Microbial physiology and diversity.

What are the types of culture vessels for liquid growth

Fernbach flask, erlenmeyer flask, cell culture flasks, kluvyer flask, bioreacter

What might you seek to control when doing a physiological study?

chemical and physical parameters like the gas phase above the culture (composition pressure, temperature, light/dark).

When doing a physiological study what type of media should be used

Defined media (controllable and reproduceable)

Defined vs. Undefined media

Defined (the exact chemical composition of the media is known) Undefined (the exact hccemical composition of the media is not known).

What is a growth factor

Organic molecules needed in the media for the growth of a specific microorganism. Carbon/nitrogen/sulfur/phosphorus can not be growth factos.

Why is e.coli not a glucose autotroph

glucose is a carbon source for e.coli

what is a minimal media media

a minimal media is a media were only the chemicial compounds necesay for growth are in the media.

What is a vitamin

a vitamin is an ORGANIC molecule needed at low levels to support the growth. Typically these are cofactos.

Additional growth factors

Typically at higher concentrations (amino acids/purines and pyrimidined)

Auxotroph

Organism requires a growth factor

Prototroph

The organism does not need any growth factors.

Trace elements

Metals and ions that are needed at low concentrations, these are typically cofactors (they are NOT growth factors because they are not organic).

Cofactors

A cofactor is a non-protein opart of a protein required for function. The can be organic/inorganic.

Metalloprotiens

Contain an inorganic metal cofactor.

What is the metalloprotein we cover most extensively in class?

The c-type cytochrome. The contian the organic/ metal cofactor heme (iron) Heme is bound covalently to the cytochrome protein. (Apoptosis and an electron carrier).

What are two key parameters of microbial growth

(specific growth rate and Molar growth yeild)

What is the formula for binary fission

N_t=N_o x 2ⁿ (N is the number of cells at time (t) and n is the number of generations

How could we determine an increase in cell number?

Total count in a counting chamber, viable count by plating (we don’t get all the possible surviving cells growing), Optical density via light scattering (NOT ABSORBANCE).

What equation do I used to determine the specific growth rate

u=(ln(nt)-ln(no)/(t-t0)

How do I find the doubling time

td=(ln2/u)

What are the three discernible growth phases

Log phase, lag phase, stationary phase,

How many times should the culture double to determine specific growth rate and doubling time.

at least 3

How are the specific growth rate and doubling time related

The soecific growth rate and doubling time are inverses of each other.

what is true about the specific growth rate

It is unique to an organism, and will remain the same if grown under the same conditions. (Semilogarithmic groah is used for these).

When do you determine the growth yeild

The growth yeild is determined when the culture has reached stationary phase.

What is molar growth yeild

Molar growth yeild is the total increase in cells during growth in batch culture . (maximum-initial). If a nutrient is fully depleted from the medium (becomes growth limiting)It is possible to determine the molar growth yeild. it is dry weight/mole of limiting reagent.

What does the culture look like at lag phase and stationary phase.

mixture of cells in different physiological states

What does the culture look like at exponential growth

balanced steady state growth exponential increase in growth occurs at a constant and reproducible rate. These are the average cells that are sampled in physiological studies.

Where is Halomonas sp. GFAJ-1 isolates

in the high saline Mono lake.

What was incorrecetly believed about Halomonas GFAJ-1

That was ablw to replace the biological roles of phospherous with arsenic

How was the experiment conducted incorrectly to lead researchers to this erroneous result.

The experiment first of all showed no growth in one of the curves ebcause they didn’t add glucose which would have acted as a carbon source for the organism. They also failed to account for the total removal of phosperous in their batch cultures. This allowed some growth even under supposedly no phosperous conditions becuase Halomonas sp GFJA-1 is very good at seeding phosperous sources out of its environemnt.

What are the microb of the week facts about Halomonas so, GFAJ-1

Isolated from Mono Lake, California ● Morphology: rod ● Cell envelope: Gram negative

● Phylogeny: Domain: Bacteria Phylum: Proteobacteria Class: gamma-proteobacteria Family: Halomonadaceae

● Extremophile (alkaliphile, high salinity) ● Arsenate resistant (in part due to a high-affinity and highly selective phosphate-uptake system), but it is phosphate-dependent!

what is the difference between collecting Wet weight vs. Dry weight

To determine the wet weight the batch culture is centrifuged/filtered with washing steps and weighed. In dry weight the centrifuged cell pellet is put into the oven.

The difference in wet weight vs Dry weight show us the water content is this high for microorganisms

70% in prokaryotes and 90% in eukaryotes.

What are the four main elements that compose the dry weight of a cell?

Carbon, Hydrogen, oxygen, and nitrogen (95%)

What other elements are always present

Sulfur, potassium, magnesium, iron, zinc , phosporus

WHat elements are typically but not always present in cells?

