cels191 microbiology

what are the main 3 shapes of bacteria?

• cocci (sphere)

• rods

• spirals

what are key features that bacteria can have (7)?

1. cell wall (integrity)

2. plasma membrane (regulation)

3. ribosomes (protein synthesis)

4. nucleoid (circular chromosomes)

5. glycocalyx (extracellular protection)

6. flagella (movement)

7. fimbrae/pili (attatchement)

what is the genomic structure in bacteria?

• typically a single nucleoid - circular chromosome

• no nuclear membrane

• plasmids - other small circular self-replicating DNA molecules can be found in the cytosol (separate to main chromosome) highly variable in number

what is the cell wall structure of bacteria?

made of peptidoglycan - a mesh like structure

function:

• rigid macromolecular layer that provides strength to the cell

• protects cells fro osmotic lysis and confers cell shape

(type of prokaryotes that lack cell walls = mycoplasmas)

what is the structure of peptidoglycan? (structure of cell wall)

repeating units of NAG/NAM (carb backbone), cross linked with transpeptidase → forms rigid cell walls

what is a gram stain?

a test for gram- positive (purple) and gram-negative (pink) bacteria

1. apply crystal violet (stains all)

2. apply iodine

3. alcohol wash (gram-neg decolourizes)

4. apply safranin (counter stain)

works due to differences in structure of cell wall (peptidoglycan)

what is the structure of a gram positive cell wall?

• thick peptidoglycan layer (20-80nm) on the outside of the plasma membrane

• peptidoglycan traps crystal violet

what is the structure of a gram negative cell wall?

• thin layer of peptidoglycan (5-10nm) between the plasma membrane and outer layer of cell wall

• crystal violet easily rinsed away → shows red safranin dye

what is bacterial flagella, what and how does it function?

what: structure that allows motility to some bacteria in liquid medium

how:

• long, flexible appendage (like ‘tails’) act like a propeller

• proteinaceous 10-20nm in diameter 5-10 per cell

• number and location of flagella on cell varies

allows for chemotaxis

what is chemotaxis?

bacteria moving along a concentration gradient towards a chemical attractant (+) or away from repellent (-)

(through flagella ‘swim and tumble’)

what are 2 bacterial adherence features?

1. fimbriae

   • function: are structures with adhesive properties that cause bacteria to adhere to surfaces

   • inherited trait (not all have)

   • much shorter and more numerous than flagella

   • 100-1000 per cell, 2-8nm diameter, 1 micrometer in length

2. pili

   • attachment to other bacteria

   • transfer of genetic material from one cell to another called conjugation (a form of horizontal gene transfer

what is glycocalyx?

a gelatinous polysaccaride and/or polypeptide covering - forms a sticky meshwork of fibres

• if tightly packed and organised, attached firmly to cell wall = capsule (has affects on cell):

  • virulence factors - protecting bacteria from phagocytosis and engulfment by immune cells

  • prevents drying out

• if loose and disorganised, attached loosely to cell wall = slime layer

what are bacterial endospores?

• formed during unfavourable growth conditions → protects cells from stress (nutrient starvation/high cell density)

• only present in some gram-positive bacteria

• dormant stage of bacterial life cycle - everything it need to survive later is packaged tightly until conditions are favourable and can divide again

why are prokaryotes so dominant

because they have a very high growth rate (13min doubling rate) → they evolve/adapt fast (eg antimicrobial resistance)

what is the process of binary fission?

process of prokaryote asexual replication as one cell splits into 2 identical cells

1. chromosome replication begins

2. one copy of the 2 origins is at each and of the cell

3. replication finishes → 2 daughter cells

what are the characteristics of bacterial growth in a ‘closed’ batch culture system

microbes need building blocks to grow → these are very similar to the ones mammals require (however in different amounts)

thus, if we provide them will all the required materials, they can reproduce

Closed batch system

• type of cell culturing

• defined/specific amount of nutrients is provided → once used up the cells cannot grow

→ standard method of studying microorganism in a culture

how do you read closed batch culture results?

