BS1040- Bacterial genomes, cloning and AMR

What is the bacterial genome

Term coined by Hans Winker

• Current usage - all DNA within a cell

• Chromosome, plasmids, other genetic elements

What is genomics

• Genomics

• The study of genomes and genome sequences

• Mapping, sequencing and characterising genomes

Bacterial chromosome structure

-Usually one circular chromosome

-condensed into the nucleoid

Size and complexity

• link to structural complexity and biosynthetic capability

The E. Coli chromosome

• E.coli cell:

- ~0.5 μm wide x 1-2 μ m long: ~1-2 μm3 vol

•Chromosome:

- single dsDNA (double stranded DNA) molecule = 430 μm circle (~500x cell

width)

- 4.639 x 10^6 bp

- 1.5mm long! (ie. ~1000x cell length)

The bacterial nucleoid- structure

- occupies ~ 1/5 cell (~ 0.2-0.4um3)

• no membrane boundary -more diffuse in slow growing cells

- Compaction is dynamic, linked to growth rate

• in growing cells, typically in centre & bi-lobed

• Chromosome organised into large supercoiled domain loops.

• DNA has variety of proteins bound to it

- Histone-like protein HU very abundant structural protein

How do bacterial cells divide

• Bacterial cells divide by a process known as BINARY FISSION

• Single cell divides into 2 identical daughter cells

• Bacteria increase in length and mass

• Nucleoid expands - semi-conservative replication of the chromosome

• Synthesis of a septum at the midcell - divides the cell into two compartments

Semi conservative DNA

Semi-conservative DNA replication means that when DNA is copied, each new DNA molecule has one original (parent) strand and one newly made strand.

Origin of replication (oriC) and termination point (terC)

• Replication of the bacterial chromosome is BIDIRECTIONAL (processed in two directions at the same time)

• Starts at oriC - formation of two replication forks (Enzymes like DNA helicase unwind the double helix at oriC, creating an open replication bubble.

• From this point, two replication forks form and move in opposite directions around the circular chromosome.)

• Replisome complexes - replicate DNA at each fork

• Two replisomes approach each other at the terC site

terC is the termination site (the "end" of replication).

• The two replication forks move around the circular chromosome and meet at the terC region on the opposite side of the circle from oriC.

• When they meet, replication stops, and you get two identical circular DNA molecules

Chromosome replication and cell division

• Chromosome is attached by the oriC at the equator of the cell envelope

• Formation of 2 replication forks

• Two new oriC sites move apart at the cell expands

• The terC stays in the middle of the cell

• Completion of replication triggers FtsZ to form the Z-ring complex

• DNA chromosomes are separated and cell divides

Growth rate

• Chromosome replication time independent of growth rate.

• Time to replicate E. coli chromosome is 40 minutes

• But E. coli can divide every 20 minutes!

• Time to replicate chromosome can be longer than time for cell division

• The next round of replication can be initiated before the first is complete

• Faster growing cells have multiple replication forks

• The DNA inheritied by the daughter cells is already partially replcated

Plasmids, structure and function

• Found in most bacteria

• Size varies

• 1000s to 100,000s bp

• Like "mini-chromosome"

• Genes encoding protein and RNA

• Not normally essential for host growth

• Adaption. Selective advantage in specific

niche conditions

• Often contain multiple IS and Tn

Plasmid regulation points

• Autonomous replication: independent of

chromosome;

• fixed origin of replication; oriV

• some encode own initiator protein; oriV-specific

• Many control systems use functional RNAs

• use host DNA-synthesis functions. eg. DNA Pol

• semi conservative replication

• short replication time; 6 sec for 5kb, 3 min for 150kb

• Distributed on cell divisio

Plasmid regulation maintenance

• Self-regulating

• characteristic fixed copy number per cell;

• plasmid determined genetic property

• F @ 1 copy

• R6 @ 5-6 copies

• ColE1 @ 15 copies

• copy-control genes linked to initiation of replication at

oriV; Basic replicon

• related plasmids cannot coexist in same cell

(Incompatibility)

Role of plasmids

• Roles

• Conjugative plasmids eg F & P

• Transmissible

• Sequence transfer- evolution & AMR spread

• Tumour induction

• Agrobacterium Ti plasmids

• Resistance plasmids eg R

• Resistance (antibiotics, metals) and

antimicrobial production

• Metabolic capacity

• Rhizobium N fixation in legumes (symbiosis)

• Virulence

• Recombinant DNA technology

Bacteriophages

• "Eaters of bacteria"

• Twort & d'Herelle, 1920s

• Protein/membrane coat containing phage genome which is a nucleic acid

• Very small

• Presence detected by plaques which are Holes on lawn of bacteria (where they have died)

• causes Cell lysis

• OR insert into the chromosome called prophage

What are Insertion Sequences and Transposons

Insertion sequences (IS) and transposons are types of mobile genetic elements that can move within and between genomes.

