Microbiology - Bacterial and functional genomics

The e.coli chromosome

Circular

oriC

Genes are densely packed

Contains operons

Only ~118 b between genes

Sizes range from 112kbp to 14 Mbp

Haploid

No introns

Common mobile genetic elements

Plasmids

Transposons (jumping) and insertion sequences (both often have resistance genes)

Integrons (accumulate 'useful;'genes e.g resistance))

Bacteriophage (lytic/lysogenic)

Plasmids are only 2.5% of the size of the bacterial chromosome

Extrachromosomal - conjugation

Plasmids confer diverse phenotypes:

Extrachromosomal but can be integrated

Varied size range

Often mobile (conjugation)

Can confer accessory functions

Bacterial genomes vary between strains of the same species:

Core genome - all strains of a species will contain these genes

Accessory genome - includes mobile elements (vary)

All the genes found across all different isolates of a species is called the pangenome

Pathogenic bacteria frequently possess pathogenicity islands - clusters of genes of foreign consent present in certain strains (not core) and correlated with virulence

types of genome sequencing

Sanger - slow and expensive but very accurate

Illumina - highly scalable sequencing of short fragments of DNA

Sequence assembly: bioinformatics

Overlapping sequences are aligned

However repetitive sequences interfere with assembly

Complete genome assembly:

Compare with known sequences

Use long-read sequencing methods (oxford nanopore)

Infectious disease and epidemiology and sequencing:

Pre-screen patients for problem organisms

Treat or quarantine patients before hospitalisation is necessary

Can identify all the virulence factors and antibiotic resistances in a single patient sample and you can prescribe appropriate treatment using combination (precision medicine)

Identification of new threats and rapid response

Sequence isolates from multiple patents

Determine epidemics

Track spread of disease and identify the origin of new variants

Metagenomics

allowing for new ways of studying the diversity of bacteria because many bacteria are unculturable difficult to isolate

The study of genetic material recovered directly from environmental samples

Gut microbiome

skin microbiome

Ways we use bacteria everyday (3)

Food industry: bacteria generate useful properties such as flavour and texture in every food products

Water treatment facilities:

Activated sludge in water treatment plants contain microbes that metabolise organic matter to CO2

Therefore cleaning out waste organic material from the water

Can be reused to "seed" the aeration tank

Composting is a natural process of decomposing heterogenous wastes in a controlled, microbially active environment - can be highly optimised or rudimentary - basic ingredients are carbon, nitrogen, oxygen and water

Bioremediation

Bacteria used in environmental applications

A process that uses mainly microorganisms, planta or microbial/plant waste to detoxify contaminants in the soil or other environments

Rapidly form spherical biofilms (to increase SA) around oil droplets which expedite degradation

Hydrocarbon degrading bacteria occur naturally

Xenobiotic pollutants often are resistant to natural degradation

Microbes that can deal with them can often be found and exploited

For example, pesticide de-chlorination using oxygenases

New bacteria is being discovered and characterised - particularly with a focus on methods to degrade plastics

Biotechnology

Bacterial cell factories - living organisms which produce medically or commercially useful biomolecules a(e.g antibiotics, enzymes, drugs, hormones)

The main advantages is that cell does the hard work

Bacterial cell cultures grow quickly and easily, input material often cheap and environmentally friendly, whereas chemical synthesis can be difficult and expensive

Antibiotics

Naturally occurring secondary metabolites produced naturally by bacteria

Ongoing quest for new biosynthetic gene clusters which may produce new drugs

Discovery of previously "unculturable" microbes may lead to new drug discovery

E.g tetracycline/gentamicin

vitamins (e.g b12)

Essential for DNA synthesis and metabolism

Very complex

Only produced by certain bacteria and archaea and in certain foods e.g

Hence a popular supplement

hard to produce synthetically

Genetic modification of bacteria:

Expressing a mammalian or a mutant protein

Using recombinant DNA technologies, these proteins can be made in large quantities bny factorial fermentation too

First, we clone the gene of interest into an expression vector, then transform into E.coli

Generates a microbial "factory" for the gene of interest

E.coli factories are grown by large scale fermentation

Expression of the gene of interest is induced

Cells are lysed and the expressed protein is purified for research, commercial or medical

recombinant therapies

Huge range of actual and potential application

Well understood theoretical and practical path to commercialisation

Small scale, high value products and high value, low cost commodities

Synthetic biology and bio-engineering:

Synthetic biology is the design and construction of new biological parts, devices and systems, and the re-design of existing, natural biological systems for useful processes

Wide ranging definition - Multidisciplinary field

often uses engineering concepts

Biological components of bio-engineering:

Components need to be modular and well-characterised

Can be put together in any order

Allow for complex designs and predictable outcomes

Components can be replicated by symbols (like in electrical engineering)

Gene regulation recap: (brief)

Units of DNA encoding genes are controlled by the activity of genetic elements including the promoter, the RBS, the terminator and the DNA binding transcription factors

The classic example is the lac operon- the lac repressor and it switching "on" in the presence of lactose (allolactose preventing the repressing characteristic)

Optogenetics:

Optogenetic circuits from MIT

Different wavelengths of light trigger the production of different coloured compounds by E.coli

Control of gene expression though light

Induction of bacterial gene expression in tissue via small molecule signalling

Potential therapeutic delivery tool

Temperature-controlled gene expression

Gene expression can respond to temperature

E.g Piraner et al - genes expression turned on between 40-45 degrees

The circuits dictate the colour response on the e.coli

Potential applications in temperature triggered therapeutics (e.g inflammation and infection)