4 phases:

1. lag (time required to get biosynthetic reactions running)

2. exponential (cells actively dividing and nothing limiting cell growth - population is doubling in a constant time interval

3. stationary (cells stop growing and cryptic growth is observed - equilibrium between growing and dying cells)

4. death (equilibrium is skewed towards death)

no growth means death and growth rate is in balance

what do prokaryotes need to multiply?

1. carbon source - building blocks for macromolecules

2. energy source - electrons to drive anabolic and catabolic reactions in the cell

3. reducing power - electron energy carriers (eg NAD+/NADP+)

how do microbes harvest energy?

• chemical energy is stored in bonds

• broken chemical bonds release energy that can be captured in new bonds (ATP)

• ATP can be broken to release that energy

  • this red/ox of coupled compounds can be applied to many compounds and forms the basis of redox reactions

simple transformations lead to big repercussions

what are the 4 tropic groups (nourishment) in microbiology?

defined by where they get compounds and what strategies they use for survival:

• energy

  • light (photo-)

  • chemicals (chemo-)

• carbon source

  • carbon dioxide (auto-)

  • organic compounds (hetero-)

rubric of these four lead to:

1. photoautotrophs

2. chemoautotrophs

3. photoheterotrophs

4. chemoheterotrophs

what is the difference between growth as a individual vs a community member?

limitations to pure cultures when studying communities:

• auxotrophs (organism that cannot grow unless growth factor provided) cannot be cultured unless limiting factors provided

• 98% of all microorganisms sequences lack essential pathways or key genes for the synthesis of amino acids

thus

• cross- feeding occurs (one species gains metabolic products off another species)

• this benefits one or both of the species

• auxotrophs are common in nature and explain our inability to culture most microbes

what are the pros and cons of culture dependent vs independent approaches?

having to grow the organism in the lab (pure or reduced cultures) or analysing straight from the environment

pros of culture dependent:

• allows access to phenotype

• can study one organism at a time

• can manipulate conditions to see response of organism

cons:

• not all organisms can be cultured

• too many species to grow them all

• culturing requires precise conditions to match microbes needs

• does not match real world conditions

pros of culture independent:

• allows access to genotype

• can study many organisms at a time

• shows communities as they are in nature

• can target non-culturable organisms

• provides access to unknown species

cons:

• no pure culture so no ability to manipulate

• expensive and complex methods

what does it mean by ‘traditional microbiology was driven by pathology’?

• tens of thousands of microbes were cultured and charactized (pure samples)

• mostly identified pathogens or fast-growing organisms

• ignores complex interactions and how organisms live in communities

what is microbial ecology?

the study of microorganisms and their interactions with each other and the environment

• 20-50% of the earths biomass are prokaryotes - most organisms cannot be or have not been isolated in pure culture

• identification done using molecular fingerprints - millions of 16s rRNA sequences (molecular barcodes) in databases

• the majority are from uncultivated bacteria

→ the uncultured microbial world is far greater than the cultured world

what do the terms ‘population’ and ‘communities’ mean in the context of microbes?

individual microbial cells of a species that proliferate (live/thrive/multiply) = population

populations interact/communicate to form communities

what is the definition of microbiome

all microorganisms and their genes, within a particular environment

what are the basic components of microbial metabolism/energy/ and carbon acquisition?

process of breaking down food can lead to 2 different goals:

1. harvesting building blocks

2. harvesting energy

this process can run in reverse → you need both to energy to create a bond and a building block to attach to that bond

this is done through redox (every reaction has equal and opposite reaction)

• energy harvested from the environment is converted to ‘local currency’ inside the cell

• NADH/NADPH serve as intermediates to transfer energy inside of cell

• NAD+/NADH facilitate redox without being consumed, they are recycled

how does carbon based tropic groups work? (building block)

autotrophs

• primary producers

• fix CO2

  • self sufficient, does not require carbon

heterotrophs

• decomposers

• need fixed carbon, cannot use CO2 directly

  • dependent on primary producers

how does energy based tropic groups work?

chemotrophs

• use chemical energy from either

  • organic = carbon compounds (glucose)

  • inorganic = non-carbon compounds (hydrogen sulfide)

phototrophs

• use solar energy

how do microbes exploit different environments?

they can utilise different wavelengths of light to reduce competition

light = common resource

what is a microbiota?

individual microbial species in a biome - bacteria, fungi, archaea, and viruses

what is the human microbiome project?