K, Ca, Cl, Na, Co, Cu, Ni, Mn, Mo, Se

what does the relative chemical elemntal composition of the cell tell us?

The dry weight ration between all of the elemnts. PUT THIS IN BRACKETS

What are the typical compositions of the cell

15% Nitrogen (varies) and 50% carbon (just an average)

What microbe was found in 2014 that would only grow if additions of mudpot water were added to the batch culture.

Methyloacidiphilum fumariolicum

What does aerobic methanotrophy look like

Done by methane monooxygenase. Converts methane, oxygen, 2 electrons, and 2 H⁺ into methanol and water. (Draw out the actual physical reaction).

What are the types of monooxygenases

There is the iron dependent sMMo and the copper dependent pMMO they are isozymes and metalloproteins.

After the methane monooxygenase catlayzes the formation of methanol, hwat happens

methanol dehydrogenase oxidizes it into an aldehyde (This is either done by the calcium dependendt Mxfal or lanthanum dependent enzyme xoxf

What is methano/methyltrophy

methane/methanol is the carbon/energy source. The final formaldehyde product of methanol dehydrogenase can either be oxidized and used as an energy source or is can be assimilated into biomass as cellular carbon.

What enzyme has a lanthanum cofactor to methanol oxidation

XoxF

In what way is methylobacterium extorquens a good model organism

It helps us understand methylotrophy. It can help us study organisms that use C-1 compounds like methanol as a carbon and an energy source. It has two different methanol dehydrogenases, one is calcium dependednt and the other is lanthanum dependent. it is also a facultative methyltroph it is able to use succinate and methanol as a carbon source. It will NOT use glucose.

methylobacterium extorquens

Morphology: Short rod ● Cell envelope: Gram negative ●

Phylogeny: Domain: Bacteria Phylum: Proteobacteria Class: alpha-proteobacteria Family: Methylobacteriaceae

● soil, on (or in) plants leaves ● pigmented (pink) due to carotenoids

What does PQQ do?

Coordinates the metal in the Xoxf or MxafI enzyme

Prosthetic group (PQQ)

Non-protein part of a protein that is essential to it’s biological activity (covalently bound)

Which type of methanol dehydrogenase is more abundent in nature

XoxF is more abundent in nature, lanthanide are required for growth of many many methylotrophs.

Proteins

Polypeptides of amino acids linked by peptide bonds

Ribonuleic acids

Polymers of ribonucleotieds linked by phosphodiester bonds

Carbohydrates/polysaccharides

polysugars/glycans.polymers of sugars and sugar derivatives. linked by glycosidic bonds.

Lipids

Broad definition, hydrophobic part, fat-soluble, mainly hydrocarbons.

Deoxyribonucleic acids

Polymers of 2’-deoxyribonucleic acids linked by phosphodiester bonds.

What is the difference between lipids in eukaryotes/bactera and archea

ester bond between glycerol and fatty acids in eukarytoes/bacteria. Archea have an ether bond between glycerol and isoprenoids

What is genomics

Determination of the nucleotide sequence of all deoxyribonucleic acid molecules as part of the organism. It is the determination of the full set of genes in an organism (even plasmid dna)

What is the typical genome size for bacteria/archea

3-5 million base pairs codind for close to 3000-5000 proteins. (ORF is close to 1000 base pairs).

Forward genetics

Going from a phenotype to a genotype. Random mutations altering the genome can lead to a mutant with an observable trait (phenotype) different from the wild type. The genes mutated will be mapped (genotype) to propose a posisble function based on the phenotype this is used for HYPOTHESIS FORMING

Why do cells that contain mxaF with knockout xoxf still show really low/no growth in cultures where not lanthium is added (mxafI should be entirely calcium dependent)

xoxf presense is required to activate the system and begin transcription of the mxa operon.

Genome annotation

Allows the prediction of what proteins (and RNA) may be present in a cell and aims to predict the function of what proteins (and RNA) may be presnt in the cell.

Reverse genetics

Specifc gene is deleted/modified from the organism and phenotype is observed.

What is the transcriptome

The transcriptome is the complete set of labile RNA (sRNA and mRNA) within the cell under specific conditions. This will absolutely change based on environmental conditions.

How are transcriptomics done

Stable RNA like rRNA or tRNA is removed and the labile RNA is reverse transcribed into DNA (Deep RNA seq)

What is the proteome

The entire set (Type and concentration) of proteins present in the cell under specific conditions. This changes based on the conditions the cell is in.

What is the volcano plot an example of

Comparative proteomics

x=Log2Fc

y=Statistical value

What is a prequesite to doing proteomics

The genomic sequence it allows a good idea of what proteins will be present when you do peptide mass finger printing

what is peptide mass finger printing

peptide mass finger printing is digesting the protein in specific places usually with trypsin. It cleaves at specific places, leaving identifiable mass finger prints for a spefici set of proteins in an organism.

