Bacteria - diversity and methods of categorisation

Fundamentals of bacteria that cause the patterns in its populations

Asexual nature

Reproduce by binary fission

‘Vertical’ inheritance of genes (with some HGT)

What do bacterial populations often form (in terms of mapping phylogenies)

Tree-like phylogenies

What enables diversity in populations that reproduce asexually

Accumulation of mutations

Diversity seen even in ‘genetically monomorphic’ pathogens

What does the progressive accumulation of mutations in asexual bacterial populations lead to

Diversity

Tree-like phylogenies

Linkage disequilibrium

What is linkage disequilibrium

Non-random association of genes, inherited together more often than chance

What does this generalised tree-like phylogeny model assume

No loss of diversity

No recombination

What is the clonal population model

Fundamental population structure in asexual organisms

Shows diversification of population due to asexual reproduction with mutations

But diversity reduction occurs

What diversity reduction can occur in clonal population structures

Period selection → maintains genotype as the ‘most fit’

Bottlenecking → the next generation survive due to chance, not the ‘most fit’

What are the different types of selection forces

Diversifying (directional/positive) → changes phenotype

Stabilising (negative) → retains phenotype and fitness

Neutral variation → no impact on fitness phenotypes

What causes particular patterns to appear in bacterial lineages

Mutational changes causing different patterns in different lineages

Contradiction with the progressive accumulation of mutations in bacteria

Should produce exponential diversity → but bacterial populations are not that diverse

What two factors mean that bacteria populations are not that diverse

Muller’s ratchet

HGT

What is Muller’s ratchet

Small, asexual populations are vulnerable to accumulating deleterious mutations - causing reductive evolution

What is HGT

Horizontal gene transfer between bacteria - higher than expected in bacteria

Leads to chromosomal mosaic gene as genes are introduced and then homologous recombination adds them to the chromosome

Through transformation, transduction, and conjugation

What is the impact of HGT on bacterial population structure

Leads to varying population structures, breaks down clonal population structure

Needs to be modified to have a non-clonal population structure model

Clonal vs non-clonal population structures

Clonal: linkage disequilibrium, tree-like phylogeny, congruence

Non-clonal: linkage equilibrium (random allele combinations), net-like phylogeny, incongruence

How could a non-clonal population become clonal, and what could happen after

Reproductive isolation can lead a non-clonal population to become clonal.

Then it can be affected by period selection and bottle-necking

History of bacterial genome sequencing

First genome sequence was of a bacteria in 1995

Genome sequencing ability has continued increasing

But many in the database are incomplete, are ‘drafts’ or ‘high quality drafts’ relying on inferences

Features of the bacterial genome

Mostly singular, circular chromosomes (exceptions of multiple and linear chromosomes)

Have episomal (part of DNA that can replicate itself) elements like phages and plasmids

Bacterial genome replication

Proteins bind to initiation site

Two helicases unwind the DNA bidirectionally (leading to replication forks)

Recruit DNA primase to synthesise RNA primers

Synthesis begins with DNA polymerase

Comparison of bacteria and eukaryotic genome

Bacteria genome is smaller, more compact, very little non-coding/junk DNA, have operons which eukaryotic genomes don’t have, usually don’t have introns

Why do bacteria have a linear relationship between genome size and number of genes

Very compact and very little ‘junk’ DNA

As a generalised rule, what does the increased genome size lead to in bacteria

Increased metabolic capacity

Basic cellular machinery genes remain largely the same for all bacteria, so the added genes usually related to metabolic processes

Features of the bacteria leading strand

Continuously replicated

Essential genes and coding sequences preferentially located on the leading strand

Selection consequence against transcription fork and transcriptional machinery collisions, and toxic truncated products

Key difference of bacteria and human genomes

Many bacteria genomes are ‘open’ while humans always have ‘closed’ genomes

Features of the bacteria open genome

Formed of a core and accessory genome

Core → encodes genes for essential processes e.g. DNA replication, ribosomes, cell envelope etc.

Accessory → specific to each bacteria, can include alternative metabolic pathways / transport systems etc.

Accessory genome is formed of mobile DNA (phages, conjugative plasmids, transposons, integrons, insertion sequences) which can move from bacteria to bacteria

What is the gene pool / pangenome

All the genes / every genome available to a bacterium

Features of mobile genetic elements

Can promote their own spread by associating with adaptive traits in the gene pool, so they are passed down.

Can have a core and accessory genome - the accessory genes which can give their host a selective advantage like antibiotic resistance / virulence / metabolism etc.

