MICROBIOLOGY 120.

Microbial Physiology
Course description: Physiological processes in microorganisms including a study of structure, energy production, macromolecular biosynthesis, nutrition and growth Credits: Prerequisite: 3 units (3 hrs of lecture per week) MCB 1 and CHEM 160

Course Objectives: 1) To identify the structure, chemistry and functions of major structures in a bacterial cell; 2) To elucidate the requirements of microbial growth; 3) To identify the various metabolic pathways present in different microorganisms; 4) To understand enzyme regulation present in microbial cells; 5) To relate the principles of microbial physiology to the industrial applications of microorganisms.

OUTLINE
I. II. Introduction The bacterial cell: structures and functions 1. Major cellular structures 2. Chemistry and synthesis of cellular structures First long exam

III.

Microbial Growth 1. 2. 3. 4. 5. Definition of growth Measurements of growth Growth Physiology Steady-state Growth and Continuous Factors affecting growth

IV. V.

Microbial Nutrition and Solute Transport Bioenergetics in the Cytosol Second long exam

VI. Bacterial Metabolism 1) Central metabolic pathways a) b) c) d) e) Glycolysis Pentose Phosphate Pathway Entner-Doudoroff pathway Tricarboxylic Acid Cycle Glyoxylate Cycle

Third long exam

2. Fermentation 3. Photosynthesis 4. Metabolism of lipids, nucleotides, amino acids and hydrocarbon 5. Inorganic metabolism 6. C1 metabolism Fourth long exam

REFERENCES
ATLAS, R.M. 1995. Principles of Microbiology. Mosby-Year Book Inc., Missouri. MADIGAN, M., MARTINKO, J. and PARKER, J. 2012. Brock Biology of Microorganisms. 13th edition. Prentice Hall, New Jersey. NEIDHART, F.C., INGRAHAM, J.L. and SCHAECHTER, M. 1990. Physiology of the Bacterial Cell. A Molecular Approach. Sinauer Associate, Inc., Massachusetts. STRYER, L. 1995. Biochemistry. W.H. Ferman and Co., New York. WHITE, D. 2007. The Physiology and Biochemistry of Procaryotes. Oxford University Press, New York Specific journal articles as cited On-line materials as cited

GRADING SCALE 1.0 1.25 1.5 1.75 2.0 2.25 2.5 2.75 3.0 4.0 5.0 95.6 - 100 91.1 - 95.5 86.7 - 91.0 82.2 - 86.6 77.8 - 82.1 73.3 - 77.7 68.9 - 73.2 64.4 - 68.8 60.0 - 64.3 55.0 - 59.9 <55.0

ATTENDANCE AND MAKE-UP EXAM

1. A general make-up exam which is comprehensive in scope will be given towards the end of the semester to students who missed any of the 4 long exams due to a valid reason (an official excuse slip must be presented). A student who incurs at least 10 absences in the lecture will be automatically dropped from the course. Attendance sheets will be passed around for monitoring of the student's attendance in class. If majority of the absences are excused, the student shall be given a grade of DRP. If majority of the absences are unexcused, the student shall be given a grade of 5.0. 3. Excuse slips must be presented not later than the second class session following the student's return.

2.

LECTURERS INFORMATION Name: DR. RINA B. OPULENCIA MS Microbiology: University of Queensland, Australia PhD Microbiology: University of Illinois, UrbanaChampaign, USA Office: B-302 or D-333 Consultation hours:
WED and FRI: 9:00 AM-11:00 AM; 2:00 PM - 4:00 PM TUES: 2:00 PM - 4:00 PM

INTRODUCTION

Physiology - study of the functions of living organisms and their physicochemical parts and metabolic reactions cytology (physical and chemical structures) biochemistry (enzymes and chemical reactions) nutrition genetics Microbial Physiology - study of life processes of microorganisms

Importance of Studying Microbial Physiology

1. Knowledge of microbial physiology can be applied to other fields. 2. Microorganisms can serve as models to understand life processes.

