Books, Homework Please try to get your books for next week, especially the Biology of Microorganisms text (used is best I think – a few bucks…) Homework:

Books, Homework• Please try to get your books for next week,

especially the Biology of Microorganisms text (used is best I think – a few bucks…)

• Homework: Make a 1-page diagram of a bacterial or archeal cell, include all pieces and what they are made of as well as their primary function(s)

• Due September 14

Field Trip

• Ely Mine, Central VT

• Friday afternoon departure to campground at Thetford State Park

• All day Saturday – sampling and analysis of different areas of microbial activity

• Return Saturday evening

• Possible Dates: 09/22-23, 09/29-30, 10/6-7

• Preferences??

Microbes and Thermodynamics

• First and foremost, the basic tenet relating microbial activity with thermodynamic descriptions of physical and chemical systems is:

Equilibrium = Death

• Why then are microbes on seemingly every corner of the planet’s surface? Why might we expect to find them on other planets?

Cell Metabolism

• Based on redox reactions– Substrate (food) – electron is lost from this

(which is oxidized by this process)– that electron goes through enzymes to

harness the energy for the production of ATP

– Electron eventually ends up going to another molecule (which is reduced by this)

Nutrition value

• Eukaryotes (like us) eat organics and breathe oxygen

• Prokaryotes can use other food sources and acceptors

Redox gradients and life

• Microbes harness the energy present from DISEQUILIBRIUM

• Manipulate flow of electrons

O2/H2O

C2HO

Other nutrients needed for life

• Besides chemicals for metabolic energy, microbes need other things for growth.– Carbon– Oxygen– Sulfur– Phosphorus– Nitrogen– Iron– Trace metals (including Mo, Cu, Ni, Cd, etc.)

• LET’S PUT THIS TOGETHER INTO A MICROBE….

Cell Composition• 70-90% water

• Organic chemistry key to the construction of cells is inherently linked to the properties of water vs. organic compounds

• Consider 4 groups of monomers (a single, repeated ‘building block’):– Sugars– Fatty Acids– Nucleotides– Amino Acids

Polysaccharides

Lipids

Nucleic Acids

Proteins

Macrom

olecules

Macromolecules• Informational macromolecules: They carry

information because the sequence of monomer building blocks is specific and carries information = Nucleic Acids and Proteins

• Non-informational macromolecules: The sequence is highly repetitive and the sequence has no function to carry information

• composition and how exactly the sequences are structures delineate different functionality

Small molecules present in a growing bacterial cell.

Monomers Approximate ## of kinds

Amino acids, their precursors and derivatives  120

Nucleotides, their precursors and derivatives 100

Fatty acids and their precursors  50

Sugars, carbohydrates and their precursors or derivatives 250

quinones, porphyrins, vitamins, coenzymes and prosthetic

groups and their precursors 

300

Molecular composition of E. coli under conditions of balanced growth.

MoleculePercentage of dry weight

Protein  Total RNA  DNA  Phospholipid Lipopolysaccharide  Murein  Glycogen  Small molecules: precursors, metabolites, vitamins, etc. Inorganic ions  Total dry weight 

55 20.5 3.1  9.1  3.4  2.5  2.5  2.9

1.0 100.0

Inorganic ions present in a growing bacterial cell.

Ion  Function

K+ Maintenance of ionic strength; cofactor for certain enzymes

NH4+ Principal form of inorganic N for assimilation

Ca++ Cofactor for certain enzymes

Fe++ Present in cytochromes and other metalloenzymes

Mg++ Cofactor for many enzymes; stabilization of outer membrane of Gram-negative bacteria

Mn++ Present in certain metalloenzymes

Co++ Trace element constituent of vitamin B12 and its coenzyme derivatives and found in certain metalloenzymes

Cu++ Trace element present in certain metalloenzymes

Mo++ Trace element present in certain metalloenzymes

Ni++ Trace element present in certain metalloenzymes

Zn++ Trace element present in certain metalloenzymes

SO4-- Principal form of inorganic S for assimilation

PO4---  Principal form of P for assimilation and a participant in many metabolic

reactions

What controls element and species availability in the environment??

