Bacterial structure metabolism and growth

The lecture prepared by

Danuta Kunikowska, Ph.D.
Medical University of Gdańsk Microbiology Division Department of Molecular Microbiology and Serology National Salmonella Centre

Bacterial structure, metabolism and growth
 Bacterial cells belong to procariotic cells in opposition to

eucariotic cells
 Each cell contains DNA genome – the genetic basis for

reproduction. It is kind of biochemical machinery for transcribing genetical information into mRNA and translatig the mRNA into proteins. This machinery is used for biosynthesis, cell divission and replication.
 The structure of bacterial cell is specific, because of the diffrent

environment in which the cell lives, the source and means of the cell’s energy production, the nature and requirement for cell interreaction.

E.coli strain 157:H7(division SEM x 22,810)

Bacterial structure, metabolism and growth
 Bacterial cells belong to procariotic cells (Greek for ‘primitive’

nucleus’) in opposition to eucariotic cells ( Greek for ‘true nucleus’)
 Each cell contains DNA genome – the genetic basis for

reproduction. It is kind of biochemical machinery for transcribing genetical information into mRNA and translatig the mRNA into proteins. This machinery is used for biosynthesis, cell divission and replication.
 The structure of bacterial cell is specific, because of the diffrent

environment in which the cell lives, the source and means of the cell’s energy production, the nature and requirement for cell interreaction.

Major Characteristics of Eucariotes and Procariotes
Characteristics
Major groups Size (approximate) Nuclear structures: Nucleus Chromosomes Cytoplasmic structures Mitochondria Golgi bodies Endoplasmic reticulum Ribosomes (sedimentation coefficient) Cytoplasmic membrane Cell wall

Eucariote
Algae, fungi, protozoa, plants, animals > 5 µm Classic membrane Strands of DNA Diploid genome

Procariote
Bacteria 0.5 to 3 µm No nuclear membrane Single, circular DNA Haploid genome Absent Absent Absent 70S (50S + 30 S) Does not contain sterols Is a complex structure containing protein, lipids and peptidoglycans Asexual (binary fission) Simple flagellum, if present Via cytoplasmic membrane

Present Present Present 80S(60S+40S) Contains sterols Is absent or is composed of chitin Sexual and asexual Complex falgellum, if present Via mitochondria

Reproduction Movement Respiration

Major features of procariotes and eucariotes

Bacterial Ultrastructure
Bacteria Gram positive and Gram negative have similar internal, but different external structures.

Specific structure of bacterial cells helps to overcome very extreme conditions ( temperature, low osmotic pressure, diverse energy sources) survive and invade the host

The cytoplasm
 Composition: About 80% of the cytoplasm of bacteria is

composed of water. Within the cytoplasm can be found nucleic acids (DNA and RNA), enzymes and amino acids, carbohydrates, lipids, inorganic ions, and many low molecular weight compounds. The liquid component of the cytoplasm is called the cytosol.  Functions: the cytoplasm is the site of most bacterial metabolism. This includes catabolic reactions in which molecules are broken down in order to obtain building block molecules for more complex molecules and macromolecules, and anabolic reactions used to synthesize other molecules and macromolecules. The chemical reactions occuring within the bacterium are under the control of endoenzymes.  Bacterial cytoplasm also contains helical actin-like proteins that, along with the cell wall, contribute to cell shape.

The cytoplasm contains:
 DNA – single, double stranded circle  Plasmids are smaller, circular extrachromosomal DNA. Mostly

found in Gram negative bacteria. They confer to the resistance to one or more antibiotics.
 Bacterial ribosome – consists of 30S + 50 S subunits ( forming

70S ribosome) unlike 80S (40S + 60S) eucariotic cell.Ribosomes are major target for antibacterial drugs.  Cytoplasmic membrane – has a lipid bilayer structure
 Many functions: electron transport, energy production. C.m. contains

transport proteins ( allows for uptake of metabolites and release of other substances), ion pumps to maintain membrane potential, enzymes. Coiled C.m.- mesosome acts as an anchor to bind and pull apart daughter chromosomes during cell division.

Cell wall
 The components of Gram+ and Gram –

bacteria cell wall are quite different.
 Cytoplasmic membranes of most

procariotes are surrounded by rigid peptidoglycan layer.

