Bacterial Genetics
Horizontal Gene Transfer
Horizontal gene transfer can lead to the rapid dissemination of antibiotic resistance gene or convert a relatively harmless microbe into a deadly one!
Bacterial Transformation — Uptake of “Naked” DNA from Environment
Food (carbon) source
As a means to repair damaged DNA
Acquire new traits to adjust to changes
Transformation Requires Competence
Competence is regulated by a quorum sensing mechanism to ensure the bacteria is likely to encounter DNA
Transformation Process
Double stranded DNA is bound to surface proteins
Enzymes cleave one strand of the DNA while proteins coat the other strand
The single strand of DNA is brought into the cell and incorporated by homologous recombination mechanism
Homologous Recombination
Requires long stretch of homologous (similar) DNA sequence such as in crossing over during meiosis
The incoming single strand is coated with RecA protein and forms a temporary triplex with chromosomal DNA; eventually displacing one original strand
Differences between incoming strand and original strand (heteroduplex) are fixed by replication or DNA repair processes
Natural Transformation
Can be used to vary surface antigens of microbes as a way to avoid our immune system
Bacteria may possess various genetic versions for the sample molecule but only one is expressed at a time
When a cell dies, its DNA enters the environment where it may be picked up by neighboring bacteria and incorporated
Bacteria population now expresses different surface molecules from original population
Bacterial Conjugation — Exchange of Plasmid DNA Requiring Cell to Cell Contact
Plasmid: small circular piece of DNA, not essential for bacteria but provides beneficial genes such as antibiotic resistance
Bacteria may have different types of plasmids to add to their genomes
Q: You are setting up a conjugation experiment using the two bacterial strains listed below (BB4 and SCSC1). You mix the two bacterial strains together in liquid broth media overnight. Would genes go from BB4 to SCSC1 or from SCSC1 to BB4? Explain
A: BB4 to SCSC1 because of plasmid
Q: How would you select for the transconjugant in the experiment above? i.e. what media / what would you add to the media in order to select only for the bacteria with new traits
Bacterial Conjugation — Hfr Strains Allow the Transfer of Chromosomal DNA
Most plasmids are independent episomes. They replicate independent of the chromosome, and they do not need to integrate into the chromosome
However, the F plasmid can insert itself into the host chromosome. The resulting cellis called Hfr for high frequency recombination
The conjugation genes still function even though plasmid is inserted into the chromosome
DNA transfer starts in the middle of the plasmid and moves around the chromosome
Typically only parts of the chromosome are transferred but these could be incorporated by homologous recombination
Only part of plasmid DNA is transferred so recipient remains F-
Prior to DNA sequencing, Hfr strains were used to map the position of genes in the E. coli chromosome
Gene positions were reported in minutes which corresponds to the amount of time required to transfer the gene to the recipient cell
F Prime
Another variation of the F system
The F plasmid can pop out of the chromosome just as easily as inserting
On rare occasions the separation isn’t perfect and some chromosomal DNA becomes part of the plasmid
Conjugation occurs as normal and the plasmid with extra DNA is transferred to the recipient
Recipient would become F’ because it has the plasmid containing extra DNA
Generalized Transduction
Viruses mistakenly package degraded chromosomal DNA into capsid. DNA is transferred to new host and incorporated by homologous recombination
Specialized Transduction
Lysogenic phage has an imprecise excision event taking some chromosomal DNA
Which Mechanism of Horizontal Gene Transfer is Responsible?
Prototrophic bacteria: strains that can grow in minimal media with only
Carbon, nitrogen, phosphorus, vitamins, ions, nutrients
Have genes required to MAKE everything else
Auxotrophic bacteria: lack one, multiple genes encoding enzymes required for synthesis of AA, nucleotides, substances not add
Addition of DNAse to mixture tube could be used to rule out transformation
If colonies still occur on selective media, then horizontal gene transfer occurs by other means
Blocking Cell to Cell Contact Would Prevent Conjugation
Plate on media that selects for the recombinant bacteria. The presence of colonies indicates that horizontal gene transfer still occurred (ruled out conjugation)
The lack of colonies suggests that conjugation is necessary to make recombinant
Horizontal Gene Transfer and Species Designation
What is the common biological definition of a species?
How would bacteriologists define species
High degree of relatedness in “house keeping” genes (i.e. informational genes)
These genes should be orthologs (common origin & function) not paralogs (common origin but different function)
Common ecotype (ecological niche) habitat and life history
Genetic relatedness of bacterial species is determined byy
Total genome DNA hybridization > 70%
SSU rRNA similarity > 97%
Average nucleotide identity of orthologs is >95%
Q: How would this model affect the species designation of humans?
A:
Difficulties of Species Designation
Heliobacter pylori genomes can differ by as much as 7%, but their ecotype is identical living in stomach and causing gastritis
Bacillus anthracis (anthrax) has a nearly identical genome to Bacillus thringiensis (insect pathogen-Bt) but very different ecotype
Phage Therapy vs Antibiotic Resistance & Horizontal Gene Transfer
Phage PRD1 is “male-specific” phage that attaches to proteins for mating pair formation
RP4 plasmid is multi-drug resistant plasmid
Addition of PRD1 on day 1 along or every day causes loss of RP4 plasmid
CRISPR + Phage Therapy vs Antibiotic Resistance & Horizontal Gene Transfer
CRISPR CAS system inserted into lysogenic (temperate) phage genome
Guide RNAs to target antibiotic resistant genes ndm-1 & ctx-M-15
CAS / CRISPR phage targets antibiotic resistance genes on plasmid causing destruction of plasmid
Also engineered to provide protection against phage used in phage therapuy
Cells that are resistant to CAS / CRISPR phage will be killed by 2nd phage
Cells will either lose plasmid and become sensitive to antibiotics which can be used to kill them, or the 2nd phage will kill them
Lysogenized bacterial cells containing complete CRISPR / CAS phage cannot be transformed with resistance plasmid pNDM or pCTX