Microbiology – Chapter 9 – Microbial systems biology
9.1 Introduction to Genomics
Microbial genetics; focused on a single gene (or a group of releated genes)
Microbial genomics; major advances in the sequencing technology made it possible
to look at the whole genome.
Genome; entire complement of genetic information
Genomics; mapping, sequencing, analyzing and comparing genomes.
Genomics; then and now
First one in 1976; 3569 nucleotides of RNA virus MS2
Second one 1977; 5386 nucleotides of DNA virus FX174
First bacterial genome in 1995; Haemophilus influenzae
First human genome in 2001 (3165000000 nucleotides)
Over 50000 sequences of Bacteria, Archaea and viruses are available in databases.
9.2 Sequencing and annotating genomes
Sequencing; determining the precise order of nucleotides in a DNA (or RNA)
molecule
Genome annotation; converting raw sequence data into a list of genes present in the
genome
Bioinformatics; the use of computers to store and analyze the sequences and
structures of nucleic acids and proteins.
DNA sequencing
First generation (classical Sanger method);
Dideoxy method, the sequence is determined by making a copy of the original single-
stranded DNA. Mixture of normal deoxyribonucleotides (dNTP) and small amounts of
the dideoxy ribonucleotides (ddNTP), one for each of the 4 bases. Capillary
electrophoresis of fragments and detection of fluorescent labels.
Sanger method was dependent on primers binding to a known sequence and was
limited around 800 nucleotides.
Second generation (Pyro-sequencing);
DNA polymerase make a growing strand from a template
strand. DNA is amplified via PCR. After incorporation
sulfurylase converts AMP into ATP. Luciferase consumes
ATP and emits light.
Generates100x faster than Sanger method
Third generation; single DNA molecules sequencer, no DNA amplification any more
Fourth generation; not optical detection anymore
Genome assembly and annotation
Sequences must be assembled before they can be analyzed.
, Genome assembly; putting the fragments in the correct order and
eliminating overlaps.
Assembly occurs by the computer. The computer looks for overlaps.
Entire genome is clones and the clones are sequences, much of the
sequencing is redundant (overbodig). Generally 7 to 10 fold coverage
(dekking)
From the genome map the annotation process can begin, annotation in order to
identify genes and other functional regions.
Bioinformatics; the use of computers to store and analyze the sequences and
structures of nucleic acids and proteins.
Finding and identifying ORFs
ORF; open reading frame that codes for a protein. Bacteria
and Archaea consist of this.
Computer search for ORFs; look for start/stop codons and
look for ribosome binding sites (RBS/shine-dalgarno site).
Codon bias; some codons are more frequently used than
others.
Genomic analysis; The final tally
Many genes encode proteins whose function remains
unknown.
70% of the total ORFs detected can be clearly identified as
genes with certain functions.
Hypothetical proteins; uncharacterized ORFs; proteins that exist but whose function
is unknow. They are likely to encode non-essential genes. They encode regulatory or
redundant (overtollige) eiwitten.
9.3 Genome size and gene content in Bacteria and Archaea
Small genomes
The smallest genomes belong to parasitic or endosymbiotic bacteria.
• Parasites range from 490 kbp to 4400 kbp
• Endosymbionts can be smaller 139 kbp (symbionts of insects, they are totally
dependent on their insect host cell for survival and nutrients in turn symbionts
provide insect with essential amino acids and other nutrients that insect
cannot synthesize)
• The minimum number of genes for a viable cell is 250-300 genes
Large genomes
Some bacteria have genomes that are as large as those of some eukaryotic
microbes.
Eukaryotes have significant amounts of non-coding DNA and bacteria do not. Some
bacterial genomes have more genes than eukaryotes, despite have less DNA.
Largest bacterial genomes
• Sorangium cellulosome (14,8 Mbp)
9.1 Introduction to Genomics
Microbial genetics; focused on a single gene (or a group of releated genes)
Microbial genomics; major advances in the sequencing technology made it possible
to look at the whole genome.
Genome; entire complement of genetic information
Genomics; mapping, sequencing, analyzing and comparing genomes.
Genomics; then and now
First one in 1976; 3569 nucleotides of RNA virus MS2
Second one 1977; 5386 nucleotides of DNA virus FX174
First bacterial genome in 1995; Haemophilus influenzae
First human genome in 2001 (3165000000 nucleotides)
Over 50000 sequences of Bacteria, Archaea and viruses are available in databases.
9.2 Sequencing and annotating genomes
Sequencing; determining the precise order of nucleotides in a DNA (or RNA)
molecule
Genome annotation; converting raw sequence data into a list of genes present in the
genome
Bioinformatics; the use of computers to store and analyze the sequences and
structures of nucleic acids and proteins.
DNA sequencing
First generation (classical Sanger method);
Dideoxy method, the sequence is determined by making a copy of the original single-
stranded DNA. Mixture of normal deoxyribonucleotides (dNTP) and small amounts of
the dideoxy ribonucleotides (ddNTP), one for each of the 4 bases. Capillary
electrophoresis of fragments and detection of fluorescent labels.
Sanger method was dependent on primers binding to a known sequence and was
limited around 800 nucleotides.
Second generation (Pyro-sequencing);
DNA polymerase make a growing strand from a template
strand. DNA is amplified via PCR. After incorporation
sulfurylase converts AMP into ATP. Luciferase consumes
ATP and emits light.
Generates100x faster than Sanger method
Third generation; single DNA molecules sequencer, no DNA amplification any more
Fourth generation; not optical detection anymore
Genome assembly and annotation
Sequences must be assembled before they can be analyzed.
, Genome assembly; putting the fragments in the correct order and
eliminating overlaps.
Assembly occurs by the computer. The computer looks for overlaps.
Entire genome is clones and the clones are sequences, much of the
sequencing is redundant (overbodig). Generally 7 to 10 fold coverage
(dekking)
From the genome map the annotation process can begin, annotation in order to
identify genes and other functional regions.
Bioinformatics; the use of computers to store and analyze the sequences and
structures of nucleic acids and proteins.
Finding and identifying ORFs
ORF; open reading frame that codes for a protein. Bacteria
and Archaea consist of this.
Computer search for ORFs; look for start/stop codons and
look for ribosome binding sites (RBS/shine-dalgarno site).
Codon bias; some codons are more frequently used than
others.
Genomic analysis; The final tally
Many genes encode proteins whose function remains
unknown.
70% of the total ORFs detected can be clearly identified as
genes with certain functions.
Hypothetical proteins; uncharacterized ORFs; proteins that exist but whose function
is unknow. They are likely to encode non-essential genes. They encode regulatory or
redundant (overtollige) eiwitten.
9.3 Genome size and gene content in Bacteria and Archaea
Small genomes
The smallest genomes belong to parasitic or endosymbiotic bacteria.
• Parasites range from 490 kbp to 4400 kbp
• Endosymbionts can be smaller 139 kbp (symbionts of insects, they are totally
dependent on their insect host cell for survival and nutrients in turn symbionts
provide insect with essential amino acids and other nutrients that insect
cannot synthesize)
• The minimum number of genes for a viable cell is 250-300 genes
Large genomes
Some bacteria have genomes that are as large as those of some eukaryotic
microbes.
Eukaryotes have significant amounts of non-coding DNA and bacteria do not. Some
bacterial genomes have more genes than eukaryotes, despite have less DNA.
Largest bacterial genomes
• Sorangium cellulosome (14,8 Mbp)
