Molecular Basis of Bacterial Infections Evelien Floor
Bacterial genomics
Introduction into genomics
The genome is all the DNA in a cell. In bacteria this consists of the chromosomal and plasmid DNA.
The genome includes genes, intergenic sequences and repeats. Genetics is the study of inherited
phenotypes and genomics is the study of genomes. However, genetics is a major tool for functional
genomics.
Two definitions for genomics:
An operational definition:
o Genomics is the application of high throughput automated technologies to study
sequences, gene organization and mutations at the DNA level
A philosophical definition:
o A wholistic or systems approach to study the information flow within a cell and
evolutionary relatedness
Genomics has led to a new scientific vocabulary: transcriptome, proteome, secretome, virulome,
metabolome, resistome and interactome.
There is a huge interest in genomics because it provides a comprehensive list of genes (and the
proteins they encode) for the entire organism: genome wide understanding of systems and networks
can be initiated. It also provides a global picture of genome organization: clustered functions in
operons. It allows identification and distribution of gene families between phylogenetic lineages.
With genomics it is possible to compare the global genetic composition of different organism that
occupy the same niche/different niches: genotype-phenotype comparisons.
The creation of the field of genomics was made possible by the development of new technologies
that made it possible to sequence entire genomes. The costs per Mb of DNA have declined over the
last 20 years while the output of Mb DNA per day have increased in the last 20 years.
There is a difference between functional and comparative genomics. The comprehensive study of
microbial pathogenesis and the interaction between pathogens and their host belongs to functional
genomics. Just like the selection of potential candidates for the rational development of new
therapeutic agents and vaccines. Identification of molecular targets suitable for microbial
identification, typing, and for use as markers of e.g. anti- microbial resistance, risk and severity of
disease belongs to functional and comparative genomics.
Functional genomics
Functional genomics is deducing global information about the function of DNA sequences. There are
two types of experiments: RNA-seq and TnSeq.
RNA-seq
With RNA-seq changes in the expression of
different genes between different
experimental conditions can be compared.
First bacterial cells are treated, and a control
condition is set. Then the RNA is extracted,
and cDNA is made. This cDNA will be
sequenced. Subsequently the data will be
analysed; the more reads the higher the
expression of a gene. Key genes or hits can
be validated with qPCR. After validation
mechanistic studies can be performed.
1
, Molecular Basis of Bacterial Infections Evelien Floor
Another form of RNA-seq is dual RNA-seq. With dual RNA-seq the interaction between bacteria and
the host cell is examined, so both cell types are used.
TnSeq
With TnSeq the function of genes between different experimental conditions can be compared. With
TnSeq it is possible to find out which genes are important for a certain phenotype or for survival in a
certain niche. This is done by disruption of genes or gene libraries.
With TnSeq a transposon is inserted into the
genomic DNA of bacteria with transposase.
Genes are randomly hit by the transposon; in
every cell there is a single insertion. The library
will be exposed to two conditions: a challenge
and control condition. The challenge condition
is for instance with a particular antibiotic. In
the challenge condition some bacteria died
because the gene needed for this condition
was absent. After a while the DNA is isolated
and sequenced for both conditions. This way,
genes that are important for survival under
particular conditions can be identified.
After functional genomics it is possible to study identified targets in more detail via gene knockouts.
This evaluation is possible in vitro or in vivo in mutant vs WT
Comparative genomics
With comparative genomics it is possible to find sequence signatures to infer gene functions or
functions in non-coding regions. This can be done with BLAST. This way, homologs in other organisms
can be found to determine the function of the gene. It is possible to use specific databases with
virulence and resistance genes.
With comparative genomics it is also possible to look at variation in bacterial genome size. There is a
wide range in genome sizes of bacteria: 0.6 to 9 Mb. The vast majority of a bacterial chromosome
consists of coding sequences (~80% of the DNA molecule). The basic blueprint is very similar:
numerous operons, few repeated genes (compared to eukaryotes) with the exception of parasitic
DNA elements like IS-elements.
Changes in genome size reflect differences in gene content, in biochemical capabilities and, hence, in
the range of environments available to particular microbial lineages.
Core, accessory and Pangenome
The genes that are present in all strains
of a bacterial species are called the
core genome. Genes that aren’t
present in all the strains but are
present in some strains are called the
accessory genome. The core genome
plus the accessory genome together is
the Pangenome. The more strains are
sequenced the more the Pangenome
increases and the core genome
decreases.
