Lecture 1- microbial genome structure an function Flashcards
what are the characteristics of bacterial chromosomes?
Contain housekeeping genes
Most abundant genes are involved in metabolic processes
Also involved in transport, replication, translation, pathogenicity
Coding regions are usually continuous, no introns (normally) *this is also the case in certain parasitic protozoans such as trypanosomes (single cell eukaryotes)
Genes can be grouped into function
what are genes to increase virulence?
Example: Pathogenicity islands 10-200 Kilobases
Salmonella, Shigella, enteropathogenic E. coli, Staphylococcus aureus
Examples: Attachment to host cells, secretion of virulence factors (toxin genes, uropathogenic proteins)
Secretion systems are transport systems in bacteria and has a role in pathogenesis. Salmonella have several path islands and some can also be found on plasmids. They are thought to be conferred by horizontal gene transfer.
Roles in attachment and uses secretion systems to inject virulence factors such as toxins
how is DNA replicated in E.coli?
DNA polymerases catalyze the addition of nucleotides (A,T,G,C)
DNA synthesis begins at the origin of replication in prokaryotes
DNA synthesis is bidirectional in prokaryotes
Two replication forks moving in opposite directions
DNA Polymerase III adds 1,000 nucleotides per second
what is the process of replisome?
- Helicase unwinds parental DNA
- Single Stranded Binding proteins keep strands apart
- DNA Gyrase breaks tension in strands
- DNA primase will synthesis RNA primer
- DNA pol III synthesises new strand
- Replisome moves along strand
Leading strand replicates continuously
what is E.colis replication machinery?
> 30 proteins forming complex called replisome
Helicase unwinds parental DNA
Single stranded DNA binding proteins (SSBs) keep strands apart
DNA gyrase (topoisomerase) breaks tension in strand (drug target-essential in bacteria)
Primase synthesises RNA primer
DNA pol III attaches and synthesises new DNA strand
what are characteristics of plasmids?
Found in prokaryotes-additional genetic element
Replicate independently of the host chromosome
Many double stranded DNA and circular
Variable abundance
Relatively few genes (<30)
Plasmids can be made artificially-genetic engineering
what information do plasmids carry?
Examples:
Antibiotic resistance plasmids, e.g. penicillinase
Virulence plasmids
-Bacteria acquire new traits as an advantage to survive-in soil, living amongst fungi & bacteria that produce antibiotics
Why are bacterial plasmids so useful in labs?
Expression of proteins
Sequencing DNA
Stability
Transferring information: Knock-ins and Knock-outs
How does an artificial plasmid work?
Origin of replication
Antibiotic resistance selection marker
Multiple cloning site: DNA sequence that contains several restriction enzyme recognition motifs
Promoter: initiates transcription of the gene of interest
Protein tag –visualisation or purification
Why are we all so interested in sequencing microbes and what do we do with all this data?
Gene prediction…discovery of new genes unique to microbes, annotation/bioinformatics
Understanding genomic structure/mechanisms
Diagnostics/Identification
Microbiome studies
what is bacterial conjugation? CELL TO CELL CONTACT part 1
Only mechanism that is cell to cell.
The transfer of F+ to F- is directional, (from an F+ to a F- cell). It is thought that there is only one F plasmid per cell.
F plasmid is a specific, large plasmid, often found in Enterobacteria, commonly E.coli. The F-plasmid contains F-factor that codes for the sex pilus, and therefore conjugation transfer.
F plasmids associated with virulence factors and antibiotic resistance genes.
Sex pilus makes contact
with F– recipient cell.
Sex pilus contracts,
bringing cells together.
Type IV secretion system
is constructed and joins
two cells.
what is bacterial conjugation part 2-
Relaxosome is a complex of proteins encoded by the F-plasmid.
Relaxosome makes a nick at origin of transfer
Begins to separate one DNA strand.
Intact strand is replicated by the rolling-circle mechanis.m
Accessory proteins of the
relaxosome are released.
DNA/relaxase complex is recognized by the coupling
factor and transferred to the secretion system.
what is bacterial conjugation part 3-
The secretion system pumps the DNA/relaxase
complex into the recipient cell.
As the DNA enters, the F plasmid DNA is replicated to become double stranded
what is bacterial transformation?
There are many factors involved in this process such as how DNA can persist in soil, if the DNA is degraded in the environment or inhibited by soil components. So this process doesn’t happen all the time. How frequently DNA is released from cells and what conditions are required for DNA to bind, take up and possibly express the DNA. It is multi-factorial.
how does gene cloning and protein expression use microbiology?
Microbes have turned out to be incredibly useful in all molecular studies. Whether or not you are interested in genetic diseases, cellular processes sports science, or Infection biology, all fields in biology are now using molecular methodologies.
I see molecular biology as a toolkit for studying interesting questions.
Gene cloning studies-sequencing to investigate mutation/population based SNPs
Not talking dolly the sheep-talking about cloning a single gene
Gene expression studies to investigate genes/proteins from any biological system-bacterial proteins to mammaliam proteins
how do you clone a gene of interest?
step 1 = DNA amplification –> by DNA then PCR then agarose gel
step 2 = cloning –>Restriction endonuclease makes staggered cut at recognition site.
Sticky ends used to join DNA into plasmid with DNA Ligase
step 3 = transformation
step 4 = antibiotic selection –> Cells selected –only containing Plasmid with recombinant DNA. only a cell with the plasmid will survive
