapping in (a).

17. The method of claim 16 , wherein the method further comprises:
(c) performing de novo assembly of genomes of each strain of the final generation progeny bacteria; and
(d) determining structural variants by comparing the assembled genomes of the final generation progeny bacteria to the genomes of the starting bacterial strains.

18. The method of claim 1 , wherein the population-wide analysis comprises a variant calling step that comprise mapping the genotypes of the final generation progeny bacterial strains to the genotype of the starting bacterial strains.

19. The method of claim 1 , wherein the population-wide analysis comprises a pruning step to cluster away variants that have indistinguishable association to a given phenotype via a parameter sweep function that scans every variant as a potential start position for pruning.

20. The method of claim 1 , wherein the population-wide analysis comprises a haplotype calling step.

21. The method of claim 1 , wherein the population-wide analysis comprises performing a Continuous Wavelet Transform Analysis.

Assignees

• Ut Battelle Llc

Inventors

• Michener Joshua K
• Jacobson Daniel A
• Vasileva Delyana
• Streich Jared

Classifications

• C12Q1/02

  involving viable microorganisms · CPC title

• G16B20/00

  ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations · CPC title

• C12N15/03

  Bacteria · CPC title

• G16B25/00

  ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression · CPC title

• C12N15/902

  using homologous recombination · CPC title