advanced microbial physiology
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Advanced Microbial Physiology. Lecture 1 Minimum Bacterial Genome. Definitions:. Genome – the sum of total genes within a species of an organism Essential genes – genes absolutely required for growth and survival - PowerPoint PPT PresentationTRANSCRIPT
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Advanced Microbial Physiology
Lecture 1Minimum Bacterial Genome
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Definitions: Genome – the sum of total genes
within a species of an organism Essential genes – genes absolutely
required for growth and survival Non essential genes – genes whose
destruction does not lead to significant growth defects in a cell.
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Why study essential genes?
Essential genes are important for cellular function and physiology; to study them will reveal details about microbial physiology
Practical application: essential genes encode essential proteins which are excellent drug targets to develop new antibiotics
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Strategies for Essential ID
Saturated transposon mutagenesis Antisense expression controlling
gene expression Systematic gene knock-out (or
inability to knock-out)
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Nonhomologous recombination
No requirement for two DNAs being of the same or similar nucleotide sequences
Needs enzymes that recognize specific regions in DNA
Mechanisms include: transposition phage integration and excision resolution of cointegrates
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Transposition Transposons – DNA elements that can hop
(transpose) from one place in DNA to another
Transposons are known to exist in all organisms on earth
Movement by a transposon is called transposition, catalyzed by enzymes called transposases
Transposons usually encode their own transposases
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Transposition Many transposons are essentially cut
out of one DNA and inserted into another
Other transposons are copied and then inserted elsewhere
Donor DNA and target DNA
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Structure of a Bacterial transposon
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Structure of Bacterial Transposons
All contain repeats at their ends, usually inverted repeats (IR)
Presence of short direct repeats in the target DNA that bracket the transposon
The sites of insertion are different among target DNAs
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Types of Bacterial Transposons
Smallest bacterial transposons are called insertion sequence elements (IS elements); they only encode transposase enzymes
Composite transposons – formed by two IS elements of the same type, bracketing other genes
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Composite transposons
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Antisense expression
Antisense RNA expression. Random cloning and expression of short pieces of genomic DNA on a plasmid in an microorganism to elucidate the function of the genes
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Conditional Antisense Inhibition
of Protein Synthesis
Antisense cell
Noprotein
XAntisense RNA
Inducible promoter
mRNA
Normal cell
Protein
mRNA
Plasmid DNA
DNADNA
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Shotgun Antisense Expression Determines Essentiality of Genes
Non essential geneblocked by antisense
Essential geneblocked by antisense
Millions of random DNA fragments
No cell growthmRNA
Essential Protein
DNA
Pathogen genome
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Ultra-Rapid Functional Genomics
Identify >100 essential gene drug targets per monthAntisense
(+ inducer)No antisense(- inducer)
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Gene Knock-out
Gene replacement (knock-out): also known as reverse genetics. The purpose is to remove (knock-out) most of one gene and see what happens to the phenotype of the organism. Suicide vector is used.
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Number of essential genes determined for various bacterial species.
Species No. of Essential Genes Methods Used ReferencesBacillus subtilis 271 Gene disruption 43
Mycoplasma genitalium 265-350 Transposon Mutagenesis 39Streptococcus pneumoniae 113 Gene disruption 72
Haemophilus influenzae 478 Transposon Mutagenesis 1Escherichia coli 620 Transposon Mutagenesis 28
E. coli (PEC data base) 250 Various methods 52Staphylococcus aureus 150 Antisense expression 41Staphylococcus aureus 168 Antisense expression 22Typical bacterial species 206 Theoretical analyses 32