Peptidoglycan
 is a thick rigid layer that is found in both G+ and G- cells. It

   

composed of a overlapping lattice of 2 sugars that are crosslinked by amino acid bridges. The exact molecular makeup of these layers is species specific. The two sugars are N-acetyl glucosamine (NAG) and Nacetyl muramic acid (NAM). NAM is only found in the cell walls of bacteria and no where else. Attached to NAM is a side chain generally of four amino acids. Many bacterial cell walls have been looked at and the crossbridge is most commonly composed of... L-alanine D-alanine* D-glutamic acid* diamino pimelic acid (DPA)

Chemical structure of peptidoglycan

Structure of peptidoglycan layer

NAM=NAcMur

NAG=NacGlc

Gram positive and Gram negative bacteria

Without the peptidoglycan the bacteria succumb to the large osmotic pressure differences across the cytoplasmic membrane and lyse. Removal of the cell wall Gram positive bacteria with lysosyme forms protoplast

Gram positive cell wall
 Peptidoglycan is thick, multilayered (150 – 500 Å)

meshlike exoskeleton, but sufficiently porous to allow diffusion of metabolites to plasma membrane.  Peptidoglycan is essential for the structure, replication and survival in hostile conditions. During infections peptidoglycan can interfere with phagocytosis, is mitogenic ( stimulates mitosis of lymphocytes) and has pyrogenic activity (induces fever).  The peptidoglycan can be degraded by treatment of lysosyme* * (enzyme in human tears and mucus – also produced by bacteria and other organisms).  Lysosyme degrades the glycan backbone of the peptidoglycan.

Gram positive cell wall (2)
Gram positive cell wall may include:  Teichoic acids – covalently linked with peptidoglycan  Lipoteichoic acid – have fatty acid, anchored in cytoplasmic membranes.  These acids promote attachment to other bacteria and adhesion to mammalian cells. Base for differentiation bacterial serotypes.

Gram negative cell wall
 Gram negative cell wall: is more complex than Gram

positive cell wall.  Immediately external to cytoplasmic membrane is peptidoglycan (only 5-10% cell wall by weight).  External to peptidoglycan layer is outer membrane  periplasmic space ( compartment containing a variety of hydrolytic enzymes: proteases, phosphatases, lipases, nucleases and carbohydrate - degrading enzymes. Part of these enzymes act as virulence factors.  The outer membrane: - maintains the bacterial structure - is the permeability barrier to large molecules (e.g. lysosyme) and hydrophobic molecules - protects from adverse environmental conditions( as digestive system – important to Enterobacteriaceae).

Gram negative cell wall (2)

Gram negative cell wall (3)
The outer membrane is asymetric:
 The inner leaflet contains phospholipids normally found in bacterial

membranes.
 Outer leaflet is composed of amphipatic ( both hydrophobic and hydrophilic)

molecule called LPS ( lipopolysaccharide). LPS is also called endotoxin  powerful stimulator of immune response. LPS activates B cell, macrophages and other cells to release IL-1, IL-6, TNF and other cytokines and inflammatory mediators.

 During infection large amounts of LPS are found in the blood. LPS causes

fever and symptoms. LPS consists of Lipid A, core and oligosaccharide units.
 OPS (oligosaccharide unit) is very useful for serological identification

Lipopolysaccharide
Repeating units ( up to 40) – O antigen

Core ( polysaccharide)

Lipid A (fatty acids, disaccharide diphosphate) KDO- Keto-deoxy-octanoate NAG - NAcetylglucosamine

Gram negative cell wall(4)
 Variety of proteins ( called transmembrane proteins) are

found in the outer membrane. Among them porins ( form pores) - allow the diffusion of hydrophilic molecules less than 700Da in mass through membrane.  The outer membrane and the porin channel allow for the passage of metabolites and small hydrophilic antibiotics, but the outer membrane is a barrier for large or hydrophobic and antibiotics and proteins such as lysosyme.  The outer membrane contains structural proteins and receptor molecules for bacteriophages and other ligands. The outer membrane is connected to the cytoplasmic membrane at adhesion sites and is tied to the peptidoglycan by lipoprotein.