• 5 year project from 2008 ($150 million)

• they used culture independent methods of microbial community characterization as well as whole genome sequencing of individual bacterial species

• emphasis on different parts of the body

• 10,000 microbial species in the microbiome - 500-1,00 in the gut alone

goals

1. develop reference set of microbial genome

2. explore relationship between disease and changes in microbiome

3. develop new tools for computational analysis

4. establish resource repository

5. study ethical/legal/social implications of research

results:

strong neiche specialization within and among individuals → different sites different microbes & diversity and abundance widely varies

everyone has same function but with different communities

what are the known functions of the human genome?

competition by commensal (+/o) microbes protects from pathogens

• stops pathogens form being successful

• blocks colonization niches

• competes for nutrients

• modifies environment to change virulence factor expression

• makes environments hostile: producing bacteriocins and short chain fatty acids

• lower pH

• thickened mucus layer on host

• host upregulates antimicrobial peptides

• primes host neutrophils and macrophages

what are examples of human gut microflora?

different sites = different bacteria in healthy humans

main 4 phylum:

1. firmicutes

2. bacteroidetes

3. actinobacteria

4. proteobacteria

few bacterial groups but many more strains

what are some functions of the gut microbiome?

• the gut microbiota creates SCFAs that modulate our metabolisms and affects our defense against pathogens

• the microbiome can:

  • synthesise vitamins

  • modulate the immune response

  • alter drug delivery (good or bad)

what is a functional food?

food that claimed to have a health promoting or disease preventing property beyond the basic function of supplying nutrients

most target intestinal health

what is a probiotic?

live microorganisms eg fermented foods/yoghurt

• lactic acid bacteria and bifidobacteria are most common types as they can survive transit though stomach and duodenum

• potential benefits = treatment for chronic inflam intestines/pathogen induced diarrhoea/urogenital infections

what are prebiotics?

an ingredient that beneficially nourishes the good bacteria already in the large bowel or colon (stimulate the growth of probiotics)

• the body cannot digest these so it acts as fertiliser for good bacteria

• mostly obtained through oligosaccharide carbs like whole grains/bananas/onions/garlic/honey

do pro and prebiotics work?

not really - poor scientific evidence

how does modifying our microbiome influence us and treat diseases?

microbes are just trying to live/thrive/survive

• we are a colonized ecosystem full of good/bad/neutral colonies

• all are simply extracting carbon and energy

fecal matter transplants can be treatment for clostridium difficile infection of the gut

what are the similarities and differences between C. difficile and Lactobacillus?

both use sialic acids from mucins (main structual component of the mucus layer in the gut) as carbon/energy source → heterotrophs

speed of growth and presence of accessory genes are the only factor making C. difficile a pathogen

what areas of the body did the human microbiome project emphasize?

oral, skin vaginal, gut, and nasal/lung

what are viruses?

Acellular organisms that cannot survive without a host: they have no metabolic abilities of their own

• use biosynthetic machinery of infected cell to multiply

• can infect all types of cells

• most abundant entities on earth

what is the composition of a virus?

viruses must have:

1. genetic material - RNA or DNA

2. capsid - protein coat that surrounds and protects the genetic material

some viruses are:

3. enveloped - envelope of lipids that surrounds the protein coat when they are outside a cell and facilitates entry into the cell

(if virus does not have an envelop = naked)

what is the capsid of a virus?

