4 & 5. Microbial Physiology.pdf function metabolism

microbial physiology

1. 4 & 5. MICROBIAL PHYSIOLOGY
-PROKARYOTIC & EUKARYOTIC STRUCTURE, FUNCTION,
METABOLISM & GROWTH
-VIRUSES (A SPECIAL LOOK AT HIV REPLICATION)
1

2. Prokaryotic Vs. Eukaryotic Cell














2 (Figure 2.11; Madigan et al.,
2010)

3. Prokaryotic Cells Vs. Eukaryotic Cells



3 Property/Structure Prokaryote Eukaryote
1. Size Typically 0.2-2.0 μm Typically 10-100 μm
2. Membrane enclosed
organelles
Absent
Present. Include mitochondria,
endoplasmic reticulum, Golgi,
lysozymes, chloroplasts.
3. Nucleus Absent
True nucleus which has nuclear
membrane plus nucleolus (an
area of condensed chromosome
within nucleus where rRNA is
synthesized)
4. Chromosome (DNA)
DNA is not membrane enclosed.
DNA is aggregated in a mass
called a nucleoid.
Usually single circular
chromosome; linear in some.
Usually no histones.
DNA is found in the cell
nucleus, which is membrane
enclosed in nuclear membrane.
Usually linear, multiple
chromosomes.
DNA assoc. with histones.
5. Cell wall
Present in most. Chemically
complex, containing
peptidoglycan. Exceptions:
Mycoplasma; some Archaea
Usually present. Chemically
simple.
6. Cell division
Binary fission (DNA copied & cell
splits into two).
Mitosis
7. Sexual
recombination/reproduction
None, only DNA transfer Meiosis

4. 4
Prokaryotic structure, function & growth

1.Cell shape, size and arrangement (morphology)
–Cells usually 0.2-2.0 μm dia. and 2-8 μm in length
–Basic shapes:

Spherical (coccus, pl. cocci)
Rod-shaped (bacillus, pl. bacilli)
Spiral (vibrios, curved; spirilla, helical; spirochetes)

Variations:
Diplococci (paired cocci), streptococci (chained cocci), diplobacilli, streptobacilli, etc.

Pleomorphic (more than one shape)

–Cell division can be in 1, 2, 3 or multiple planes

5. 5
(Figs 4.2, 4.3, &4.4 Tortora et al., 2010)

6. 2.Cell wall
–Surrounds cytoplasmic membrane
–Semi-rigid
–Confers cell shape
–Major function is to protect cell from desiccation
–Main composite:
Peptidoglycan- disaccharides (glycan backbone), interlinked with
polypeptides. Disaccharides:
-Composed of the sugars N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM) joined by β-1,4 glycosidic linkages.
-Linkages susceptible to certain antibiotics, and enzymes such as lysozyme.
–Gram + or – based on gram reaction
6

7. Structure of the repeating unit in peptidoglycan, the
glycan tetrapeptide. (Fig 4.18)

7 (Madigan et al., 2010)

8. 8
Figure 4.19 Peptidoglycan in
Escherichia coli and Staphylococcus
aureus. (a) There is no interbridge
present in Gram-negative bacteria.
(b) The glycine interbridge in S.
aureus (gram-positive). (c) Overall
structure of peptidoglycan. G, N-
acetylglucosamine; M, N-
acetylmuramic acid.
(Madigan et al., 2010)

9. Gram positive cell wall
•Composed of many layers of
peptidoglycan (90% of wall),
thick, rigid
•Teichoic acids (ribitol or
glycerol phosphate residues)
linked to peptidoglycan
layers
Two types: Lipoteichoic and
wall teichoic acids
Teichoic acids confer negative
charge to cell surface and
antigenic properties

Gram negative cell wall
•Thin layer of peptidoglycan
•Has outer membrane of
lipopolysaccharide (LPS)
Three constituents:
Lipid A, core polysaccharide, &
O-polysaccharide (function as
defence to phagocytosis &
barrier from antibiotics and
lysozyme)
•Peptidoglycan layer bonded
to outer membrane via
lipoproteins and imbedded
in periplasm
•No teichoic acids
•More susceptible desiccation