Gram negative cell wall (5)

Gram negative cell wall (6)

The outer membrane is held together by divalent cation (Mg+2 and Ca+2) linkages on LPS molecules and hydrophobic interactions between LPS and proteins. This stiff membrane can be disrupted by antibiotics (e.g.polymyxin) or by removal Mg and Ca ions with EDTA ethylnediaminetetraacetic acid). Disruption of the outer membrane weakens the bacteria and allow the permeability large, hydrophobic molecules. The addition of lysosyme to cells treated in this manner produces spheroplasts, which like protoplast are osmotically sensitive

EXTERNAL STRUCTURES
 Some bacteria ( Gram+ or Gram-) are closely

surrounded by loose polysaccharide or protein layers called capsules. When the layer is loose, nonuniform in thickness and density is called slime layer or glycocalyx. Bacillus anthracis, exception to this rule, produces polypeptide capsule.  Capsules and slimes are unnecessary for the growth of bacteria but are very important for survival in the host.

Capsule
The capsule is: - poorly antigenic - antiphagocytic - is a major virulence factor. (e.g., Streptococus pneumoniae) - barrier to toxic, hydrophobic molecules (e.g. detergents) - promote adherence to other bacteria

Capsule surrounding cells of Streptococus sp.the capsule is about diameter of the cell

Flagella
 

Atrichous
 

Lacking in flagella. Having a single flagellum at one end of the cell. Having two flagella, one at either end of a cell. Having two or more flagella at a polar attachment point. A tuft of polar flagella. Having a large number of flagella located various (many) places about
the cell

Monotrichous

Amphitrichous

Lophotrichous
 

Peritrichous

Flagella (2)
 Ropelike propellers composed of helically 

  

coiled protein subunits (flagellin). Anchored in bacterial membranes through hook and basal body structures that are driven by membrane potential. Flagella provide motility and allow bacteria to swim (or to tumble) Chemotaxis: toward food and away from poisons. Express antigenic and strain determinants

Flagella (3)
Relative speeds of organisms Organism Km/h Body length/sec

cheetah Human Bacterium

111 37.5 0.0001 5

25 5.4 10

The structure of flagella in a G- bacteria

Fimbriae (pili)
 Hairlike structures on outside bacteria;

composed of protein subunits (pilin).  Fimbriae are smaller in diameter than flagella usually not coiled  Generally, several hundred fimbriae are arranged peritrichously (uniformly) over the entire surface of the bacterial cell. They may be as long as 15 – 20 mm or many times the length of the cell!

Fimbriae (pili) 2
Transmission electron micrograph of Aquaspirillum hydrophila showing flagella ( thick, long structures) and pili (thinner fibers)

Fimbriae (pili) 3
 - promote adherence to other

bacteria or to the host ( adhesins) – important virulence factor for E.coli, colonization and infection of urinary tract, Neisseria gonorhoeae and other bacteria.  - F pili ( sex pili) - promote transfer of large segments of bacterial chromosome between bacteria. These pili are encoded by a plasmid (F).

Bacterial Exeptions
 Mycobacteria  have peptidoglycan layer

(different structure), covalently attached to an arabinogalactan polymer and surrounded by waxlike lipid coat of mycolid acid, cord factor, wax D and sulpholipids. These bacteria are described as acid-fast staining.  Corynebacterium and Nocardia  also produce mycolic acid lipids.  Mycoplasmas don’t have peptidoglycan cell wall and incorporate steroids from the host into their membranes.

CELL DIVISION also triggers the The replication of the bacterial chromosome

initiation of cell division.  The production of two daughter bacteria requires the growth and extension of the cell wall components followed by the production of septum ( cross wall) to divide the daughter bacteria into two cells. Septum consists of two membranes separated by two layers of murein. The septum grows from opposite sides towards the center of the cell, causing cleavage of the daughter cell.
 This process requires special transpeptidase and other enzymes.

Several kinds of bacterial division are distinguished:

CELL DIVISION 2
Binary fission occurs when an expanding cell splits with one
cell becoming two. Prior to division an indented ring in the cell wall and membrane, called a septum, forms around the middle of the cell's length. As the time of division grows nearer, the septum contracts, eventually pinching off and separating the two daughter cells. During this process the cytoplasm is divided and the newly replicated chromosomes are partitioned into separate daughter cells.

CELL DIVISION 3

CELL DIVISION 4

A second method of cell division is budding, in which a small protrusion expands outward from a mother cell, forming a daughter cell. The daughter cell increases in size until it breaks off from the mother cell. This type of division is common in yeast cells and in certain strains of bacteria, especially stalked bacteria such as Caulobacter. During budding, a copy of the chromosome from the mother cell must pass into the daughter cell before the bud breaks off.