• List:

Cell Composition• 70-90% water

• Organic chemistry key to the construction of cells is inherently linked to the properties of water vs. organic compounds

• Consider 4 groups of monomers (a single, repeated ‘building block’):– Sugars– Fatty Acids– Nucleotides– Amino Acids

Polysaccharides

Lipids

Nucleic Acids

Proteins

Macrom

olecules

Construction, Part 1…• Sugars (aka carbohydrates) can be linear or cyclic

(if >5 C)• Sugars start out with 4,5,6, or 7 carbons:• Pentoses (C5) are critical to DNA, RNA (form the

‘backbone’)– Hexoses (C6) are crucial to cell walls

• Polysaccharides contain hundreds of sugars or more held together with glycosidic bonds with either or orientations

• Cn(H2O)n-1 where n is typically 200-2500

Polysaccharides:• Glycogen – C and energy storage

• Starches – C and energy storage ( poly)

• Cellulose – cellular wall material ( poly)

• Extracellular polysaccharides (aka glycoproteins or glycolipids) - pathogenic component of some cells, also useful for attachment and solubilization

Construction, Part 2• Fatty Acids – long chains with hydrophobic

and hydrophilic parts

• Lipids are made of fatty acids put together

The chemical characteristics of the fatty acids and subsequently the lipids make them ideal for membranes

Construction, Part 3

• Bases – Two types:Pyrimidine Purine

• Derivatives

Cytosine, C Uracil, U Thymine, T Adenine, A Guanine, G

DNA C,T,A,GNo U

RNA C,U,A,GNo T

•DNA is double-stranded (double helix), while RNA is single stranded•RNA has a slightly different sugar backbone – ribose instead of deoxyribose•RNA has a lot of turns and kinks, more chaotic structure, but some sections are closer to the outside than others…

DNA

RNA

Construction, Part 4• Amino acids monomer units of proteins

All amino acids have 2 functional groups – one carboxylic acid group (COO-) and one amino group (NH3)

Some amino acids have hydrophobic ends, others are acidic, some hydrophilic, or ionzable

Bonds between the C and N form a peptide bond, which helps form proteins

Proteins – ‘key and lock’ concept

Peptidoglycan (aka Murein)

• Polymer consisting of both sugars and amino acids

• Rigid material and serves a structural role in cell wall

Cell Construction• OK – using the building blocks we have

described, let’s make a microbe…

FlagellaFunction(s) Swimming movement

Predominant chemical composition

Protein

Pili

Sex pilus Mediates DNA transfer during conjugation Protein

Common pili or fimbriae

Attachment to surfaces; protection against phagotrophic engulfment

Protein 

Capsules (includes "slime layers" and glycocalyx)

Attachment to surfaces; protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients or protection against desiccation

Usually polysaccharide; occasionally polypeptide

Cell wall

Gram-positive bacteria

Prevents osmotic lysis of cell protoplast and confers rigidity and shape on cells

Peptidoglycan (murein) complexed with teichoic acids

Gram-negative bacteria

Peptidoglycan prevents osmotic lysis and confers rigidity and shape; outer membrane is permeability barrier; associated LPS and proteins have various functions

Peptidoglycan (murein) surrounded by phospholipid protein-lipopolysaccharide "outer membrane"

Plasma membrane

Permeability barrier; transport of solutes; energy generation; location of numerous enzyme systems

Phospholipid and protein

Ribosomes Sites of translation (protein synthesis) RNA and protein

Inclusions Often reserves of nutrients; additional specialized functionsHighly variable; carbohydrate, lipid,

protein or inorganic

Chromosome Genetic material of cell  DNA

Plasmid Extrachromosomal genetic material DNA

Prokaryote Structure

Cell wall

membrane

Nuclear material

Membrane is critical part of how food and waste are transported - Selectively permeable

Phospholipid layerTransport proteins

Cell Membranes• The membrane separates the internal part of the cell from

the external that these environments remain separate, but under CONTROLLED contact is a key to life

Membrane Components:•Phospholipid bilayer

•Hopanoids, which provide additional structural stability (similar to sterols (cholesterols) which provide rigidity to eukaryote cells)

•Proteins – direct transport between outside and inside the cell

~ 40% lipid, 60% protein

Eubacteria vs. Archaebacteria

Bacterial cell structure Archaeal cell structure

Difference??