Insertion Sequences and Transposons

Move (transpose) around genome

• Moves as unit

• No "free" form

• Transposition mediated by Transposase

• Transposition tightly controlled

• Inverted repeat sequences at ends to be recognised by transposase

• Can insert into chr, b'phage and

plasmid

• Moves genes between genetic elements

in genome

• Cargo genes (additional genes) eg:

• Anti-microbial resistance common

• Spread of AMR

• can have a mutagenic effect eg:

• inactive a gene it is insterted into

Horizontal Gene Transfer (HGT) - how

bacteria acquire new DNA

• Bacterial can exchange DNA within and between species

• Selective advantage

• Rapid acquisition of new DNA sequence and potentially functions

• Important source of genetic diversity

What are the 3 mechanisms of genetic exchange in bacteria

conjugation, transformation, transduction

Conjugation

• Transfer of DNA directly between bacterial cells via cell-cell contact

• Usually plasmid DNA

• Direct contact through pilus formation

Transformation

Transformation

• Bacteria take up DNA from their environment

• Natural transformation - cells must become "competent" to take up

DNA

• We can induce competence in the lab as a tool in cloning

Transduction

• Transfer of DNA between bacteria via bacteriophage

infection

• Phage infect a cell, replicate and produce new phage viruses

• Some host DNA can be packaged inside the virus - this is passed to the next bacteria when it is infected

Why is the size of bacteria important

- Link genome size to adaptive capability

• biosynthetic capability

• synthesis of nutrients

-Stress resistance

• resist environmental insults

-structural complexity

• surface structures, sporogenesis

-Regulation -sensing signals and transcriptional responses

• detect change or requirement and respond appropriately

• transcriptional regulation

Importance of genetic variation in bacteria

• Mutations

• Single nucleotide mutations and other small errors in

replication

• Error rate =10-5/bp with repair =10-10/bp

-Genetic Recombination

• Rearrangements: changes in organisation and deletions

-Genetic elements (plasmids, bacteriophage, Tns

etc)

• Acquisition of new genetic material

• Recombination with genetic elements

• Post-transformation recombination

-Core and non-core genome content

What is core and non core genome

Pangenome is entire set of genes found in all the strains of a species.

• Set of genes found in all strains is the core genome

• Genes found only in some strains is the non- core genome

• Non-core genome are the variable or accessory genes

• High level of horizontal gene transfer(HGT) promotes more non-core genes (open pangenome)

• Low level or no HGT promotes fewer non-core genes (closed pangenome

Lecture 2

DNA cloning

the process of making multiple, identical copes of a particular

piece of DNA

• Core technique that is essential for many areas of biology and medicine

What is a genome library

• Collection of DNA fragments that have been cloned into vectors

• Each DNA fragment can be identified and isolated for further study

• Genomic libraries - collection consists of overlapping DNA fragments that together make up the total genomic DNA of an organism

• cDNA library - collection of cloned DNA sequences that are

complementary to the total mRNA extracted from an organism -

representative of all of the genes that are expressed

Cloning vectors- key features

-Maintenance (replication) in host cell

• plasmid DNA

• Other types of vectors are available...

-Restriction enzyme site

• insertion of foreign DNA fragment

• unique

• multiple cloning site

• create RECOMBINANT DNA molecule

-Method to in2troduce into host cell

• transformation (plasmids)

-Genetic selection marker

• small proportion transformed

• need to recover cells containing vector/clones

• antibiotic resistance -direct selection

- Identify recombinants

• system/screen for recombinants

• Insertional inactivation

How to construct a genome library

-Isolate chromosomal DNA

-Digest with restriction

enzyme

• Digest chromosomal DNA

• Digest vector DNA

-Ligate chromosomal DNA

with vector DNA

• Transform ligated DNA into E. coli

• Select cells containing vector- eg. Antibiotic containing

medium

• Transformation is relatively inefficient

• Not all ligated DNA maintained in E. coli

• Not want all ligated products

Outcome of transformation

• Linear molecules destroyed in E. coli by nucleases

• Transformant will not replicate on antibiotic-containing medium

• Transformant will survive as vector plasmid has antibiotic resistance gene

• Transformant will survive as recombinant=vector plasmid has antibiotic resistance gene

Blue white screening for recombinants

lacZ encodes β-galactosidase

• β-galactosidase converts X-Gal (colourless) to blue compound

• X-Gal

• 5-bromo-4-chloro-3-indolyl β-D- galactopyranoside

• Vector containing lacZ fragment

• lacZα gene

• Insert DNA fragments into sequence encoding lacZα

• Insertional inactivation

• β-galactosidase no longer produced, X-Gal not converted

• SCREEN for recombinants

No lacz activity= white, insertional inactivation

LacZ activity= blue

What can you do with a DNA library

• Each DNA fragment can be identified and isolated for further study

• We can SCREEN the library to identify specific clones that we may be interested in

Protein product based approaches

-Assumes DNA is expressed

-Use antibody specific for target protein product

• Find clone antibody binds to

-Use specific assay for protein product

• Assay for enzyme activity

-Complementation

• Complement a mutant in the gene

DNA sequence based approaches

DNA sequence-based approaches:

-Utilise base sequence complementarity

• Hybridisation to labelled DNA probe

-Utilise known DNA sequence

• Use PCR amplification

How to make a genomic library

• Representative Library

• There is a clone in the library for every part of the genome

• Reality is some parts will be missing- Why?

