Advanced Microbial Physiology

Lecture 1Minimum 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

Antisense cellAntisense cell

Noprotein

XXAntisense RNAAntisense RNA

Inducible promoterInducible promoter

mRNAmRNA

Normal cellNormal cell

Protein

mRNAmRNA

Plasmid DNA

DNADNA

Shotgun Antisense Expression Determines Essentiality of Genes

Shotgun Antisense Expression Determines Essentiality of Genes

Non essential geneblocked by antisenseNon essential geneblocked by antisense

Essential geneblocked by antisenseEssential geneblocked by antisense

Millions of random DNA fragmentsMillions of random DNA fragments

No cell growthNo cell growthmRNAmRNA

Essential Protein

DNA

Pathogen genome

Ultra-Rapid Functional GenomicsUltra-Rapid Functional Genomics

Identify >100 essential gene drug targets per monthIdentify >100 essential gene drug targets per monthAntisense

(+ inducer)Antisense(+ inducer)

No 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.

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