Compositions and methods for evading bacterial defense mechanisms
The present invention features modified polynucleotide sequences that mimic host cell DNA and methods of using such sequences for the genetic engineering of bacteria that are otherwise genetically intractable.
1. A method for obtaining a syngenic polynucleotide, the method comprising:
(a) detecting, in silico, based on genomic sequences of a bacteria of interest and using epigenetic information of methylated DNA sequences of a polynucleotide sequence of the bacteria of interest, recognition sites for Restriction Modification (RM) systems and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in a heterologous polynucleotide sequence; and
(b) modifying the heterologous polynucleotide sequence to alter a plurality of said recognition sites to no longer be recognition sites in the bacteria of interest, wherein the plurality of said recognition sites includes at least one RM system and at least one CRISPR system, and thereby obtaining by mutagenesis or de novo synthesis the syngenic polynucleotide having the recognition sites that are no longer recognitions sites in the bacteria of interest and that resists restriction endonuclease degradation and CRISPR degradation, when transformed into the bacteria of interest,
wherein the bacteria of interest is selected from the group consisting of Actinobacteria, Armatimonadetes, Aquificae, Bacteroidetes, Chlamydiae, Chloroflexi, Caldiserica, Chlorobi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus - Thermus, Dictyoglomi, Elusimicrobia Euryarchaeota, Firmicutes, Fusobacteria, Fibrobacteres, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes , SRI, Synergistetes, Tenericutes , TM7 , Thermodesulfobacteria, Thermomicrobia, Thermotojae , and Verrucomicrobia.
2. The method of claim 1 , wherein a coding region sequence of the heterologous polynucleotide sequence is modified by synonymous codon substitution.
3. The method of claim 1 , wherein a noncoding region sequence of the heterologous polynucleotide sequence is modified by one or more single nucleotide polymorphisms.
4. The method of claim 1 , wherein the syngenic polynucleotide is selected from the group consisting of a plasmid, replication origin, antibiotic resistance cassette, promoter, repressor, terminator, protein coding domain, transposon, operon, linear DNA knockout cassette and a bacterial genome.
5. The method of claim 1 , wherein the syngenic polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
6. The method of claim 1 , wherein modifying the heterologous polynucleotide sequence is relative to a reference sequence.
7. The method of claim 1 , wherein the genomic sequences of the bacteria of interest, epigenetic information of methylated DNA sequences of a polynucleotide sequence of the bacteria of interest, or both are identified by Single Molecule Real Time (SMRT) sequencing of the bacterial genome of the bacteria of interest.
8. The method of claim 1 , wherein the syngenic polynucleotide is a replicative plasmid.
9. The method of claim 1 , wherein the syngenic polynucleotide recapitulates the preferential codon bias of the bacteria of interest.
10. The method of claim 1 , wherein methylations in the heterologous polynucleotide are modified via synonymous codon substitution using splicing by overlap extension (SOEing).
11. The method of claim 1 , wherein methylations in the heterologous polynucleotide are modified via enzyme that methylates adenine residues.
12. The method of claim 1 , wherein the bacteria of interest is a probiotic bacteria selected from the group consisting of any one or more of Lactobacillus species, Lactococcus species, Bifidobacterium species, Entercoccus species, Streptococcus species, Pediococcus species, Leuconostoc species, Bacillus species, and Escherichia coli species.
13. The method of claim 12 , wherein the bacteria of interest is a probiotic bacteria selected from Prevotella.
14. The method of claim 13 , wherein the bacteria of interest is P. intermedia.
15. The method of claim 1 , further comprising detecting epigenetic information of methylated DNA sequences by detecting each methylation site in the polynucleotide sequence.
16. The method of claim 1 , wherein modifying the heterologous polynucleotide sequence comprises altering all of said recognition sites to no longer be recognition sites in the bacteria of interest.
17. The method of claim 1 , wherein the bacteria of interest is a gram positive bacteria selected from the group consisting of any one or more of Pasteurella species, Staphylococci species, and Streptococcus species.
18. The method of claim 1 , wherein the bacteria of interest is a gram negative bacteria selected from the group consisting of any one or more of Escherichia coli, Pseudomonas species , and Salmonella species.
19. The method of claim 1 , wherein the bacteria of interest is any one or more infectious bacteria selected from the group consisting or any one or more of Borelia burgdorferi, Legionella pneumophilia, Mycobacteria species, Staphylococcus aureus, Neisseria gonorrheae, Nesseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridians group, Streptococcus faecalis, Streptococcus bovis, Streptococcus anaerobic species, Streptococcus pneumoniae, Enterococcus species, Haemophilus influenzae, Bacillus anthracis Corynebacerium diphtheriae , other Corynebacterium species, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia , and Actinomyces israelli.