Lectures 1 & 2

Phages: Bacterial Viruses

• Phages are viruses that attack bacteria and archaea.
• There are an estimated 10^{31} phages on Earth.
• There are more phages than all other organisms combined.
• For every grain of soil there are trillions of phages.
• Phage genomes can be composed of DNA or RNA, single-stranded or double-stranded.
• Phages can be specialists targeting specific species, strains, or isolates.
• Phages can also be generalists with a broad host range, infecting multiple species.
• Phages are considered harmless to humans due to their host specificity.
• Bacteriophages are the focus.

Bacteria and Phages in the Environment

• Bacteria exist everywhere and have champions of adaptation.
• Phages exist wherever bacteria are present because they prey on bacteria.
• The name "phage" means "eater".
• Bacteriophages outnumber bacteria by tenfold.
• Multiplicity of infection (MOI) is the ratio of phage particles to bacteria in a laboratory setting.
• Phages initiate infection by adsorbing to bacteria, either to the cell body, flagella, or pili.
• After adsorption, phages inject their genome into the bacterial cytoplasm.

Phage Infection Cycles

• After genome injection, phages can enter a non-productive (dormancy) or productive phase.
• In the lysogenic cycle (non-productive phase), phage DNA integrates into the bacterial host genome, becoming a prophage.
• Prophages replicate along with the bacterial genome.
• During the productive cycle, more viral particles are produced, which can occur through the lytic or chronic cycle
• In the lytic cycle, new phages are produced, the bacteria lyses, and phages are released.
• In the chronic cycle, phages are released without killing the host cell.
• Temperate phages can switch between lysogenic and lytic cycles based on environmental conditions.
• Temperate phages enter the lysogenic cycle when nutrients are scarce, as there are not enough host cells to kill

Phage Types and Persistence

• Filamentous phages undergo chronic cycles, but can sometimes integrate into bacterial genomes.
• Lytic phages always kill the host cell upon infection.
• Temperate phages can switch between lysogenic, lytic and chronic cycles.
• Phages can exist in a state called pseudo-lysogeny.
• Phage attaches to the cell and injects its genome, but no phage particles are produced immediately.
• The phage genome persists as single, extra-chromosomal DNA in the cytoplasm and primes host cell towards lysis.
• Pseudo-lysogeny can last for extended periods until progeny production begins.
• Infected cells produce immune factors, which protects them from secondary viral infections, called superinfection exclusion.
• Cells without phages in the cytoplasm remain susceptible to phage infection
• Pseudo-lysogeny has been shown for DNA phages. Recent studies indicate RNA phages can also persist within the cytoplasm.

Phage Replication: One-Step Growth Curve

• Phage replication follows a one-step growth curve, characteristic of the lytic cycle.
• The stages includes:
  • Attachment (adsorption) to the bacterial cell.
  • Genome injection
  • Latent period until phage particles are produced and released.
  • The latent period consists of eclipse and maturation.
  • Eclipse is the preparation phase for new particle production, involving viral genome transcription and phage protein synthesis.
  • Maturation stage involves assembly of phage particles.
  • Cell bursts, releasing progeny into the environment.
• Burst size: number of phages produced per phage particle during infection cycle, ranging from 100 to thousands, depending on the phage.

Tools for Studying Phages

• Metagenomics sequencing is a technique to describe phage diversity by getting a sample and sequencing it.
• Single virus genomics is used to study genetic variation within phage species.
• Laboratory techniques include cultivation, microscopy, and phage assays.
• Petri dishes with agar plates are used to cultivate bacteria, the clear zones (plaques) indicate bacterial lysis by phages.
• Spotting assays and soft agar assays are preformed to count phages and assess their success in infecting and lysing bacterial populations

Phage Diversity and Morphology

• Double-stranded DNA phages are the most common type, they consists a capsid, tail, and adsorption apparatus (fibers).
• Phages can be tailed or non-tailed, this slide shows the diversity of morphology.
• Some phages contain circular DNA genomes.
• CrAss-like phages are prevalent in the gut microbiome.
• Single-stranded DNA phages have varied morphologies, they can be capsid-containing or filamentous.
• Double-stranded RNA phages resemble coronaviruses with an envelope and spike proteins.
  • Their genomes are segmented into small, medium, and large segments
  • They attack appendices and attach to type four pili of their host.
• Single-strand RNA phages also attach to pili.

