MCB 120 Exam 1A

MICROBIOLOGY 120. Microbial Physiology

Course description: Physiological processes in microorganisms including a study of structure, energy production, macromolecular biosynthesis, nutrition and growth

Credits: 3 units (3 hrs of lecture per week)

Prerequisite: 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. Introduction

II. The bacterial cell: structures and functions

1. Major cellular structures

2. Chemistry and synthesis of cellular structures

First long exam

III. Microbial Growth

1. Definition of growth2. Measurements of growth3. Growth Physiology4. Steady-state Growth and Continuous5. Factors affecting growth

IV. Microbial Nutrition and Solute Transport

V. Bioenergetics in the Cytosol

Second long exam

VI. Bacterial Metabolism

1) Central metabolic pathways

a) Glycolysisb) Pentose Phosphate Pathwayc) Entner-Doudoroff pathwayd) Tricarboxylic Acid Cyclee) Glyoxylate Cycle

Third long exam

2. Fermentation3. Photosynthesis4. Metabolism of lipids, nucleotides, amino

acids and hydrocarbon5. Inorganic metabolism6. C1 metabolism

Fourth long exam

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 theBacterial 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 UniversityPress, New York

Specific journal articles as cited

On-line materials as cited

REFERENCES

1.0 95.6 - 1001.25 91.1 - 95.51.5 86.7 - 91.01.75 82.2 - 86.62.0 77.8 - 82.12.25 73.3 - 77.72.5 68.9 - 73.22.75 64.4 - 68.83.0 60.0 - 64.34.0 55.0 - 59.95.0 <55.0

GRADING SCALE

ATTENDANCE AND MAKE-UP EXAM

1. A general make-up exam which is comprehensive in scope will be given towards theend of the semester to students who missed any of the 4 long exams due to a validreason (an official excuse slip must be presented).

2. A student who incurs at least 10 absences in the lecture will be automatically droppedfrom the course. Attendance sheets will be passed around for monitoring of thestudent'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 thestudent's return.

LECTURER’S INFORMATION

Name: DR. RINA B. OPULENCIA

MS Microbiology: University of Queensland, Australia

PhD Microbiology: University of Illinois, Urbana-Champaign, 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

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

INTRODUCTION

1. Knowledge of microbial physiology can be applied toother fields.

2. Microorganisms can serve as models to understand lifeprocesses.

Importance of Studying Microbial Physiology

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

Lipids: alcohols ether-linked toglycerol Cell Wall: variable, some havepseudo-PG Genome: eukaryotic-typehistones; DNA organized intonucleosome-like structures Transcription machinery: RNAPhas 8-10 subunits, like eukaryotes;RifR

Lipids: fatty acids ester-linkedto glycerol Cell Wall: Peptidoglycan (PG) auniversal component Genome: histone-like proteins,but not organized like nucleosomes

Transcription machinery: RNAPhas 4 subunits; is RifS

ARCHAEA vs BACTERIA

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

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

Translation machinery:Use f-Met as initiatoramino acid; sensitive totranslational inhibitors,e.g., Tet, Cm

ARCHAEA vs BACTERIA

MICROBIOLOGY 120. MICROBIAL PHYSIOLOGY

majority of the topics is “bacterial” physiology

studies done on Escherichia coli or Bacillus subtilis

Model organisms provide basis for understandingimportant biological principles but not all bacteria are the same.

The Prokaryotic Cell

Brock Biology of Microorganisms 10/eMadigan/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/eMadigan/Martinko/Parker

2003 Benjamin Cummings

GRAM POSITIVE CELL WALL

Characteristics

A. Thick layer of peptidoglycan (murein; mucopeptide)

•• a polymer of disaccharide linked by polypeptidea polymer of disaccharide linked by polypeptide

•• insoluble, porous, big polymerinsoluble, porous, big polymer

•• > 50% of the cell wall> 50% of the cell wall’’s dry weight; 15 - 40 nm thicks dry weight; 15 - 40 nm thick

•• isolatable as murein sacculusisolatable as murein sacculus

Peptidoglycan Subunit

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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