2
Bacterial genomics
Introduction into genomics
The genome is all the DNA in a cell. In bacteria this consists of the chromosomal and plasmid DNA.
The genome includes genes, intergenic sequences and repeats. Genetics is the study of inherited
phenotypes and genomics is the study of genomes. However, genetics is a major tool for functional
genomics.
Two definitions for genomics:
An operational definition:
o Genomics is the application of high throughput automated technologies to study
sequences, gene organization and mutations at the DNA level
A philosophical definition:
o A wholistic or systems approach to study the information flow within a cell and
evolutionary relatedness
Genomics has led to a new scientific vocabulary: transcriptome, proteome, secretome, virulome,
metabolome, resistome and interactome.
There is a huge interest in genomics because it provides a comprehensive list of genes (and the
proteins they encode) for the entire organism: genome wide understanding of systems and networks
can be initiated. It also provides a global picture of genome organization: clustered functions in
operons. It allows identification and distribution of gene families between phylogenetic lineages.
With genomics it is possible to compare the global genetic composition of different organism that
occupy the same niche/different niches: genotype-phenotype comparisons.
The creation of the field of genomics was made possible by the development of new technologies
that made it possible to sequence entire genomes. The costs per Mb of DNA have declined over the
last 20 years while the output of Mb DNA per day have increased in the last 20 years.
There is a difference between functional and comparative genomics. The comprehensive study of
microbial pathogenesis and the interaction between pathogens and their host belongs to functional
genomics. Just like the selection of potential candidates for the rational development of new
therapeutic agents and vaccines. Identification of molecular targets suitable for microbial
identification, typing, and for use as markers of e.g. anti- microbial resistance, risk and severity of
disease belongs to functional and comparative genomics.
Functional genomics
Functional genomics is deducing global information about the function of DNA sequences. There are
two types of experiments: RNA-seq and TnSeq.
RNA-seq
With RNA-seq changes in the expression of
different genes between different
experimental conditions can be compared.
First bacterial cells are treated, and a control
condition is set. Then the RNA is extracted,
and cDNA is made. This cDNA will be
sequenced. Subsequently the data will be
analysed; the more reads the higher the
expression of a gene. Key genes or hits can
be validated with qPCR. After validation
mechanistic studies can be performed.
1
, Molecular Basis of Bacterial Infections Evelien Floor
Another form of RNA-seq is dual RNA-seq. With dual RNA-seq the interaction between bacteria and
the host cell is examined, so both cell types are used.
TnSeq
With TnSeq the function of genes between different experimental conditions can be compared. With
TnSeq it is possible to find out which genes are important for a certain phenotype or for survival in a
certain niche. This is done by disruption of genes or gene libraries.
With TnSeq a transposon is inserted into the
genomic DNA of bacteria with transposase.
Genes are randomly hit by the transposon; in
every cell there is a single insertion. The library
will be exposed to two conditions: a challenge
and control condition. The challenge condition
is for instance with a particular antibiotic. In
the challenge condition some bacteria died
because the gene needed for this condition
was absent. After a while the DNA is isolated
and sequenced for both conditions. This way,
genes that are important for survival under
particular conditions can be identified.
After functional genomics it is possible to study identified targets in more detail via gene knockouts.
This evaluation is possible in vitro or in vivo in mutant vs WT
Comparative genomics
With comparative genomics it is possible to find sequence signatures to infer gene functions or
functions in non-coding regions. This can be done with BLAST. This way, homologs in other organisms
can be found to determine the function of the gene. It is possible to use specific databases with
virulence and resistance genes.
With comparative genomics it is also possible to look at variation in bacterial genome size. There is a
wide range in genome sizes of bacteria: 0.6 to 9 Mb. The vast majority of a bacterial chromosome
consists of coding sequences (~80% of the DNA molecule). The basic blueprint is very similar:
numerous operons, few repeated genes (compared to eukaryotes) with the exception of parasitic
DNA elements like IS-elements.
Changes in genome size reflect differences in gene content, in biochemical capabilities and, hence, in
the range of environments available to particular microbial lineages.
Core, accessory and Pangenome
The genes that are present in all strains
of a bacterial species are called the
core genome. Genes that aren’t
present in all the strains but are
present in some strains are called the
accessory genome. The core genome
plus the accessory genome together is
the Pangenome. The more strains are
sequenced the more the Pangenome
increases and the core genome
decreases.
2
