Isolation, Cultivation, and Nutrition of Bacteria

1. Isolation, Cultivation, and Nutrition of Bacteria • Nutritional categories: • A. Energy source: • ORGANOTROPH = organic molecules (e.g. sugars, amino acids) • LITHOTROPH = inorganic molecules (e.g. H2S, NH3) • PHOTOTROPH = Light • B. Macronutrients: Cell requires ~ 30 elements. Needs 6 in large amounts (CHNOPS), some only in trace amts, some in between • C-source; Can be • CO2 = AUTOTROPH, or • organic molecules = HETEROTROPH • N-source: Typical bacterium requires 15% nitrogen • Most bacteria obtain N from amino groups (-NH2) or ammonia (NH3), or nitrate (NO3--) • Some bacteria can fix atmospheric nitrogen (N2) to form ammonia (NH3).

2. 3. P-source • Generally available as phosphate ion (PO4---) or organic molecules (e.g. RNA). • Many bacteria secrete phosphatase enzymes to remove phosphate from organic molecules and use it for their own growth • 4. S-source • Obtained from 2 amino acids and from some vitamins. • Generally available as sulfate (SO4--) or sulfide (S--). • C. Micronutrients • K, Mg, Ca, Fe are required in small but significant amounts. • Act as cofactors for many enzymes and cell structures • Trace elements: Co, Zn, Mo, Cu, Mn, Ni, W, Se -- usually required by a small number of enzymes.

3. Membrane Transport Membranes are permeability barriers; they maintain equilibrium "inside" and "outside" of cell. Active cells must continually move food and nutrients in, and wastes out. Requires specific transport carriers. Types of Membrane Transport: Passive Diffusion. Lipophilic solutes cross the membrane freely by dissolving in the lipid bilayer Facilitated Diffusion. Protein carrier is required, and will speed up movement of substrate across membrane. No energy required, no preferential direction is necessary Active Transport -- substance is imported without chemical change, and energy is expended.

4. Culture Media • Media must include source of C, N, P, S, 4 of the 6 major nutrients (CHNOPS), and micronutrients. • Can be liquid or solid. Use for different purposes: • Liquid media: easiest to prepare and use. Good for growing quantities of microbes needed for analysis or experimentations. • Unless inoculated with pure culture, cannot separate different organisms. • Solid media: usually made by adding. 1.5% agar.

5. Types of culture media: • Synthetic of Defined Media: Relatively simple media, all components are known • Complex Media: Composition of media not completely known. Often made from inexpensive organic materials such as slaughterhouse wastes (tryptic digests called tryptone, trypticase, etc.) • Selective Media: Media favors the growth of one or more microbes: Example: The use of bile salts to inhibit growth of most gram-positive bacteria and some gram-negative bacteria. • Differential Media: Media allows distinguishing between different bacteria that are growing. MacConkey agar has color indicator that distinguishes presence of acid which is a product of bacteria that ferment a particular sugar.

6. Microbial Genetics Every living organism has DNA = cell database. Bacteria have single chromosome and no nucleus Central dogma: Information is encoded in DNA. To express this information, RNA is transcribed with same coding, then translated into amino acid sequence which folds to form active proteins • DNA is made from subunits called nucleotides • Each nucleotide contains: • Purine (Adenine or A, Guanine or G) or Pyrimidine (Cytosine or C, Thymine or T) bases • Deoxyribose sugar • 1, 2, or 3 phosphate groups

7. Nucleotides are named according to # of phosphates: e.g., dATP = deoxy adenosine triphosphate, whereas dAMP = deoxy adenosine monophosphate Watson & Crick discovered structure of DNA by analyzing X-ray data from Franklin & Wilkins. DNA is double helix, "backbone" consists of alternating units of: deoxyribose -- phosphate -- deoxyribose -- phosphate – Purine & Pyrimidine bases are attached to deoxyribose sugar, free to rotate. In DNA, form specific base pairs: A with T (2 H-bonds), G with C (3 H-bonds). Two chains of DNA face in opposite directions, called antiparallel In a linear DNA molecule, one strand has free 3'-end, other (complementary) strand has 5'-end. 5'-CAGCTAGAGTCATCG-3' 3'-GTCGATCTCAGTAGC-5'