Let’s look more closely at the membrane, though only 8 nm thick, it is the principle difference between these 2 groups of microbes

Archaea vs bacteria membranes• Principle difference between these two is

the membrane• In archaea, lipids are unique they have

ether linkages instead of ester linkages

Membrane function• SELECTIVELY PERMEABLE

– Passive diffusion Gases (O2, N2, CO2, ethanol, H2O freely diffuse through layer

– Osmosis because solute concentration inside the cell are generally higher (10 mM inside the cell), water activity is lower inside, H2O comes in – increased water results in turgor pressure (~75psi)

– Protein-mediated transport selective and directional transport across the membrane by uniporters and channel proteins, these facilitate diffusion – still following a gradient and does not require an energy expenditure from the cell

Membrane function 2 • Active transport proteins that function to move

solutes against a gradient, this requires energy• Uniport, Symport, and Antiport proteins guide

directional transport of ions/molecules across membrane – different versions can be quite selective (single substance or class of substances) as to what they carry

Membrane and metabolism• As the membrane is the focus of gradients, this is where

electron transport reactions occur which serve to power the cell in different ways

• Many enzymes important to metabolic activity are membrane bound

H+ gradients across the membrane

• Proton Motive Force (PMF) is what drives ATP production in the cell

Figure 5.21

Membrane functions (other)

• In addition to directing ion/molecule transport and providing the locus for energy production, membranes are also involved in:– Phospholipid & protein synthesis for membrane– Nucleoid division in replication– Base for flagella– Waste removal– Endospore formation

• Though very small, the membrane is critical to cell function Lysis involves the rupture of this membrane and spells certain death for the organism

Cell Wall• Cell wall structure is also chemically quite

different between bacteria and archaea• Almost all microbes have a cell wall –

mycoplasma bacteria do not• Bacteria have peptidoglycan, archaea use

proteins or pseudomurein• The cell wall serves to provide additional

rigidity to the cell in order to help withstand the turgor pressure developed through osmosis and define the cell shape as well as being part of the defense mechanisms

• Cell wall structure• Two distinct groups of bacteria with very different

cell walls– Gram negative has an outer lipid membrane (different

from the inner, or plasma membrane) – Gram positive lacks the outer membrane but has a

thicker peptidogycan layer

Peptidoglycan layer• This layer is responsible for the rigidity of the cell wall,

composed of N-Acetylglucosamine (NAG) and N-acetylmuramic (NAM) acids and a small group of amino acids.

• Glysine chains held together with peptide bonds between amino acids to form a sheet

Outer membrane – Gram (-)

• Lipid bilayer ~7 nm thick made of phospholipids, lipopolysaccharides, and proteins

• LPS (lipopolysaccharides) can get thick and is generally a part that is specifically toxic (aka an endotoxin)

• LPS layers are of potential enviornmental importance as a locus of chelators and electron shuttles

• Porins are proteins that are basically soluble to ions and molecules, making the outer layer effectively more porous than the inner membrane, though they can act as a sort of sieve

External features

• Glycocalyx (aka capsule – tightly bound and adhering to cell wall, or slime layer – more unorganized and loosely bound) – helps bacteria adhere to surfaces as well as provides defense against viruses

• Flagella – ‘tail’ that allows movement by rotating and acting as a propeller

• Pili – thin protein tubes for adhesion (colonization) and adhering to surfaces

Inside the cell• Cytoplasm – everything inside the membrane• Nucleoid/Chromosome – DNA of the organism – it

is not contained by a nuclear membrane (as eukaryote cell)