9

10. 10 (Madigan et al., 2012)

11. 11
Gram positive cell wall
Gram negative cell wall
(Madigan et al., 2012) Porin

12. Gram staining method:

–Crystal violet primary stain, enters cytoplasm in
both cell types (G+ and G-), leaving purple
–Iodine applied, forms crystal violet-iodine complex
in cell
–Complex extracted by alcohol in G-, but not G+
–Safranin counter stain leaves G- red (pink) and G+
purple




12

13. Explanation of the Gram Staining Mechanism
13 (Campbell et al., 2008)

14. 14
Cell wall (continued)
Atypical cell walls
The wall-less bacteria
E.g. Mycoplasma (the smallest of prokaryotes) and Thermoplasma spp.
Bacteria resemble protoplasts, but are more resistant to osmotic lysis – they contain sterols in their
cytoplasmic membrane.
Acid-fast cell wall (acid-fastness)
Unique property of Mycobacterium – due to presence of mycolic acids bound to cell wall peptidoglycan.
Mycolic acids are only found in this genus. A staining technique is used to identify this property is called
the acid-fast (or Ziehl Neelson) stain. Steps:
-Primary stain (carbolfuchsin) is driven into cells by slow heating.
-After washing with water and decolourising with acid-alcohol, cells are counterstained with
methylene blue.
-Cells that are acid-fast retain red colour; the non-acid-fast appear blue.
The Archaea
A major distinction from the Bacteria is the absence of peptidoglycan in Archaeal cell wall. Cell wall may
be absent or unusual in composition. E.g.
-Polysaccharide pseudomurein (N-acetylmuramic acid replaced by N-acetyltalosaminuronic acid with
a β1,3 link instead of β1,4. Lysozyme insenstive.
-S-Layers or paracrystalline surface layers. Composed of protein and glycoprotein arranged in distinct
hexagonal, tetragonal, or trimeric symmetries. Most common cell wall type among Archaea – found
all major groups including halophiles, methanogens, and hyperthermophiles.
-A distinct and thick polysaccharide cell wall made up of various sugars and acetate with no
pseudomurein. Found among the extreme halophiles.
-Cell walls of glycoprotein found among the haloalkaliphiles, the Archaea from extreme alkaline saline
environments.

15. 3.Structures external to the cell wall

–Capsules or slime layers
E.g.
•Encapsulation in Streptococcus pneumoniae
•Biofilm formation in S. mutans & Pseudonmonas
aeruginosa
•Adhesins in P. fluorescens

–Fimbriae and Pili
•Fimbriae utilised by E. coli, Salmonella spp., &
Neisseria gonorrhoeae
•Pili – genetic exchange





15

16. 4.Cytoplasmic membrane

–Structure
Four main features
oPhospholipid bilayer
oMembrane proteins
oAttachments to outer membrane
oSterols (lack of)

–Functions
oSelective barrier
oEnergy conservation (ATP synthesis; photosynthesis)
oProtein anchorage
oMovement of substances (simple diffusion & protein
transporter systems)







16

17. 17
Protein transporter systems in detail:

1. SIMPLE TRANSPORT (single integral or
membrane protein)

Example: The Lac Permease of Escherichia coli
•Symporter
•Transports lactose into cell – involved in lactose
metabolism
•Transport is energy driven by the proton motive force
•Cotransport of lactose and protons results in
accumulation of lactose in the cytoplasm against
concentration gradient

18. Protein transporters – by function
(Fig 4.12; Madigan et al 2010) 18
Uniporter: single molecule is transported uni-directionally – in or out
Antiporter: two molecules transported simultaneously in opposite directions
Symporter: co-transport of a molecule with another substance, e.g. proton

19. •In fermentation, ATP is
produced by substrate level
phosphorylation.