8. RNA (in prokaryotes) • Components: Ribose,Phosphate,Purine bases A, G; Pyrimidine bases C, U • U has same base pairing properties as T (forms U=A base pairs) • RNA is not double stranded (except in some viruses) • RNA can have modified bases (after transcription) -- such as inosine, pseudouridine. • Types of RNA: • Messenger RNA -- Carries codons to RNA • Ribosomal RNA -- Part of ribosome structure, catalyzes peptide bond formation • Transfer RNA -- Small RNAs (about 60 diff. types in E. coli), transport amino acids to ribosome for incorporation into growing polypeptides

9. Transcription Requires enzyme RNA polymerase Opens up DNA helix for short stretch (~ 15 base pairs), selects one of two strands as template strand RNA synthesized in 5' to 3' direction Promoters: sites on DNA that are recognized as "start" signals for RNA synthesis. Typical promoter has "TATA..." base sequence (consensus sequence -- some variation) at -10 bases (upstream from actual site of RNA synthesis). Terminators: can be stem-loop structures with poly-U runs, or certain sequences recognized by rho factor. Antibiotic effects: Rifamycin (prokaryotic): affects beta subunit of RNA polymerase Actinomycin: (acts on both prokaryotic and eukaryotic cells): binds to DNA at GC pairs, blocks attachment of RNA polymerase

10. Many genes occur in operons: • Several structural genes (for protein) controlled by a single promoter • mRNA is polycistronic: has multiple start & stop signals, codes for multiple polypeptides

11. Transcription & Translation are coupled • Protein synthesis begins before transcription ends • Multiple ribosomes bind to each mRNA • All control at level of transcription • Once m-RNA is made, it is translated. • Protein synthesis (in prokaryotes): • Role of mRNA :Carries coding information for amino acids = codons, 3 adajacent nucleotide bases Example: AAA, AGU, etc. • leader sequence on mRNA (called Shine-Dalgarno sequence) binds to complementary sequence on small ribosome subunit. • Role of ribosome • acts as a "decoding box" or "tape player" for the information in mRNA • Role of tRNA • Anticodon site: recognizes codon on mRNA • AA added by enzyme: AA-tRNA activating enzymes • ATP required, forms AA-AMP + PP, then AA-tRNA + AMP

12. Gene Transfer Mechanisms • Types of Horizontal Gene Transfer: • Transformation: • Natural transformation = uptake of DNA fragments from medium surrounding cell. Requires specific proteins in cell membrane and energy. • Artificial transformation = laboratory technique to enhance DNA uptake of cells that do not have genetic machinery for transformation. • Electroporation: pulsed electric fields produce short-lived membrane pores, allows DNA movement (either in or out of cell). Useful way to move small pieces of DNA and plasmids between cells. Conjugation: Plasmid-directed transfer of DNA from one cell to another.

13. Generalized Transduction: • DNA bacterial viruses = bacteriophages normally replicate in cell, produce many copies of phage DNA. • Many copies of phage head coat proteins, finally assemble phage heads by packing phage DNA into new phage heads. Attach tail (if present), open cell, release progeny. • Host DNA often degraded. But occasionally, piece of partially degraded bacterial DNA is correct size to be packed inside phage coat proteins, phage erroneously packs up a "mistake" = transducing phage.

14. Biofuels and Biofuel Cells from Microorganisms Photosynthetic microorganisms, such as micro-algae and cyanobacteria are able to harness low-intensity solar energy and store it as latent chemical energy in the biomass. Photo-biological hydrogen production: Chloroplast of some photosynthetic microorganisms such as the green alga chlorella in the presence of suitable electron acceptors is capable of producing H2 and O2 through direct photolysis of water. a) Alcohol (ethanol) Production: Yeast cultures, (saccharomyces), are very efficient in converting sugars into ethanol, i.e. cost competitive and are not as strongly inhibited by high ethanol concentrations as are other microbes.

15. b) Methane production: Energy-rich fuel Large amounts of methane can be produced by anaerobic decomposition of waste materials In microbial production of methane, naturally occurring mixed anaerobic bacteria population is always used to convert organic compound to methane gas Cells are normally retained within the digester for continuous digestion During the fermentation process, a large amount of organic matter is degraded, with a low yield of microbial cells, while about 90% of the energy available in the substrate is retained in the easily purified gaseous product CH4. The end product is a mixture of methane gas and CO2 (also called biogas)