Importance of Prokaryotes

ubiquitous exhibit great metabolic and genetic diversity comprise the majority of organic matter major environmental determinants on earth

Whitman, W. B. 1998. PNAS USA. 95: 6579

Whitman, W. B. 1998. PNAS USA. 95: 6579

White, D. 2006
Whitman, W.B. 2009. J. Bacteriol. 491:2000-2005

ARCHAEA

vs

BACTERIA

Lipids: alcohols ether-linked to glycerol Cell Wall: variable, some have pseudo-PG Genome: eukaryotic-type histones; DNA organized into nucleosome-like structures Transcription machinery: RNAP has 8-10 subunits, like eukaryotes; RifR

Lipids: fatty acids ester-linked

to glycerol Cell Wall: Peptidoglycan (PG) a universal component Genome: histone-like proteins, but not organized like nucleosomes Transcription machinery: RNAP has 4 subunits; is RifS

ARCHAEA

vs

BACTERIA

Translation machinery: Use Met as initiator amino acid; insensitive to translational inhibitors that affect bacteria; require EF-2 like eukaryotic ribosomes

Translation machinery: Use f-Met as initiator amino acid; sensitive to translational inhibitors, e.g., Tet, Cm

UNIQUE:

Light-driven ion pumps (halophiles) Unique coenzymes (methanogens)

MICROBIOLOGY 120. MICROBIAL PHYSIOLOGY

majority of the topics is bacterial physiology

studies done on Escherichia coli or Bacillus subtilis Model organisms provide basis for understanding important biological principles but not all bacteria are the same.

The Prokaryotic Cell

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

CELL WALL

fairly rigid layer that lies outside the plasma membrane Importance: - confers shape - protects the cell from osmotic lysis - anchors the flagellum - adds to pathogenicity of the cell - protects the cell from toxic substances and pathogens Bacteria can be divided into two big groups based on cell wall structure.

BACTERIAL CELL WALL

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

GRAM POSITIVE CELL WALL Characteristics A. Thick layer of peptidoglycan (murein; mucopeptide) a polymer of disaccharide linked by polypeptide insoluble, porous, big polymer > 50% of the cell walls dry weight; 15 - 40 nm thick isolatable as murein sacculus

Peptidoglycan Subunit

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

Peptidoglycan Interbridge
Type I. Direct D-alanyl-R3 peptide bond - found in E. coli and other gram negative bacteria - also found in many bacilli Type II. Pentaglycine or Other L- or D- amino acid sequences - varies from organism to organism Type III. A bridge composed of one to several peptides, each having the same amino acid sequence as the peptide unit attached to muramic acid Type IV. A bridge extending between carboxyl groups belonging to either D-alanine or D-glutamic acid and a diamino acid residue or a diamino acid containing short peptide

Peptidoglycan Interbridge

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

Peptidoglycan Interbridge

Peptidoglycan Polymer

Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker 2003 Benjamin Cummings

Peptidoglycan Synthesis: Synthesis of UDP-derivatives

Enzymes 1 2 3 4 glutamine:fructose-6-P aminotransferase glucosamine-P transacetylase N-acetylglucosamine phosphomutase UDP-N-acetylglucosamine pyrophosphoryalse

Peptidoglycan Synthesis
Enzymes 1 enoylpyruvate transferase 2 UDP- N-acetylpyruvoylglucosamine reductase 3 each amino acid is added by separate enzyme

http://micro.digitalproteus.com/pics/peptidoglycansynthesis.jpg

Synthesis of peptidoglycan occurs in three phases: assembly of precursor in the cytoplasm, transport across the inner membrane, and polymerization. The lipidlinked muropeptide (lipid I) is generated in the cytoplasm from amino acids and UDP-MurNAc (MurNAc is depicted by orange squares). Transfer of Nacetylglucosamine (blue squares) from UDP-GlcNAc completes formation of the precursor lipid II. Translocation across the inner membrane occurs, and subsequently, the chain polymerizes while attached to the lipid carrier. The unit is then transferred to existing peptidoglycan. (PEP) Phosphoenolpyruvate; (mDAP) meso-diaminopimelic acid. http://www.ncbi.nlm.nih.gov/bookshe lf/br.fcgi?book=glyco2&part=ch20

