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 Homeworks10% Discussion participation20% Mid term exam20% Final Exam20% Lab/Field Project and Paper30%

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. 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. 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

10. Inorganic ions present in a growing bacterial cell. Ion Function K+K+ Maintenance of ionic strength; cofactor for certain enzymes NH 4 + 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 SO 4 -- Principal form of inorganic S for assimilation PO 4 --- Principal form of P for assimilation and a participant in many metabolic reactions

11. Molecular composition of E. coli under conditions of balanced growth. Molecule Percentage 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 2.9 1.0 100.0

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: PyrimidinePurine Derivatives Cytosine, CUracil, UThymine, TAdenine, AGuanine, 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 nucleoside containing 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 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

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  m 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. 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 3 100 µm20 µm0.5 µm

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

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

28. 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

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 (O 2, N 2, CO 2, ethanol, H 2 O freely diffuse through layer –Osmosis  because solute concentration inside the cell are generally higher (10 mM inside the cell), water activity is lower inside, H 2 O 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