Phage Genomes and Mutation Rates

• Phage genomes vary in size from encoding three genes to several hundreds.
• Single-stranded RNA phage genomes are around 5 kilobases long with genes for replication protein (RNA-dependent RNA polymerase), coat protein, maturation protein, and lysis protein.
• Double-stranded DNA phage genomes can be 50 kb long. There is a huge diversity between the genomes
• RNA phages have high mutation rates compared to DNA phages.
• RNA-dependent RNA polymerases lack proofreading activity, meaning higher mutation rates for RNA phages.

Databases and Phage Classification

• Most scientific knowledge is based on DNA phages because databases contain more than tens of thousands DNA phage genomes while foreign exchange genomes are much less.
• The International Committee on Taxonomy of Viruses (ICTV) classifies virus taxonomy and diversity. They recently accepted new RNA phage species.
  • Different databases (e.g. virus-host database, NCBI virus database) provide varying information due to ongoing updates because of of new knowledge.
• Phage research has focused on DNA phages because techniques are cheaper, protocols are optimized, DNA is more stable, and there is extensive knowledge in public databases.
• RNA phages have high mutation rates, making them easily missed if scientists don't know what they are looking for.
• Scientists are applying non-biased detection methods and computationally demanding analysis routines to identify new phages.

Phage-Bacteria Coevolution: The Red Queen Hypothesis

• The Red Queen hypothesis describes phage-bacteria coevolution, where species must constantly evolve and adapt to maintain their relative fitness.
• The Red Queen said that she can only stay in a specific place if she runs.
• The Red Queen hypothesis is used in evolutionary theory, mutually interactive species that are competing constantly need to evolve and adapt.
• Starting with equal densities of phage and bacteria. The numbers of phage and bacteria change.
• Bacteria numbers drop because bacteria are the phage host. Certain mutations arise, rendering some bacteria unsusceptible to the phage. Mutations can include strategies such as rejecting the receptor for their absorbance, or some anti-phage strategies.
• When those mutations pop up in bacterial populations the bacterial number rises and phage number decreases.
• The phage then evolves different strategies to adapt to that phage.

Bacterial Defense Mechanisms Against Phages

• Restriction-modification systems protect bacterial host DNA by methylation and cleave unmethylated foreign DNA (e.g. phage DNA).
• Bacteriophage exclusion (BREX) systems contain methylation enzymes and block phage replication without degrading phage DNA.
• CRISPR-Cas systems protect against foreign material.
• Toxin-antitoxin systems contain enzymes that degrade RNA, inhibit DNA replication, or impede cell synthesis.
  • Some toxin-antitoxin systems use abortive infection, where infected bacterial cells undergo altruistic suicide to prevent phage reproduction.

CRISPR-Cas Systems

• CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and CAS is CRISPR-associated proteins.
• Classified into two classes and six types based on mechanisms and structural properties.
• Present in at least half of all sequenced bacterial genomes and act as anti-phage defense mechanisms.
• The CRISPR-Cas array consists of spacers (memory of past viral or phage genomes) separated by repeats under a leader promoter with upstream Cas genes.
• System has two stages: immunisation and immunity.
  • In immunisation which occurs if the bacterial cell never saw a virus before, phage DNA is chopped and integrated into bacterial genomes.
  • The second time the Crispr CAS array is being used, guide RNAs are transcribed from the spacer region, and they guide CAS to specific phage genome, cleaving the DNA.

Phage Counter-defense Strategies

• Phages can produce proteins that counteract CRISPR or restriction systems, called anti-CRISPR or anti-restriction.
  • This involves protein-protein interactions where phage proteins bind and inhibit defense proteins.
• Phage proteins can be enzymes that modify bacterial defense proteins, e.g. by post-translational modifications thus preventing proper activation.
• Phages target signaling cascades involved in defense mechanisms, including secondary messengers or signal molecules.