GRAM POSITIVE CELL WALL

B. Presence of Teichoic acids - polymers of repeating units of glycerol or ribitol joined by phosphates - amino acids (D-ala) or sugars (glu) are attached to glycerol/ribitol - covalently linked to murein through muramic acid - connected/embedded in PG layer or to membrane lipids lipoteichoic acid
- linear polymers of 16-40 phosphodiester-linked glycerophosphate residues covalently linked to the cell membrane

GRAM POSITIVE CELL WALL: TEICHOIC ACID

Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker 2003 Benjamin Cummings

TEICHOIC ACID: Properties and Importance

1. highly antigenic 2. anchors the wall to the cell membrane 3. provides high density of regularly oriented charges 4. storage of phosphorus 5. facilitates attachment of bacteriophage 6. inhibits activity of autolytic enzymes which hydrolyze the murein

TEICHOIC ACID: Variations A. Glycerol Teichoic Acids

1. Glycerol2. Glycerolalanyl 3. Glycerolglucosaminyl alanyl

P P

TEICHOIC ACID: Variations B. Ribitol Teichoic Acids 7. ribitol- P 8. ribitol- P ala glucosyl 9. ribitol- P ala NAG

Other substances which may be found on the cell wall:

Teichuronic acids acidic polysaccharides containing uronic acids Neutral polysaccharides important in classification of some Gram (+) Other glycolipids may substitute for whatever function of the LTA Mycolic acids waxy lipids found in Mycobacterium

Substances active against peptidoglycan synthesis

1. Phosphonomycin (Fosfonomycin) - prevents synthesis of UDP-NAM from UDP-NAG 2. Cycloserine - inhibits the formation of the pentapeptide 3. Bacitracin - inhibits incorporation of lysine into the peptidoglycan - prevents dephosphorylation of the carrier lipid 4. Vancomycin, Tunicamycin, Ristocetin - inhibits translocation step of peptidoglycan 5. Penicillin - prevents cross-linking

Beta-lactam antibiotics

Beta-lactam antibiotics

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

Beta-lactam antibiotics Mode of action:

inhibition of final stages of assembly of the peptidoglycan must penetrate the cell wall and operate at the periplasmic space bind to penicillin-binding proteins in the outer leaf of the cell membrane enzymes which are responsible for the final stages of assembly of the peptidoglycan molecule

Properties of penicillin-binding proteins

PBP 1A or 1B 2 3 4 5 6 1B1C, 7, 8 Molecules per cell Known Enzyme Activities Possible Function murein synthesis during cell elongation growth in rod shape; cell elongation murein synthesis during septation

100 (each) transglycosylasetranspeptidase 20 50 110 1,800 600 transpeptidase transglycosylasetranspeptidase

DD-endopeptidase, Cross-link hydrolysis DD-carboxypeptidase during cell elongation DD-carboxypeptidase Destruction of unutilized pentapeptide DD-carboxypeptidase Destruction of unutilized pentapeptide not known

not known not known

GRAM NEGATIVE CELL WALL

1. Peptidoglycan - thin: 1-2 layers in E. coli ( 2 - 6 nm thick) - constitute not more than 5 10% of walls dry weight - may be more of a gel than a compact layer 2. Outer membrane - located above/external to peptidoglycan layer - like the cytoplasmic membrane - other main components a. lipopolysaccharides (LPS) b. lipoproteins c. outer membrane proteins (porins)

GRAM NEGATIVE CELL WALL

Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker 2003 Benjamin Cummings

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDES

- lipids and carbohydrates - outer layer of the outer membrane - endotoxin - consists of three parts:

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDE LIPID A

- embedded in the membrane as part of the lipid bilayer - hydrophobic - composed of 2 glucosamine residues linked -1,6 (backbone) with four identical fatty acids