Why is there no universal definition of a bacterial species

Species are very difficult to classify

There’s a large disconnect between similarity of phenotype and similarity of genomes.

Dogs look very different but have similar genomes, whereas bacteria often look very similar with very different genes

Why couldn’t you classify bacterial species by what disease they cause

Again huge disconnect

Meningitis is caused by several different bacteria

E. coli causes multiple diseases - based on different accessory genes and its host it can turn from commensal to pathogenic

What approach is needed for studying classification of bacteria

‘Polyphasic approach’

What does a polyphasic approach entail

Looking at all phenotypic, genotypic and chemotypic data cohesively

Aiming for a monophyletic classification that reflects evolution

Need to look at molecular, organismal, and ecological level

Current working definition / concept for bacterial species

Phylogenetic species concept

Organisms that share characteristic traits, generally cohesive, recent common ancestor

But evolution is continuous so defining boundaries remains hard

What do scientists use to determine homology

DNA-DNA hybridisation

How are bacteria further classified after being given a species name, and why is that important

Further classified into sub-species e.g. E. coli O157:H7

Essential for dealing with infectious diseases

How does HGT affect bacterial systematics and typing

Different approaches for defining species needed based on the level of HGT

If HGT is very common then the bacteria will share a gene pool, and species can be defined by the population

If HGT is very rare/non-existent then the phylogenetic approach is best because of the little mixing of the gene pools

Different ways of studying bacteria

Microbiology

Phenotypic isolate characterisation

Phenetic characters

How is microbiology used to study bacteria

Isolation of pathogens helps diagnosis of disease

Determine the material for bacterial growth

But culture often requires bespoke reagents and equipment. And many pathogens cannot be growth cultured

How is phenotypic isolate characterisation used to study bacteria

Test characteristics with different stains

Test for antibiotic resistance

Test for antibodies (serology)

Metabolic phenotyping for species identification

How can phenetic characteristics be used to classify bacteria

Classifying bacteria based on morphological characteristics like:

Motility and shape

Biochemical characteristics (growth at certain pHs, temperatures etc.)

Different tests include gas chromatography to identify metabolites, and mass spectrometry like MALDI-TOF

What is MALDI-TOF, problems with it

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry

Rapid and inexpensive species identification

But needs a database - problem in countries with uncomprehensive databases

What is now the principle method for classification

Sequencing

What was the first sequencing method

Sanger sequencing - 1977

Importance of sequencing

Definitive results

Reproducible so can be checked

Scalable - from few base pairs to a whole genome

Manipulable - can analyse the sequences with model-based methods

Can be done from a PCR reaction or microbiome sample - don’t need an isolate

What is molecular isolate characterisation

Using increasing numbers of modern methods to determine characteristics, identify genes and sequences → all aiding classification of bacteria and other areas

Types of molecular isolate characterisation

qPCR

single locus characterisation

16S rRNA analysis

multi-locus sequence typing

qPCR

PCR with real-time quantification

PCR reaction carried out with a fluorescent agent medium. Distinguishing gene is amplified and a positive sample gives off fluorescence.

Need to know what gene you’re looking for

single locus characterisation

After gene amplification, can carry out sequencing or gel electrophoresis and then bioinformatics to determine the gene

16S rRNA gene analysis

16S rRNA is an essential, structural component of the ribosome - highly varied across organisms

Has nine variable regions that can be used to determine genus/species

It is a single gene so rapid and cheap to sequence. Doesn’t encode a protein which would complicate analysis

MLST

Multi-locus sequence typing

Looks at 7 housekeeping genes in the genome, to avoid incongruence from HGT

Uses ‘allele’ based analysis to further reduce inaccuracies from HGT → just considers whether alleles at a gene are different or not, instead of how different they are

MLST is scalable - from PCR isolates to the whole genome

Quick, standardised, sequencing method

How do the different techniques vary

In the amount and levels of information they give

Some techniques provide information up to the genus level, others up to the specific strain etc.

What is used to store information of DNA sequences and isolates

PubMLST

Has information on both DNA isolates and sequences

Has schemes for more than 100 bacteria

Compatible with MLST and whole genome sequencing data

Stores data on ribosomal MLST (MLST carried out on specific ribosomal genes)

Example of application of MLST analysis

Neisseria - MLST analysis of its core genome shows the range of the species

Can define the different species using the genetic analysis

In what area is there increased use of high resolution molecular typing - real-life example of use

Public health → understanding how different species and strains related, and their evolution within and among hosts

Quick intervention after genotyping of several isolates, surveillance, and consistent nomenclature reduced the severity of Salmonella outbreak in chocolate products