•In respiration, production of
ATP from ADP & P
i is by
oxidative phosphorylation
energised by the proton
motive force generated in
the CM.

19

20. Energy related terms of importance:

Substrate level phosphorylation

Oxidative phosphorylation

Proton motive force

Electron transport chain (general system)

Fermentation

Respiration




20

21. 21
A Comparison of Substrate-Level
Phosphorylation to Oxidative
Phosphorylation

Figure 5.14: Energy conservation in
fermentation and respiration. (a) In
fermentation, substrate-level phosphorylation
produces ATP. (b) In respiration, oxidative
phosphorylation produces ATP by way of the
energized proton motive force.

22. 22
(Tortora et al 2010)

23. 2. GROUP TRANSLOCATION (a series of proteins)

Example: The phosphotransferase system (PTS)
in E. coli
•The PTS system in E. coli is well-studied; it transports
the sugars glucose, mannose, & fructose
•A series of five proteins is involved in transporting any
given sugar
•In group translocation, the substance transported is
chemically modified as it crosses the CM into the
cytoplasm


23

24. •Before sugar is transported into cell, the proteins are serially phosphorylated
and dephosphorylated until the final membrane protein Enz IIc
phosphorylates and transports the sugar into the cell.







•Phosphoenolpyruvic acid (PEP) – a high energy intermediate in glycolysis –
transfers its high energy phosphate to Enz I, which in turn transfers it serially to
HPr, Enz IIa, and Enz IIb.
•Enz IIb transfers the phosphate to Enz IIc, an integral protein which phosphorylates
the sugar, modifying it during transport.
24 (Fig 4.14; Madigan et al 2010)

25. Glycolysis-
PEP
production
25 (Tortora et al 2010)

26. 3. ABC TRANSPORT SYSTEMS (involve three
proteins)

Example: The ABC transporter systems in Gram
negative bacteria
•These type of transporter systems utilize 3 types of
proteins:
-Periplasmic binding proteins
-A membrane transporter
-ATP hydrolysing proteins
•The word ABC is derived from ATP binding cassette,
proteins that bind ATP in the system.
•Over 200 ABC transport systems have been recognized
in prokaryotes. Different types exist for the transport
of organic and inorganic substances, and trace metals.

26

27. Mechanism of ABC transporter
27 (Fig 4.14; Madigan et al 2010)

28. 4.Structures internal to the cytoplasmic
membrane

–Cytoplasm
(Contents-)
–Ribosomes
•Protein synthesis
•Structural differences in prokaryotes and eukaryotes
–Nucleoid & plasmids
–Cell inclusions and vesicles
•PHBs, glycogen, polyphosphate, & gas vesicles
–Endospores
•Structure & stages of formation

5.Motility
–Flagella arrangement
28

29. Endospore structure & formation
29
(Tortora et al. 2010; Madigan 2012)

30. Flagella arrangement
30
(Tortora et al. 2010)

31. 6.Prokaryotic growth

–Cell chemical composition
• Dominant elements; others

–Nutritional requirements for growth
•Carbon
•Macronutrients
•Micronutrients

–Laboratory culture
•Media types; media sterilisation; pure culture techniques

–Growth concepts
•Growth cycle
•Population growth & cell counts
31

32. Culture growth on
solid media (colonies –
are produced by both
planktonic or biofilm
growers. Each colony is
a visible population of
cells arising from a
single or a few cells.
Aseptic technique is
used for streaking
here)
32
Culture growth in liquid
media (cells grow suspend in
the liquid, in a free-floating
form referred to as planktonic
growth; the increasing optical
density coincides with an
increase in cell number)
(Sidebar & Figure 5.3; Madigan et al 2010)
(a&b) Serretia marcescens;
(c) Pseudomonas aeruginosa;
(d) Shigella flexneri
Liquid E. coli cultures

33. Aseptic technique (for pure culture)
33
Aseptic transfer
Streak plating for pure cultures
(Figures 5.4 & 5.5; Madigan et al 2010)

34. Growth Concepts
The growth cycle
In microbiology (bacteriology), growth is defined as an
increase in the number of cells through a process of cell
division called binary fission - a cell divides into two. A
population of cells growing in an enclosed vessel exhibits a
growth curve depicting the lag, exponential (log), stationary,
and death phases of growth.