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDE CORE

Outer core - shows high to moderate variability - consists of hexoses Inner core - shows low structural variability - consists of 2-keto-3-deoxyoctonate (KDO), heptose, ethanolamine and phosphate

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDE O-ANTIGEN

short polysaccharide extending outward from the core consists of peculiar sugars which varies between bacterial strains not essential for viability

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDE

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

GRAM NEGATIVE CELL WALL: LIPOPOLYSACCHARIDE Importance: avoidance of host defenses (O-antigen) contributes to the negative charge on the cells surface stabilizes membrane structure acts as endotoxin

GRAM NEGATIVE CELL WALL: LIPOPROTEIN

Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker 2003 Benjamin Cummings

GRAM NEGATIVE CELL WALL: LIPOPROTEIN

- mediate interconnection between the OM and murein

OUTER ENVELOPE: PORINS

form small hydrophilic channels through the outer envelope

allowing the diffusion of neutral and charged solutes of MW <600 daltons three identical units associate to form membrane holes transmembrane

Outer membrane proteins (OMPs) of Gram Negative Bacteria

Protein Porin OmpB OmpC (1.1 nm) OmpF (1.2 nm) PhoE LamB Protein P Physiological Role Diffusion channel for various metabolites including maltose Diffusion channel for small molecules Receptor for phages Tulb, T4 Diffusion channel for small molecules Receptor for phages Tula, T2 Anion-selective diffusion channels induced under phosphate limitation Specific porin for maltose, maltodextrin Receptor for bacteriophage Anion-selective diffusion channels in Pseudomonas; induced under phosphate limitation

PERIPLASM
- A separate compartment between the cell membrane and outer membrane in Gram (-) bacteria - Seen in electron micrographs as space but should be considered an aqueous compartment - Activites: redox reactions osmotic regulation solute transport protein secretion hydrolysis

PERIPLASM: COMPONENTS AND FUNCTIONS

1. Oligosaccharides thought to be involved in the osmotic regulation of the periplasm because their amounts decrease when the cells are grown in media of high osmolarity Solute binding proteins bind to solutes and deliver solutes to specific transporters in the membrane Cytochrome cyt c Hydrolytic enzymes degrade nutrients to smaller molecules that can be transported across the membrane by specific transporter Detoxifying agents e.g. -lactamase TonB protein required for the uptake of several solutes (iron siderophores, vit B12) that do not diffuse through the porin

2. 3. 4.

5. 6.

PERIPLASM: ACTIVITIES

Protein transport Nutrient acquisition

Protein folding Disulfide bond formation

PERIPLASM IN GRAM POSITIVE CELLS? Evidences: release of putative periplasmic proteins (distinct nucleases) in protoplasts of Bacillus subtilis area outside the cell membrane of B. subtilis is bipartite (cryo-TEM)

Archaeal Cell Walls

- Lacks peptidoglycan - May contain polysaccharide, protein (S layer) or pseudopeptidoglycan

Archaeal Cell Walls: Polysaccharide

cell wall composed of glucose, glucuronic acid, acetate and galactosamine found in Methanosarcina spp.

Archaeal Cell Walls: S-layer

- protein subunits arranged in a regular array on the cell surface - found in extreme halophiles, methanogens and hyperthermophiles

Albers et al. Nature Reviews Microbiology advance online publication; published online 06 June 2006 | doi:10.1038/nrmicro1440

Archaeal Cell Walls: S-layer

Archaeal Gram negative cell wall

Archaeal Gram positive cell wall

Archaeal Cell Walls: Pseudopeptidoglycan

- also called pseudomurein - consists of glycan units linked by peptides - glycan units: N-acetylglucosamine N-acetyltalosaminuronic acid - peptide bridge contains L-amino acids rather than D amino acids
NAT: N-acetylalosaminuronic acid

Mollicutes: A Special Case

Mycoplasma; Spiroplasma Many are parasitic and pathogenic. small cell wall-less bacteria but possess distinct morphologies Have internal protein cytoskeleton that determines and maintains cell shape

Spiroplasma

Williamson, D. L. 1974. J. Bacteriol. 117:904-906.