CELL DIVISION 5
 Filamentous growth is common in the

Actinomycetes and fungi. Actinomycetes are microbes that have a life-style and appearance that is similar to that of the fungi, yet they are bacteria.
 In these organisms the microbe lengthens its

initial tubular shape as one long filament. At certain intervals the tube will branch in some species and eventually expand in a number of directions. As the microbe grows, copies of the genome are made and laid down along the filament.

SPORES
 Some Gram positive, but never Gram negative bacteria !

e.g., Bacillus and Clostridium (soil bacteria) are spore formers.
 Loss of nutritional requirement or the other harsh conditions –

bacteria convert from vegetative state to a dormant state, or spore.
 Location of spore is characteristic for bacteria and can be helpful in

identification.

Spore is dehydrated, multilayered structure. Contains a complete copy of chromosomes, minimum concentrations of proteins, ribosomes and a high concentration of calcium bound to dipicolinic acid. Spore has an inner membrane, two peptidoglycan layers and an outer keratin-like protein coat. attack by most enzymes and chemical agents. They can exist for centuries!

 DNA is protected from desiccation, intense heat, radiation and

SPORES 2

Clostridium botulinum spores

SPORES 3
Transformation of the spore into the vegetative state is stimulated by disruption of the outer coat by mechanical stress, pH, heat or another factor. It requires water and triggering nutrient (e.g. alanine). Process takes 90 min. The spore takes up water, swells, shed its coats and divide.

METABOLISM

Plants and photosynthetic microbes convert light from the sun into high energy compound that help to build cell material

When you eat a bagel, your stomach and intestines break down the compounds that make up the bagel and convert this into high energy compounds for you to use

Bacterial metabolism
 Bacteria, as all living organisms have some

basic nutrition requirements for building the structures and source of energy. They should get source of carbon and nitrogen of growth and various ions.  Some bacteria take an iron ( they produce special proteins called siderophores) to take iron ions from dilute water solutions.  The metabolism of bacteria is one of the classification criterions

Bacterial metabolism 2
Bacteria can survive in the atmosphere of different levels of saturation with O2.  Obligate anaerobes – cannot grow in the O2 atmosphere ( e.g. Clostridium perfringens)  Obligate aerobes - O2 is essential for growth and survive (e.g. Mycobacterium tuberculosis)  Facultative anaerobes – can grow either in presence or in absence of oxygen  most of bacteria

Bacterial metabolism 3
Nutritional Type Energy Source Light Carbon source CO2 Electron Source Inorganic H2O, H2S Organic compounds Inorganic compounds Examples

Photoautotrophic litothrophs

Cyanobacteria,so me purple and green bact. Some purple and green bacteria

Photoheterotrophic organothrophs Chemoautotrophic litotrophs

Light

Organic compounds CO2

Chemicals H2, NH3,H2S Organic compounds

Bacteria nd many archaea

Chemoheterotrophic organothrophs

Organic compounds

Organic compounds

Most bacteria, some archaea

Metabolism
The sum total of all reactions which occur in a cell. Metabolism consists of two types of processes catabolism and anabolism.

Catabolism
How complex molecules are broken down into smaller, simpler, molecules with the release of energy and reducing power (electrons).

Anabolism
Synthesis of complex molecules from simpler ones to form cell structures, this requires energy and often reducing power. Catabolism and anabolism are strictly interrelated!

Catabolism of proteins, polysaccharides and lipids produces glucose, pyruvate and intermediates of the TCA cycle and ultimately energy in the forms of ATP and reduced form of nicotinamide adeninonucleotide (NADH)

Glycolysis (E.M.P. pathway = Embden - Meyerhof - Parnas Pathway) Important features:
  

Glycolysis represents phase I of respiration. This pathway is common to both aerobic and anaerobic respiration. These reactions are completed in the cell cytoplasm (cytosol). Therefore, glycolysis is also called cytoplasmic respiration.  Glycolysis involves a series of reactions in which a molecule of 6-C glucose is broken down in a step-wise manner into two molecules of 3-C pyruvic acid.  Each step (reaction) is controlled by a specific enzyme and is reversible.  Reactions in the glycolysis are anaerobic in nature,( i.e. they do not require molecular oxygen). 6-C Glucose is the most commonly used organic substrate for respiratory breakdown because - It is most readily available. - It is soluble and can be easily transported and supplied Reactions of glycolysis (EMP pathway) involve : - Phosphorylation of glucose (preparatory phase), isomerization to fructose-6-phosphate, and phosphorylation to fructose-1, 6-biphosphate. - Cleavage of Fructose 1, 6 diphosphate to 2 3-C PGAL. - Formation of 3-C pyruvic acid (end product of glycolysis).