34 (Figure 6.10; Madigan et al 2010)

35. Population growth & cell counts
Exponential growth
1 cell produces 2
2 cells produce 4 cells, 4 produce 8, 8 produce 16 etc.
Also written as:
2
0
cells produce 2
1
cells, 2
1
cells produce 2
2
cells, 2
2
cells produce 2
3
cells...2
20
cells
etc..
In exponential growth, factors such as growth rate (μ), generation
time (g), and generation number (n) can be predicted mathematically.
(see examples)
Control of exponential growth
Exponential growth is an ideal and not indefinite - growth outcome is
controlled by cultural conditions - the two main types are batch and
continuous. In batch culture, of the 4 growth phases only one is
exponential; whereas continuous is a steady state where cell
numbers are kept constant.
Factors predicting growth
Temperature, oxygen, pH, & water availability



35

36. A.Total cell counts
i.Microscopic counts are used for determining total cell no.
Not always reliable
Specially designed counting chamber is used
ii.Turbidimetric methods
Spectrophotometry
Flow cytometry
Fluorescence techniques measuring the bioluminescence -
luciferase assays

B.Viable counts
i.Spread or pour plate method
Sample dilution
Sources of error
ii.MPN (most probable number)
iii.Membrane Filter Method


36

37. 37 (Figure 6.16; Madigan et al 2010)

38. 38
Eukaryotic structure, function & growth

Introduction
Eukaryotes differ from prokaryotes for having a membrane-
enclosed nucleus, as well as other organelles or membrane-
enclosed structures. The membrane enclosures are bilayers.
The cell interior is also more complex than in prokaryotes,
with structures unique only to eukaryotes.

Structures discussed:
-The cell wall and cytoplasmic membrane
-The cytoplasm
-The nucleus
-Key energy metabolising organelles
(mitochondrion/hydrogenosome)
-Other structures

39. 39
1.The cell wall and cytoplasmic
membrane

–Most eukaryotes have cell walls; they are
absent in animal cells and protists such as
protozoa
–Cell walls of eukaryotes do not contain
peptidoglycan, instead they are composed
of polysaccharides and other molecules.
E.g.
Cellulose – found in algae or plants
Chitin – found in fungal walls (polymer of N-
acetylglucosamine)
Glucans – also found in fungi, e.g. yeasts (polymers
of glucose)
–The cytoplasmic membrane of eukaryotes is
similar to that of prokaryotes, but it also
contains polysaccharides & sterols.
Transport systems are also similar, but no
group translocation is found.

The Eukaryote cell:
Note all labelled parts
– not all eukaryotes
contain mitochondria
and chloroplasts

40. 40
2.Cytoplasm
A major way in which eukaryotic cytoplasm differs from
prokaryotic is that it is interspersed with a complex arrangement
of microtubules, microfilaments and intermediate filaments that
make up the cytoskeleton. The cytoskeleton aids cell movement
and transportation of substances, and provides support and shape
to the cell.
3.The nucleus
–Often the largest structure in cell
–DNA wrapped in proteins called histones forming nucleosomes.
Nucleosomes collectively make up chromosome of cell
–No plasmids. Extra-chromosomal DNA only exists in other
organelles such as mitochondria & chloroplasts.
–Nucleolus (condensed region of nucleus) site of DNA synthesis
–Nucleus is enclosed in double-membrane called nuclear
envelope with channels called nuclear pores which control
movement of substances in and out.