• multiple units of Capsomers (building blocks)

• capsomer = subunit of the capsid arrangement in a precise and highly repetitive pattern around the nucleic acid

  • can be arranged into 3 types of symmetry

  1. helical

  2. icosahedral

  3. complex

what do viruses look like?

shape based on capsid symmetry

1. helical:

   • shaped like a hollow tube or rod

2. icosahedral

   • polyhedron with 20 faces (each face a equilateral triangle)

3. complex

   • mix of icosahedral ‘head’ and helical ‘tail’/body

what are the different types/shapes of a viral genome?

made up of nucleic acid either RNA or DNA:

• double or single stranded

shape could be:

• linear

• circular

• segmented (viruses can swap segments to create varients)

size varies 4,000-1 mill nucleotides/3-1,000s genes

what is a bacteriaphage?

viruses that infect and replicate in bacteria

• can be treatment options especially for antibiotic resistant bacteria

what is the cycle of a bacteriophage infection?

1. attach - to host cell

2. penetrate - the host cell and inject genome

3. uncoat - mobilise genome

4. genome replication and gene expression

5. assembly - of viruses

6. release - copies of the viruses (sometimes involves destruction of host cell)

what is the cycle of human infection from viruses?

1. attach - through receptor

2. penetrate

3. uncoat - genome revealed

4. gene expression and genome replication

5. assembly

6. release - host cell remains intact

what are the features of the SARS-Cov-2 virus?

1. includes RNA polymerase to poly the genome

   • this has limited proof reading to makes errors leading to different strains

2. enveloped

3. +ssRNA and linear

4. non-segmented

5. contains a spike

   • receptor binding domain

   • critical for attachment and cell entry → binds to receptor protein to infect a cell

   • vaccines target the spike

6. genome ~30kB in size and codes for the replication module and structural/accessory proteins

what is the SARS-CoV-2 replication cycle?

1.attachment - spike binds to ACE2 receptor

2/3. penetrate and release - simultaneous

4. gene expression and genome replication

• genome translation

• replicating viral genome

• transcribing viral mRNA

• translation

5. assembly

6. release - hijacks our golgi bodies to exocytose without harming our cells

replicate in the cytoplasm

what is the replication cycle of HIV? (a RNA enveloped virus)

1.attachment - spike binds to CD4 receptor on T cell

2/3. penetrate and release - simultaneous?

4. gene expression and genome replication

• uses a special enzyme reverse transciptionase to make double stranded DNA from ss RNA

• then uses special enzyme viral intergrase to integrate this DNA into human DNA

5. assembly

6. release - new viruses with viral envelope glycoproteins (from host cell) bud off

what are the properties of the bacterial genome?

1. a singular circular chromosome

2. no nuclear membrane - but chromosome is restricted to a defined region, the nucleoid

3. contains plasmids - circular, self-replicating DNA molecules seperate to the main chromosome, found in cytosol

what are the 2 types and 3 subtypes of gene transfer in bacteria?

1. vertical gene transfer:

   • parent to offspring/1 to 2 daughter cells (normal)

2. horizontal gene transfer:

   • directly from one organism to another

   • demonstrated through Griffith’s mice experiment with ‘smooth’ (harmful) and ‘rough’ (safe) strains

   • can cause:

     • virulence factors (bacteria being more harmful to humans for their survival)

     • antibiotic resistance

   1. transformation

      • dead bacteria parts around and live bacteria take it up

   2. transduction

      • through bacteriophages → issue in assembly where bacterial plasmid entered into capsid leads to genetic transfer

   3. conjugation

      • through ‘sexual pilus’ - connecting 2 of the same bacteria together and unwinding plasma to transfer it

what is phage therapy and how does it work

therapeutic use of bacteriophages (virus) to treat pathogenic bacterial infections

pros:

• safe for humans as highly specific bacterial targets

• can be used for antibiotic resistant bacteria

cons:

• lack of studies (and funding!)

• difficult to administer and not used for intracellular pathogens

• has ability to transfer toxin genes between bacteria if not careful