1. Advanced Microbial Physiology
Lecture 1
Minimum Bacterial Genome

2. 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.

3. 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

4. Strategies for Essential ID
Saturated transposon mutagenesis
Antisense expression controlling gene expression
Systematic gene knock-out (or inability to knock-out)

5. 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

6. 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

7. 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

9. Structure of a Bacterial transposon

10. 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

11. 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

12. Composite transposons

19. 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

20. Conditional Antisense Inhibition of Protein Synthesis
Inducible promoter
Normal cell
Antisense cell
Plasmid DNA No
protein
Protein
Antisense RNA X
mRNA
mRNA
DNA
DNA

21. Shotgun Antisense Expression Determines Essentiality of Genes
Pathogen genome
Millions of random DNA fragments
Non essential gene
blocked by antisense
Essential gene
One of Elitra’s target discovery tools utilizes shotgun antisense. In this process, the entire genome of a pathogen is fragmented into millions of random pieces which are then cloned into an expression vector and introduced back into the host pathogen. If the cloned fragment is a piece of a non-essential gene, then antisense expression blocks only non-essential processes. However, if the cloned fragment is a piece of an essential gene, then antisense expression may block expression of the essential protein and the cell cannot grow.
No cell growth
mRNA
Essential Protein
DNA

22. Ultra-Rapid Functional Genomics
This is a photo of real screening plates where clone expressing AS to essential gene targets are circle in red.
In the absence of inducer (and no expression of AS), the colonies will grow.
In the presence of inducer, AS is expressed and the colonies will not grow.
In order to identify all essential genes, we have developed specialized robots for the picking, scanning and analysis of up to 1.5 M colonies a month.
This has yielded in excess of 100 essential genes per month.
Identify >100 essential gene drug targets per month
Antisense
(+ inducer)
No antisense
(- inducer)

24. 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.

30. Number of essential genes determined for various bacterial species.