1. Chemicals needed for life Besides chemicals for metabolic energy, microbes need other things for growth. –Carbon –Oxygen –Sulfur –Phosphorus – Arsenic can substitute (??) –Nitrogen –Iron –Trace metals (including Mo, Cu, Ni, Cd, etc.) LET’S PUT THIS TOGETHER INTO A MICROBE….

2. 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

3. 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

4. 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

5. 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

6. 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

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. 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 C n (H 2 O) n-1 where n is typically 200-2500 

9. 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

10. Construction, Part 2 Fatty Acids – long chains of C (aliphatic) Lipids are made of fatty acids put together to form hydrophobic and hydrophilic end The chemical characteristics of the fatty acids and subsequently the lipids make them ideal for membranes

11. 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

12. 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

13. 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

15. 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

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

18. Proteins – ‘key and lock’ concept

19. Peptidoglycan (aka Murein) Polymer consisting of both sugars and amino acids Rigid material and serves a structural role in cell wall

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

21. Flagella,Pili Function(s) Swimming movement Predominant chemical composition Protein Sex pilusMediates DNA transfer during conjugationProtein 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 RibosomesSites of translation (protein synthesis)RNA and protein InclusionsOften reserves of nutrients; additional specialized functions Highly variable; carbohydrate, lipid, protein or inorganic ChromosomeGenetic material of cell DNA PlasmidExtrachromosomal genetic materialDNA

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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

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

30. Figure 5.21

31. 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

32. 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

33. 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

35. 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

36. 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

37. 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

39. 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

40. Cell structure

41. 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

42. 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 mRNA mRNA then translated into proteins.

43. 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.

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

45. 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

46. Protein Formation The ‘code’ on mRNA determines the sequence of protein assembly

47. 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.

49. Cytoplasmic inclusions Where foundCompositionFunction glycogenmany bacteria e.g. E. colipolyglucosereserve carbon and energy source polybetahydroxy utyric acid (PHB) many bacteria e.g. Pseudomonas polymerized hydroxy butyrate reserve carbon and energy source polyphosphate (volutin granules) many bacteria e.g. Corynebacterium linear or cyclical polymers of PO4 reserve phosphate; possibly a reserve of high energy phosphate sulfur globules phototrophic purple and green sulfur bacteria and lithotrophic colorless sulfur bacteria elemental sulfur reserve of electrons (reducing source) in phototrophs; reserve energy source in lithotrophs gas vesiclesaquatic bacteria especially cyanobacteria protein hulls or shells inflated with gases buoyancy (floatation) in the vertical water column parasporal crystals endospore-forming bacilli (genus Bacillus) proteinunknown but toxic to certain insects magnetosomescertain aquatic bacteria magnetite (iron oxide) Fe3O4 orienting and migrating along geo- magnetic field lines carboxysomesmany autotrophic bacteria enzymes for autotrophic CO2 fixation site of CO2 fixation phycobilisomescyanobacteriaphycobiliproteinslight-harvesting pigments chlorosomesGreen bacteria lipid and protein and bacteriochlorophyll light-harvesting pigments and antennae