Impact of Phages in Nature and Applications

• Phages affect bacterial populations, but the extent is not fully known.
• Bacteria are critical for biogeochemical cycles (nitrogen, carbon, etc).
• Phages influence taxonomic and functional composition of microbial communities and ecosystem stability.
• Phages can kill bacteria, modifying bacterial abundance; integrate into bacterial genomes, promoting diversity and horizontal gene transfer; and distribute genetic material between species.
• Phages are important in biomedicine, agriculture, and related fields.
• Phage therapy is being considered as a medical treatment against antibiotic-resistant bacterial infections which offers advantages over antibiotics due to their specificity.
• Phages are potentially useful in agriculture and food safety, to combat bacterial pathogens and extend the life of fresh food.

Phage Research and Future Directions

• UK government is discussing regulations for clinical trials to assess risks and implement phages in clinical settings.
• Phages have been used as research model systems to study gene regulation and replication machinery.
• Filamentous phages are used in phage display technology to assess protein-protein interactions.
• Most knowledge is based on double-stranded DNA phages, with less known about species diversity, genome diversity, host range, bacterial defense, or phage counter-defense strategies in other phage groups.

Quantitative and Single Cell Microbiology

Standard Microbiology Techniques

• Petri dishes are used to grow bacteria and observe colony differences.
• Quantification is necessary to compare treatment effectiveness and gene importance.
• Measuring growth is a standard technique, using absorbance at 600 nanometers to estimate bacterial population density.
• Population growth is a common concept:
  • Lag phase
  • Exponential phase
  • Stationary phase (nutrient depletion)
  • Decline phase

Quantifying Bacterial Growth

• Bacteria grow by binary fission, which doubles the population.
• Formula is : Cell
  umber {time + 1} = Cell
  umber {time } * 2
• Within the growth curve, bacterial yield (maximum cell number) and growth rate can be calculated.
• Growth rate calculation: calculated from the slope.
• Each culture contains millions of cells, and one number tells us about the variation that might occur in the population

Single Cell Techniques: Addressing Averaging Problem

• Bulk measurements mask variations within a population of bacterial cells.
• Genetically identical cells in the same environment can exhibit different phenotypes.

Genotypic and Phenotypic Heterogeneity

• Clonal populations (genetically identical cells) can express genes at different levels (cell-to-cell phenotypic variation), or change over time (temporal variation).
• Even without mutations, genetically identical populations of cells can still express different phenotypes.
• Non-genetic sources of phenotypic variation include environmental and stochastic differences.
  • Environmental stimuli triggers differences in gene expression.
  • Stochastic gene expression depends on the small number of molecules in biochemical processes.
• Individuality in bacteria refers to phenotypic differences between genetically identical cells in homogeneous environments.
• Heterogeneity can be quantified using single cell techniques and examples that were discussed include dormant cells and persister cells

Heterogeneity: Dormancy and Persistence

• Dormancy is a metabolically inactive state where cells cease to grow.
• Dormancy is a way to form persister cells.
• There are two ways how persister cells can be generated.
  • Triggered persistence in a population where bacteria are growing, bacteria are persistent in the case they're triggered by starvation, increase in cell numbers, or stress immune response .
  • Exponential cells - Some cells might undergo spontaneous switch to persistence. Rare event happening in less then 1 in a million.
• Persister cells are known for their tolerance against antibiotics and other stresses.
• Persisters are phenotypically different, and they can switch back to being a normal cell and continue dividing.
  *

Single Cell Microbiology Techniques

• Microscopy: Bright field or phase contrast microscopy for cell number, size, morphology. It uses transmitted light.
• Fluorescence microscopy: Requires fluorescent molecules or proteins like the cyanobacteria call these pigments. Information about metabolic state or viability. Fluorescence dyes hybridize for microbial species detection (FISH).
• Gene reporter systems: Fluorescent proteins introduced into bacterial genomes. We introduce the fluorophores usually for bacteria at 40x- 100x
• Environmental box for temperature control

Fluorescent Gene Reporters

• Reporter systems can report on gene expression regulated or constitutive.
• To make a reporter for gene expression, use a transcriptional fusion or translational fusion
• Transcriptional fusion occurs where the promoter remains and one transcription starts here. Reporter Gene is introduced and fluorescent from this reporter is directly correlated to the promoter activity.
• Translational fusion is where full gene is expressed where the first couple of codons remain and then there's a reporter gene. Gene is being transcribed and translated.