The metabolites are converted by one or more pathways to one common universal intermediate ( pyruvic acid) Glucose is simple sugar, very important for metabolic pathways.

Bacteria can produce energy by ( in order of increasing efficiency)

- fermentation
in the absence of oxygen

- anaerobic respiration - aerobic respiration

Metabolism of glucose
Embden – Meyerhof – Parnas pathway
   

This reaction results in conversion of glucose to pyruvate Takes place in both bacteria and eucariotic cells Aerobic and anaerobic conditions Begins in activation of glucose to form glucose – 6 phosphate ( requires 1 mole of ATP per 1 mole of glucose)

ATP is carrier of energy in the form of high energy phosphate bonds.

Transformation of ADP ( adenosine diphosphate) to generate ATP (adenosine triphosphate )  substrate level phosphorylation – with enzyme( kinase)  highly energetic phosphoric links. 4 ATP molecules are produced in glycolytic pathway.

The pyruvic acid produced from glycolysis is then converted to various end products depending on bacterial species and is called fermentation.

In yeasts – fermentation – the end product – alcohol (ethanol). Many bacteria – fermentation towards lactic acids ( different yoghurts, cabbage into sauerkraut et al.)

Tricarboxylic Acid Circle
  

Using tricarboxylic acid ( TCA) – the pyruvic acid from glycolysis ( or the other sources) can be completely oxidized ( burned) to H2O and CO2. Additional energy is produced. The process begins with oxidative decarboxylation of pyruvate ( release of CO2) to the high energy intermediate – AcetCoA). In the first step of this process the pyruvate dehydrogenase complex acts on pyruvate to form CO2 and acetyl coenzyme A (acetyl-CoA). Electrons extracted from pyruvate during this reaction are used to reduce NAD+ to NADH.

Formation of Acetyl CoA from pyruvate. One carboxyl group hilited in blue leaves as CO2. The remaining carboxyl group and methyl group of pyruvate, hilited in red, form the acetyl group or acetyl-CoA.

Acetyl-CoA is then fed into the TCA cycle:

TCA Cycle
 The TCA cycle can be

broken into 3 phases:  6 carbon phase (pink)  5 carbon phase (green)  4 carbon phase (yellow)

Acetyl CoA condenses with oxaloacetate to form citrate. The acetyl molecule is in the red box and attaches to the blue hilited carbon on oxaloacetate. Energy is extracted in the various reactions of the cycle (hilited in purple).

The subsequent reactions of the cycle: oxidation of citrate, release of energy and regeneration of oxaloacetate. In this way – oxaloacetate and the other intermediates can flow up through many turns of the cycle. One turn of TCA cycle generates: 1 GTP (guanosinotriphosphate), 3 NADH( nicotinamide – adenine dinucleotide) 1 FADP (flavin adenine dinucleotide)

TCA cycle allows the organism to generate more energy than with glycolysis alone. In addition – GTP ( equivalent of ATP) NADH and FADH may enter electron transport chain. In this chain electrons are passed through a series of donor – acceptor pairs and ultimately to oxygen.

Electron transport chain

This process is called aerobic respirations - 3 moles ATP per one molecule NADH oxidized - 2 moles of ATP per one molecule FADH2

TCA cycle is central to the metabolism many micro- and macro- organisms. Many of the intermediates in TCA cycle are also starting points for the synthesis of cellular constituents such as: - amino acids - nucleic acids - cell wall components

Aerobic respiration, Fermentation, Anaerobic respiration
 Some bacteria can use compounds other than

oxygen (e.g. NO3, SO4 -2, CO2) anaerobic respirations : little more efficient than fermentation, which uses organic compounds as electron acceptors, but less efficient than aerobic respiration.  Anaerobic organisms have neither aerobic electron transport, nor a complete TCA cycle and therefore are less efficient than aerobic organisms.  Fermentation – 2 ATP molecules per glucose  Aerobic metabolism – 38 ATP molecules.

Bacterial Growth

• lag phase – no divisions • exponential phase ( logarithmic or log phase of growth): number of bacteria will increase 2n in which n means number of generations (doublings); • stationary phase ( the medium runs out of metabolites or toxic substances are build up in the medium. Bacteria stop growing • decline – number of bacteria decreases.

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