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 Non essential genes – genes whose destruction does not lead to significant growth defects in a cell.

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

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

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

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

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

Structure of a Bacterial transposon

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

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

Composite transposons

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

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

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

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)

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.

Number of essential genes determined for various bacterial species.