Flow Cytometry

• Cells pass through a narrow channel and are hit by a light to multiple sensors for detection of specific light scattering or fluorescence.
• Analyzes and sorts bacteria populations based on fluorescence signals.
• Analyzes hundreds of thousands of cells per minute, providing good resolution.
• It measures:
  • Forward scatter (cell size)
  • Side scatter (cell complexity/content or granularity)
  • Fluorescence signal. We plot this data to the histogram.
• This gives information that the bacterial population does not respond uniformly to some stress.

NanoSIMS: Single Cell Mass Spectrometry

• Grow cells with nutrients labeled with stable isotopes (e.g., 13C-labeled glucose).
• Measures incorporation of isotopes into biomass.
• Primary ion beam screens the sample, and ions are scattered and detected.
• It can analyze different ratios. In the end the research is how this ecoli eats glucose.
• Not all cells utilize it even if single carbon source. Not everybody utilises carbon.

Summary of Single Cell Technique Measurements

• Variation in gene expression
• Cell size morphology
• Metabolism (using fluorescent dyes, reporters, NanoSIMS)
• Single cell growth (measuring elongation)
• Motility

Case Study 1: Metabolic Heterogeneity

• Methylococcus is a filosphere bacteria that grows on leaf with methanol. Uses translational reporter to the MxF gene methanol dehydrogenase.
• Bacteria are grown in 2D environment methanol solid agar form forming colony.
• Curious why there is only one C source that is high, we would expect the cells to use methanol.
• Bacteria switches between C sources since methanol is present. The switch is not easy, needing different genes being up and down regulated.
• Some cells are not fully adaptors that ensure make the second gene expressed in case nutrients change.
• Movie produces lineage tree with varying fluorescence. Some are expressing bright gfp and we know where all the sells come from
• Each time frame for florescence density helps each cell grow rate.

Case Study 2: Time-Lapse Microscopy of Growing Colonies

• This is using salmonella producing tree lineage.
• We quantify the sell started as a bright cell and had a divide with information to all the sells. The one sell becomes two becomes for it grows. In the end the cells are dim and bright producing dim and bright
• We can read a single cell growth rate with how exactly cells divided from growth rate.
• Because the transcriptional the promoter is a flood chiller needs flagella from the moves which needs infection.
• If some sales are normal tile they are protected from cells that triggered from the immune response. This is how they continue their existence, some soldiers being killed off.

Case Study 3: Microfluidics

• Cells are put in for microfluid device under microsocpe in constant media to media switch constitutive from that becomes an antibiotic, then they write on reporters.
• Worriyigly sells are not killed off in produces long fillements survive. Shows us that things that cannot be seen can be seen under microscope. Under the normal cuvette.

Microfluidic Devices and Droplets

• In the device cells can go 1D and 2D and confined space. Microfluidics helps constant conditions from the nutrient.
• Droplets are then of H2o with media with oil and then in the special chip being used in an exact way looks under 40x. It's trapped and observed better from three set ups. Has 3D effect since cells move and aggregate to what they want.
• Under what conditions with different Multi-infections of different and new averages, is what we test.
• Cells move and are in clumps in mixtures in microbial communities, that looks natural with droplets single cell levels. DNA pros are very high.
  \ It can also be quantified to and measure time to analysis population with Multi inspection

Droplet and Microfluidic Technology Summary

• Technology supports for cells growing as planking or aggregate.
• Cells are module and can be observed.
• There is a three-dimensional analysis in droplets but there are not much in fresh nutrients.
• Droplets in the microfluidics set ups can be started and be further analyzed. To have thousands of different views single setups together that were seen as six.

Case Study 4: Current Research in Heterogeneity in Response to RNA Phage

• Indicated high averages of bacteria killed. Cells are trapped in channels using micro fluidic.
• The bacteria population supports the growth in general. It's used to not waste the material. It takes time for the bacteria grow.
• Different phenotypes appear in the microfluidic channel some persist in antibiotic the longer elements stay and survives.
• A lot of cells do not see faces since two channels is made and some phases cannot persist.

Conclusions and Future Directions in Single Cell Microbiology

• Some cells persist antibiotics they not assist. Sells also see these phrases of that can persist bacteria.
• Since, Phages are used in biomedicine studies in how is not only phases lies to kill bacteria on how efficient there are there is uniform and what a sub populations means. To prevent problems can it kill sell or it be dormant. The single cell use in technique help us know.