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Madigan/Martinko/Parker

2003 Benjamin Cummings

Peptidoglycan Interbridge

Peptidoglycan Polymer

Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker

2003 Benjamin Cummings

Enzymes

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

Peptidoglycan Synthesis: Synthesis of UDP-derivatives

Peptidoglycan SynthesisEnzymes

1 – enoylpyruvate transferase2 – UDP- N-acetylpyruvoylglucos- amine reductase3 – each amino acid is added by

separate enzyme

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

Synthesis of peptidoglycanoccurs in three phases:assembly of precursor in thecytoplasm, transport acrossthe inner membrane, andpolymerization. The lipid-linked muropeptide (lipid I)is generated in thecytoplasm from amino acidsand UDP-MurNAc (MurNAcis depicted by orangesquares). Transfer of N-acetylglucosamine (bluesquares) from UDP-GlcNAccompletes formation of theprecursor lipid II.Translocation across theinner membrane occurs,and subsequently, the chainpolymerizes while attachedto the lipid carrier. The unitis then transferred toexisting peptidoglycan.(PEP)Phosphoenolpyruvate; (m-DAP) meso-diaminopimelicacid.

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=glyco2&part=ch20

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

GRAM POSITIVE CELL WALL

lipoteichoic acid

- linear polymers of 16-40 phosphodiester-linked glycerophosphateresidues 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

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: Properties and Importance

TEICHOIC ACID: Variations

A. Glycerol Teichoic Acids

1. –Glycerol- P

2. –Glycerol- Palanyl

3. –Glycerol- Pglucosaminyl

alanyl

TEICHOIC ACID: Variations

B. Ribitol Teichoic Acids

P7. –ribitol- P 8. –ribitol-

ala glucosyl

P 9. –ribitol-

ala NAG

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

Other substances which may be found on the cell wall:

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

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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 theperiplasmic 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

not knownnot knownnot known1B1C, 7, 8

Destruction ofunutilized pentapeptide

DD-carboxypeptidase6006

Destruction ofunutilized pentapeptide

DD-carboxypeptidase1,8005

Cross-link hydrolysisduring cell elongation

DD-endopeptidase,DD-carboxypeptidase

1104

murein synthesisduring septation

transglycosylase-transpeptidase

503

growth in rod shape;cell elongation

transpeptidase202

murein synthesisduring cell elongation

transglycosylase-transpeptidase

100 (each)1A or 1B

Possible FunctionKnown EnzymeActivities

Moleculesper cell

PBP

Properties of penicillin-binding proteins

GRAM NEGATIVE CELL WALL

1. Peptidoglycan

- thin: 1-2 layers in E. coli ( 2 - 6 nm thick)- constitute not more than 5 – 10% of wall’s 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. lipoproteinsc. 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 β-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 cell’s 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 envelopeallowing the diffusion of neutral and charged solutes ofMW <600 daltons

three identical units

associate to form membrane holes

transmembrane

Anion-selective diffusion channels in Pseudomonas;induced under phosphate limitation

Protein P

Specific porin for maltose, maltodextrinReceptor for bacteriophage λ

LamB

Anion-selective diffusion channels induced underphosphate limitation

PhoE

Diffusion channel for small moleculesReceptor for phages Tula, T2

OmpF (1.2 nm)

Diffusion channel for small moleculesReceptor for phages Tulb, T4

OmpC (1.1 nm)

Diffusion channel for various metabolites includingmaltose

OmpB

Physiological RoleProtein Porin

Outer membrane proteins (OMPs) of Gram Negative Bacteria

- 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

PERIPLASM:

COMPONENTS AND FUNCTIONS1. Oligosaccharides – thought to be involved in the osmotic regulation

of the periplasm because their amounts decrease when thecells are grown in media of high osmolarity

2. Solute binding proteins – bind to solutes and deliver solutes to specific transporters in the membrane

3. Cytochrome – cyt c

4. Hydrolytic enzymes –degrade nutrients to smaller molecules thatcan be transported across the membrane by specifictransporter

5. Detoxifying agents – e.g. β-lactamase

6. TonB protein – required for the uptake of several solutes (iron siderophores, vit B12) that do not diffuse through the porin

PERIPLASM: ACTIVITIES

• Protein transport

• Nutrient acquisition

• Protein folding

• Disulfide bond formation

PERIPLASM IN GRAM POSITIVE CELLS?

Evidences:

• release of putative periplasmic proteins (distinctnucleases) in protoplasts of Bacillus subtilis • area outside the cell membrane of B. subtilis isbipartite (cryo-TEM)

Archaeal Cell Walls

- Lacks peptidoglycan

- May contain polysaccharide, protein (S layer) orpseudopeptidoglycan

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 negativecell 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.