Protein engineering: (brief)

Adding new functions to proteins or improving their current functions

These proteins will change the behaviour/functionality of bacteria which express them

E.g PETase (from plastic degrading bacteria) by active site mutation leads to increased enzyme activity

Metabolic engineering:

Gene circuits and engineered proteins can be combined to alter or create new metabolic processes in bacteria

Complex, multi-system

Improves efficiency of current metabolic processes, or allow bacteria to synthesise new compounds

CRISPR DNA editing technology

Evolved as a bacterial anti-viral immune systems

Can be modified to allow precise editing of any genome

A highly powerful tool with wide ranging applications from synthetic biology to basic research to gene therapy (ethics)

If the cell gets infected by the same virus again, guide-RNAs will detect if the same virus is infecting them again due to those regions of homology. The loop attracts the cas enzymes to that interaction and will physically chop up the virus before it can infect the cell once more (a memory system - similar to the immune system). You can engineer these enzyme systems to knock-in/out genes by utilising these guide-RNAs

Detecting our microbes: (2 methods)

Culturing

Grow 'live' microbes than can be tested in lab studies (mechanistic)

Required for drug/therapy development

DNA/RNA sequencing:

Determine microbial community 'fingerprint'

Rapid and high throughput

NGS microbiome profiling approach to profiling our microbes :

Genus level profiling (16s rRNA)

Shotgun approaches:

Metagenomics (DNA)

Metatranscriptomics (RNA)

Metabolomics

Proteomics

Species vary person to person but function coded for stays the same (conserved)

Culturomics

Previously estimated could only culture <10% of the human microbiome

BUT culturomics developed to culture and identify 'unknown' microbes

rebirth of culture techniques in microbiology

very important for:

- improving reference databases for further NGS approaches

- phenotypic/mechanistic studies

- culture collections

- therapy development

The gut microbiota:

Most densely colonised microbial habitat found in nature

2500+ species

Broad range of physiological conditions creates niches along the gut

4 main bacterial phyla

Proteobacteria

Actinobacteria

Firmicutes

Bacteroidetes

Major functions of the microbiota: (2)

Breaking down complex sugars

97 genes for carbohydrate degradation

Energy

Microbiota breaks things down by fermentation, producing resistant dairy components (e.g fibre) which then can be used in energy metabolism and metabolite generation

Energy ends up in adipose tissue as fat (uptake and storage), being used in the brain for appetite regulation and for sugar metabolism in the liver

Ingestible dietary components are broken down by 'primary' degraders (an ecological network of cross-feeders)

Types of dietary fibre (soluble and insoluble) and how they are metabolised:

(only some starch is metabolised by humans)

Mammalian glycans serve as a primary nutrient source for gut bacteria

When gut bacteria degrade complex fibres that humans cannot otherwise digest, it produces short-chain fatty acids through fermentation

Plant foods > dietary fibres > gut microbial metabolism (complex carbs > monosaccharides > phosphoenolpyruvate) > metabolites

What if there's no fibre in the diet?

Healthy gut:

Bacteria cannot contact host

Expansion of the mucolytic bacteria

A fibre-deficient diet

Dietary emulsifiers

Increased osmolarity

Leads to a eroded mucus:

Mucus layer depletion

Bacterial contact, sentinel goblet cell death

Human milk oligosaccharides (HMOs)

Carbohydrates with multiple simple sugars that are found in human milk

The composition of our microbiota evolves throughout our entire lives...

Unborn:

sterile?

Baby:

Pioneer microbes

High instability

Low diversity

Toddler:

New species

Competition

Increase in diversity (diet)

High instability

Adult:

Distinct and diverse

May still change but at a much slower rate

Elderly:

Different microbiota to adult

Lower diversity

May be limited by diet

Early life factors influencing the microbiome:

During pregnancy:

Intra-uterine environment

Bacteria in amniotic fluid

Maternal exposures

Stress, weight gain, antibiotics, smoking

Length of gestation

Term vs preterm

Weight at birth

During birth:

Mode of delivery

Contact with mother/healthcare

Environment straight after birth

After birth:

Feeding modality

Breast-milk vs formula

Antibiotics

Weaning or food supplementation

Home or family setting

Rural vs urban

Home structure

Siblings or pets

^early microbiota disturbances linked to numerous diseases (e.g autoimmune and IBS)

Viral colonisation from birth

Month 0 = pioneering bacteria and phage induction

Month 1 = viruses of human cells

Alongside = breast milk = immune cells, maternal antibodies, HMOs, lactoferrin, mucin

Microbiota and colonisation resistance

Resistance to colonisation by ingested bacteria, or inhibition of overgrowth of resident bacteria normally present at low levels within the interstitial tract is called colonisation resistance

Commensal or pathogenic bacteria

Successful colonisation of the intestinal tract is very hard both for invading or beneficial bacteria

(competition for niches)

The microbiota plays a critical immune programming: (2)

1) Mucosal and systemic immune compartments

2) Stimulates both tolerance and boosts specific responses

Microbiota disturbances and disease links: (loads like way too many)

Autoimmune diseases:

Arthritis

SLE

Type I diabetes

MS

Brain-linked conditions:

Depression

Anxiety

IBS

Autism

Intestinal diseases:

Crohn's

Ulcer colitis

Colon cancer

Metabolic diseases:

Diabetes

Obesity

Malnutrition

Immune disease:

Asthma

Eczema

Rhinoconjunctivitis

Food allergies