• Ribosomes – made of ribosomal RNA and protein these are responsible for making proteins

• Vacuoles or vesicles – spaces in the cytoplasm that can store solids or gases

• Mesosomes/Organelles –a membrane system internal to the cell which facilitates protein function; there are these structures specifically for photosynthesis

Cell structure

Nucleoid

• Single strand of DNA, usually circular, usually looks like a big ball of messed up twine…

• Size – smallest organism yet discovered (Nanoarchaeum equitans) 490,889 base pairs; e. coli 4.7 Mbp, most prokaryotes 1-6 million base pairs (1-6 MBp); Humans 3300 MBp

• DNA is around 1000 m long in bacteria, while the organism is on the order of 1 m long – special enzymes called gyrases help coil it into a compact form

Ribosomes• Ribosomal RNA is single stranded • RNA is a single stranded nucleic acid

– mRNA- messanger RNA – copies information from DNA and carries it to the ribosomes

– tRNA – transfer RNA – transfers specific amino acids to the ribosomes

– rRNA – ribosomal RNA – with proteins, assembles ribosomal subunits

DNA is transcribed to produce mRNAmRNA then translated into proteins.

RNA and protein construction

• The nucleotide base sequence of mRNA is encoded from DNA and transmits sequences of bases used to determine the amino acid sequence of the protein.

• mRNA (“Messenger RNA”) associates with the ribosome (mRNA and protein portion).

• RNA (“Transfer RNA”) also required• Codons are 3 base mRNA segments that specify a

certain amino acid.• Most amino acids are coded for by more than one

codon: degenerage genetic code.• Translation ends when ribosome reached “stop codon”

on mRNA.

TranscriptionRNA polymeraze takes the DNA and temporarily unwinds it, templates the transfer RNA from that, using ribonucleoside triphosphates to assemble…

Translation• mRNA is coded for one or more specific

amino acids and moves to the ribosome to assemble amino acids into proteins

• On mRNA, codons are 3 bases, coded to specific amino acids

• On tRNA, the anticodon

latches to the codon

on the mRNA

Protein Formation

• The ‘code’ on mRNA determines the sequence of protein assembly

rRNA

• Ribosomes are made of proteins and rRNA, the tRNA and mRNA come to it andassemble the proteins

• rRNA plays a structural role, serving as a support for protein construction, and a functional role

• rRNA consists of two subunits, one 30S in size (16S rRNA and 21 different proteins), one 50S in size (5S and 23S rRNA and 34 different proteins). The smaller subunit has a binding site for the mRNA. The larger subunit has two binding sites for tRNA.

Cytoplasmic inclusions

Where found Composition Function

glycogen many bacteria e.g. E. coli polyglucose reserve carbon and energy source

polybetahydroxyutyric acid (PHB)

many bacteria e.g. Pseudomonaspolymerized hydroxy

butyratereserve carbon and energy source

polyphosphate (volutin granules)

many bacteria e.g. Corynebacteriumlinear or cyclical

polymers of PO4reserve phosphate; possibly a reserve of high

energy phosphate

sulfur globulesphototrophic purple and green sulfur

bacteria and lithotrophic colorless sulfur bacteria

elemental sulfurreserve of electrons (reducing source) in

phototrophs; reserve energy source in lithotrophs

gas vesicles aquatic bacteria especially cyanobacteriaprotein hulls or shells

inflated with gasesbuoyancy (floatation) in the vertical water

column

parasporal crystals

endospore-forming bacilli (genus Bacillus)

protein unknown but toxic to certain insects

magnetosomes certain aquatic bacteriamagnetite (iron oxide)

Fe3O4 orienting and migrating along geo- magnetic

field lines

carboxysomes many autotrophic bacteriaenzymes for autotrophic

CO2 fixationsite of CO2 fixation

phycobilisomes cyanobacteria phycobiliproteins light-harvesting pigments

chlorosomes Green bacterialipid and protein and

bacteriochlorophylllight-harvesting pigments and antennae