• Larger the genome the greater number of clones needed to be representative

• Larger the insert the fewer clones needed

• Usability vs Size

Considerations in making library of expressed genes

• Lots of non-coding DNA means many clones will not be of interest

• Not a significant issue with majority of bacteria

• Why not just focus on DNA that is expressed:

• Use the mRNA

-Ability to focus gene library on different expression patterns

• Time, place, response.....

• cDNA library (complementary DNA)

• DNA synthesised from expressed RNA is used to make a library of clones. A cDNA library

Making a cDNA library

1. Grow cells under required conditions or isolate cell type

2. Isolate mRNA

• Release from cells

3. Purify and remove any DNA

• Remove cell debris and contaminating DNA

4. Use Reverse Transcriptase to copy mRNA

• Makes RNA-DNA hybrid

• Needs 3' primer

-Remove RNA strand

• RT activity or RNase

-Synthesise second DNA strand

• Use DNA polymerase and primer

• Use to make a cDNA library

Genome vs cDNA libraries

Complete: Genomic library

-Contains all DNA in genome

• Coding and non-coding DNA

• Abundance of specific sequence matches abundance in genome

Enriched: cDNA (complementary DNA)

-Contains copies of expressed mRNA

-Contains coding sequences only

• No introns, No promoters, No intergenic regions...

-Clones in library reflect mRNA abundance

• Enrichment for highly expressed genes

• Subset of genes can constitute significant % of library

Genome library approach to sequencing a genome

• Clone based library in vector (not now approach of choice)

• Representative collection of fragments to sequence

• Simple guide to sequencing a genome:

• Chop genome up

• Make a library

• Sequence the library inserts

• Assemble the bits of DNA sequence into

contiguous sequence

• Fill in any gaps

• Find the gene

Coding sequences, ORF, and consensus sequences

• Coding sequences (genes):

These are regions of DNA that contain the instructions for making proteins.

• Open Reading Frames (ORFs):

These are stretches of DNA that start with a start codon (AUG) and end with a stop codon (UAA, UAG, or UGA) — they are potential protein-coding regions.

• Consensus sequences:

These are short, common patterns of bases found at important sites (like promoters or splice sites) that help identify where transcription or other processes start.

Example: the TATA box in promoters

How can these be studied

Bioinformatics is the use of computers and software to study biological data — mainly DNA, RNA, and protein sequences.

Scientists use it to analyze, compare, and interpret genetic information.

-Bioinformatics uses computers to scan DNA and protein sequences to find:

• Genes and coding regions (like ORFs and consensus sequences)

• Similarities (homology) between sequences to understand function and evolution.

Homology

• Homology means similarity due to shared ancestry.

• Bioinformatics tools (like BLAST) compare DNA or protein sequences from different organisms to find regions that are similar or identical.

• This helps scientists:

• Find related genes in different species

• Study evolutionary relationships

• Predict functions of unknown genes or proteins based on known ones

Transcriptonomics

The study of RNA and differences in mRNA expression across the whole genome

Profiling differences in patterns of gene expression across the genome

This means studying which genes are turned on or off in different cells, tissues, or conditions — across all genes in an organism's genome.

Lecture 3

What is AMR

Antimicrobial Resistance (AMR) occurs when bacteria,

viruses, fungi and parasites change over time and no longer

respond to medicines, making infections harder to treat and

increasing the risk of disease spread, severe illness and

death

Antimicrobials

• Compounds that stop of slow the spread of microorganisms

• Include antibiotics, anti-fungal, anti-parasitic and anti-viral drugs

• Focus for the remainder or this lecture is antibiotics -

specifically target bacteri

What is resistance

• Microorganisms that are no longer controlled or

killed by an antimicrobial

How do antibiotics work

Inhibit protein synthesis

Inhibit cell wall synthesis

Inhibit DNA synthesis

Distrust bacterial cell wall

How do antibiotics work 2

Spectrum of Activity Antibiotics target specific

bacterial structures

Choice of antibiotic depends on the type of infection

Mechanisms of antibiotic resistance

- avoid- modify target of the antibiotic

- attack- destroy the antibiotic

-remove- actively remove the antibiotic from the cell

-limit- reduce the uptake of the antibiotic

How do bacteria become resistant

• Genes that encode the resistance mechanisms

can be inherited or transferred

• Mobile genetic elements - Horizontal gene

transfer

• Spontaneous mutation

• Change in expression level

Factors affecting AMR

- globalisation

- uncontrolled use of antibiotics

Consequences of AMR

- common diseases can return to being major issues

- global pandemics