Geomicrobiology

1. Geomicrobiology

2. Course Goals • At the end of this course you will be able to… • Intelligently converse with microbiologists, geologists, environmental scientists and engineers about the role microorganisms play in the cycling of elements • Use several techniques to identify and characterize microorganisms in any environment • Relate microbial physiology, genetics, cell structure, and metabolism to the effect, role, or signature that microbes uniquely imprint on their surroundings

3. Grading • Homeworks 10% • Discussion participation 20% • Mid term exam 20% • Final Exam 20% • Lab/Field Project and Paper 30%

4. Basic Microbiology Primer • Microorganisms exist as single cells or cell clusters – almost all of them are invisible to the naked eye as individuals but can be readily seen as communities • As opposed to most ‘higher order’ life om earth, microbes can eat and breathe things besides organic carbon and oxygen  this makes them critical to cycling of compounds that are able to be oxidized or reduced in water

5. Classification of life forms: • Eukaryotic = Plants, animals, fungus, algae, and even protozoa • Prokaryotic = archaea and bacteria • Living cells can: • Self-feed • Replicate (grow) • Differentiate (change in form/function) • Communicate • Evolve Can purely chemical systems do these things? All of these things? Why do we care to go through this ?

6. 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?

7. 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 Macromolecules

8. 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

9. Small molecules present in a growing bacterial cell.

10. Inorganic ions present in a growing bacterial cell.

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

12. Construction, Part 1… • Sugars (aka carbohydrates) to polysaccharides • 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 – form starches, cellulose, glycogen, etc.

13. 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

14. Construction, Part 3 • Bases – Two types: Pyrimidine Purine • Derivatives Cytosine, C Uracil, U Thymine, T Adenine, A Guanine, G DNA  C,T,A,G No U RNA  C,U,A,G No T

15. Bases come together with a pentose sugar to form a nucleoside • A nucleosidecontaining phosphate is a nucleotide This P group has a pK 1 of ~1.0, therefore is acidic  nucleic acid

16. ATP structure • A nucleotide with 3 phosphate linkages is ATP, the principle ‘currency’ of energy  it is a high energy molecule, energy from P-P bonds drive many processes… • Different nucleotide 3 P links (GTP, CTP, UTP) also provide energy for other cell processes

17. Nucleotides are then linked together via a phosphodiester bond • Several nucleoties together = oligonucleotide  deoxyribose bonded together with the phosphodiester is the backbone… 3’ and 5’ linkages run in specific directions – assures directionality for assembling a strand of nucleosides i.e. no bonding though 3’-3’ or 5’-5’ bonding

18. Strand put together with alternating sequences • Number sequences using a shorthand: 3’ taagc-p 5’ or 5’ p-cgaat 3’

19. DNA formation • Chargaff's Law: [A] = [T], and [G] = [C] (for any DNA)  ONLY combination possible, G does NOT bond to itself, A or T – ONLY C !! • Purine –pyrimidine pairs!

20. Down-axis view

21. Construction, Part 4 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 • Amino acids  monomer units of proteins

22. Proteins • Proteins are generally either catalytic proteins (aka enzymes) or structural proteins • Proteins are polymers of various lengths (20 – 10,000 amino acid monomer units) and are folded, with a complex geometry which also helps determine function Denaturation – excess heat, outside a pH range, interaction with reactive chemicals can undo this folding – destroying protein function

23. Cell Construction • OK – using the building blocks we have described, let’s make a microbe…

24. Cell sizes and shape • Most cells are between 0.1 and 5 mm in diameter • Several shapes are common: • Rod or bacilli • Spherical or cocci • Spiral • Other forms – including square, sheathed, stalked, filamentous, star, spindle, lobed, pleomorphic forms

25. 100 µm 20 µm 0.5 µm Microbes on the head of a pin, false color SEM images, from j. Rogers, http://people.westminstercollege.edu/faculty/jrogers/V%20prokaryotes.ppt#298,3,Slide 3

26. Figure 27.3 The most common shapes of prokaryotes http://people.westminstercollege.edu/faculty/jrogers/V%20prokaryotes.ppt#298,3,Slide

27. Nuclear material Prokaryote Structure Cell wall membrane Membrane is critical part of how food and waste are transported - Selectively permeable Phospholipid layer Transport proteins

28. Eubacteria vs. Archaebacteria Archaeal cell structure Bacterial 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. H+ gradients across the membrane • Proton Motive Force (PMF) is what drives ATP production in the cell

35. Figure 5.21

36. 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

37. 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

38. 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

40. 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

41. 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

42. 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

44. Inside the cell • Cytoplasm – everything inside the membrane • Nucleoid – 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 • Organelles – structures specifically for photosynthesis – a membrane system

45. Ribosomes • 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