What is the metabolome

The metabolites of a cell under speciifc conditions (types and concentrations) POOL SIZE

What is metabolite

A building block of a polymer/cofcator/intermediate of metabolism. Identified using mass spec, liquid chromatography.

What are the challenges of metabolomics

Stability, Diversity of compounds( seperation/what to expect), identity based on mass, Quantification is limited.

What are some class given examples of metabilites

Nucleotides, aminoacids, sugars, fatty acids/isoprenoids, focfactors

What are class given examples of macromolecules

DNA, RNA, proteins, polysaccharides, lipids

Escherica coli first name

bacterium coli commune

How is e.coli relevant in the human system

Present in 90% of individuals, one of the firstbacteria to colonize the human gut microbiome, helps consume O2, anaerobic environmient for strict anaeroebes. Also has some mutualism, produces vitamin k, protects against pathogens.

Why do we test for e.coli soecifically in water contamination

easily culture-able, present in most people,

Why do we use e.coli as a model organism

Metabolic versatility (faculatative anaerobe;prototroph), Easy cultivation (37C 20-30 min doubling time), Amenability to genetic manipulation, serendipity

Why did George Beadle and Edward tatum win the 1958 Nobel Prize

for their discovery that genes act by regulating definite chemical events“ [one gene-one enzyme hypothesis, mainly using the fungus Neurospora crassa]. Biochemical genetics, (Genes control cellular metabolism, different genes different enzymes). He later works in e.coli

Why was. E.coli choosen as a model organism fr geneticists

Edward tatum used it and then other gentecists followed. we use the k-12 strain.

Why did Joshua Lederberg win a nobel prize 1958

He made discoveries concerning genetic recombination and the organization of genetic material of bacteria.

Tatum and Lederberg 1946 discovery

Serendipity, When cultivated alone auxotrophy can only be reversed one a t a time, but when cultivated with another strain, multiple auxtotrophies can be reversed, e.coli must have osme sort of sexual reproduction

What is the long term study with e.coli

analyzes the evolution of an e.coli culture across many many generations.

Who uses e.coli

Biochemical geneticists, microbial physiology, Studying evolution

What is pan-genome vs. Core-genome

Pan-genome is all genes found in all members of a species. Core genome (genes present in all members of a species). More extensive studies estimate ~2,000 genes for the core genome and 75,000 genes for pan-genome of E. coli, each strain has ~5,000 ±450 genes/genome

What is the O-antigen

O-antigen type

What is H-antigen

Flagellin type

What is K-antigen

Capsule type

What is a gram-negative cell envelope e.coli structure

Outer membrane, peptidoglycan, inner membrane

Why is the outer membrane (OM) not a typical membrane

Has unusual structural features like integral OM proteins (OMP) adopt a B-barrel conformation

Very rich in protein (OM is 80% protein in e.coli compared to IM which is 50%)

It is not a phospholipid bilayer. One sides has LPS one side has phospholipids (high permeability barrier stronger than the phospholipid bilayer).

What are the two types of membrane proteins

Alpha helical TMDS (Inner membrane) and B-stranded (TMD) (outer membrane)

What is the role of most OMP (outer membrane proteins)

To transport hydrophilic molecules across the outer membrane.

What are the two types of OMP what are they structurally like

Nonspecific porins (similar to a sieve; barrel lumen always accesible/open) Selective transporters (substrate-specific accecss to barrel lumen)

Why is the outermembrane considered asymettric

One side is LPS one side is phospholipids

How are the phospholipids in the inner leaflet structured.

They are diglycerides, Tehir head group gives them diversity and they have a phosphate group, a glycerol, and a fatty acid tail (2x glyceride)

What is the general structure of LPS

O-polysaccharde chain, Outer core, inner core, lipid A

What is unique about the O-polysaccharide chain

The O-polysaccharide chain is highly variable, It is the primary target of antibody responses agaisnt LPS, It has a long repeating chain not found in LOS

What is unique about outercore

most likely to contain hexoses/hexamines: common sugars

what is the inner core

highly conserved with unusual sugars (heptose/kdo)

What is unique about Lipid-A

Di-glucosamine backbone very highly conserved. Acyl-chain lenght/substitution pattern is primary determinant of endotoxicity. fatty acids are connected by ester and amide bonds to a glucoseamine disaccharide.

Why is LPS crucial for the main function of the outer membrane

Strong LPS-LPS lateral interactions

Saturated fatty acid chains

Hydrogen bonds

Cations (mostly Mg²⁺) neutralize bridge phosphates

more gel like than fluid like.