BTY227

1
BTY227

• Lecture 2. Microbial genomes and genes

2
The genome

• Genome - total genetic material in a cell
• includes chromosomal and extrachromosomal
  elements)
• Prokaryotic genomes
• GC content
• Size
• Comparing genomes
• DNA-DNA hybridisation
• ssDNA from two organisms is incubated at 30oC
  below Tm
• Remove residual ssDNA
• Measure dsDNA
• Sequence comparisons
• In silico alignment (BLAST)

3
Genome packaging

• Nucleoid
• Localised compacted DNA complexes
• No nuclear membrane! (c.f. eukaryotes)
• Contain 60 DNA, 30 RNA and 10 protein
• Usually single circular chromosome
• Dividing cells can have 2 chromosomes (genome
  copies)
• Some cells have more than one (Vibrio,
  Deinococcus)
• Many bacterial species have extrachromosomal
  elements (plasmids) not encoding core functions
  (degradative enzymes, antibiotic resistance etc).

4
Genome composition

• Microbial genomes vary in size and composition
• E. coli 4.6Mbp 50 Average
• B. subtilis 4.2 Mbp 43 Average
• C. jejuni 1.7Mbp 31 Low
• P. aeruginosa 6.3 Mbp 67 High
• A. aeolicus 1.5 Mbp 43 Average
• T. maritima 1.8 Mbp 46 Average
• P. furiosus 1.8 Mbp 42 Average
• M. jannaschii 1.7 Mbp 31 Low
• S. cerevisiae 13 Mbp 38 Low/average

5
Genome mapping

• Genome sequencing
• Preparation of libraries
• Clone sequencing
• Assembly
• Scaffolds and contigs
• Annotation
• HTP sequencing
• 454, Sylexa

6
Comparisons of sequencing technologies
7
Genome composition

• An average bacterial genome will have 2000 4000
  ORFs
• E. coli genome (4.6 Mbp)
• 4,288 predicted genes (ORFS) based on size,
  presence of Start and Stop codons
• 5 - 6 - cell ad membrane structure
• 12 -14 - transport proteins
• 10 - central C metabolism and energy generation
• 4 - regulatory (transcription factors, repressor
  and activator proteins)
• 8 - replication, transcription, and translation
  machinery
• 60 - unknown function
• Bacterial ORFs are aligned along the genome
• Some overlap
• Little junk DNA between ORFs
• Can be transcribed in either direction
• Genes for related functions are often scattered
  throughout the genome
• Genes for very closely related function (e.g.,
  synthesis of a product) may be assembled into
  operons, and co-transcribed.

8
Genome organisation
Transport Replication Energy metabolism Unknown
function
9
Gene (ORF) structure
RNA polymerase recognition binding (Pribnow
box)
Non-template strand Template strand
-35 -10
5 3
3 5
Promoter Antileader
Coding region
Antitrailer Terminator
Transcription
AUG
5
3
Shine Dalgarno sequence (ribosome binding site)
Direction of translation
10
Consensus sequences
11

• Operon a region of genome encoding one of more
  structural genes (ORFs) where expression is
  controlled by a single operator.
• Bacterial Lac operon (see Prescott, p278)
• Bacterial nitrile hydratase operon

12
Geobacillus smithii nitrile hydratase operon
structure
Terminator
-10
cbiN
Ferredoxin homologue
P14k

• and a ORFs encoding the b and a
  subunits of the a2b2 nitrile hydratase enzyme
• P14 Accessory protein
  involved in folding of native NHase
• Amidase Second enzyme in the NHase
  pathway
• Fd homologue Unknown function
• Cbi proteins Involved in cobalt uptake
• cbiM homology to component of putative cobalt
  uptake operon
• cbiN membrane spanning - putative anchor protein
  for cbiO
• cbiQ membrane spanning - putative anchor protein
  for cbiO
• cbiO homology to ATP hydrolysing Co-uptake
  protein