41. 41
4.Key energy-metabolising organelles (mitochondrion
vs. hydrogensome)

Mitochondrion:
–Aerobic eukaryotic cells carry out respiration and oxidative
phosphorylation in the mitochondrion
–They are the size of bacteria, & can be as many as 1000 in a cell
–The mitochondrion is enclosed in a double membrane – outer is a
mixture of proteins and lipids and is porous; inner has more protein
and is less permeable. The inner membrane is folded into
invaginations called cristae – site for respiration & ATP production
enzymes. Inner to cristae is the matrix, site of Kreb cycle enzymes.

Hydrogenosome:
–Found as energy producing organelle in some anaerobic eukaryotes
instead of mitochondrion. E.g. in Trichonomas vaginalis
–Similar in size to mitochondrion; lacks cristae & enzymes of Kreb cycle.
–Carries out oxidation of pyruvate to CO
2, H
2 & acetate. ATP is also
produced.

42. 42
5.Other structures and organelles
–Endoplasmic reticulum (ER) and Golgi complex are other
membranous structures found in eukaryotes. The ER may
be smooth or rough; it is continuous to the nuclear
membrane. Smooth ER is site of lipid synthesis and rough
is where glycoproteins are produced. The Golgi is discrete
& separate; it carries out modifications of secretory &
membranous proteins.
–Lysosome and peroxisomes are single-membraned
compartments made of proteins and lipids. Lysosome
contain enzymes that hydrolyse proteins, fats, and
polysaccharides into monomers for intracellular digestion.
Peroxisomes are specialized for oxidation of fatty acids &
alcohols.
–Some eukaryotes may also have flagella and cilia.

43. 43
Viral function (replication)

Viruses require a host to carryout many of the
functions required for survival, including replication
and metabolism.

In this section, general replication (lytic and temperate
phages) is discussed. The complex retroviral replication
strategy is covered in detail using HIV as an example.

44. Virulent cycle:
44
The replication of
the bacterial virus
shown here is a
virulent cycle; e.g. T4
infection of E. coli -
the virus infection
kills the cell.
(Figure 10.8; Madigan et al 2010)

45. 45
The replication of the
bacterial virus shown here
is that of a temperate
virus; e.g. Lambda and Pi
phages. The virus infects
the cell which enters into a
state of lysogeny, where the
virus nucleic acid (called
prophage) is incorporated
into the host DNA but is not
expressed. Some
conditions, however, may
induce the virus into a lytic
cycle.
(Figure 10.16; Madigan et al 2010)

46. 46
Replication of a human immunodeficiency virus (HIV) – a
retrovirus [Retroviruses replicate via a DNA intermediate]

•The HIV retrovirus is enveloped and contains 2 copies of an RNA genome.
The virus also carries its own reverse transcriptase and specific tRNA.
•HIV infects cells with CD4 surface proteins – mostly macrophages and
lymphocytes called T-helper cells. Macrophages are infected first.
•HIV must interact with CD4 and coreceptors on target cell for infection to
occur. Upon attachment with host cell, viral membrane fuses with
cytoplasmic membrane and enters cell. The envelop is removed and the
nucleocapsid remains, carrying enzymes and tRNA.
•In cytoplasm: One copy of RNA is reverse transcribed into ssDNA and
then converted into dsDNA by enzyme reverse transcriptase (a type of
DNA polymerase). dsDNA then enters nucleus.
•In nucleus: Viral dsDNA integrates into host genome and becomes a
permanent component of host genome. (Similar to lysogen, but
difference is it cannot be excised – integrated viral DNA = provirus)
•dsDNA is transcribed then into viral mRNA and viral genomic RNA (many
protease dependent steps follow for protein maturation)
•In cytoplasm: Assembly of nucleocapsids follows, and then budding and
release.

47. 47
(Figure 33.39 Madigan et al
2012)

48. 48
Retrovirus
replication
Chemotherapeutic
targets:

Fusion inhibitors

Nucleoside reverse
transcriptase
inhibitors

Integrase inhibitors




Protease inhibitors
(Figure 10.24; Madigan et al 2010)

49. 49

50. 50
(Madigan et al 2012)

51. 51

52. 52
Images of Mitochondrion,
Hydrogenosome, & a
retrovirus (HIV)