1

Taxonomy of

microorganisms

Taxonomy = systematics

- science of biological classification,

provides a way to identify organisms

and place them in groups with similar

characteristics

Consists of 3 parts:

1. Classification

2. Identification

3. Nomenclature

of (micro)organisms

Systematics � 1737 Carl Linné

� in Europe means mostly theoretical, in

USA mostly practical classification

Taxonomy � 1813 De Candolle

� in USA means mostly theoretical, in

Europe mostly practical classification

1. Classification

- arrangement of organisms into groups

based on mutual similarity or evolutionary

relatedness;

- ordering of organisms into groups (taxa);

- systematic grouping of microorganisms by

certain features

Classification is changing by time!

Classification can be

phenotypic, genotypic or analytic

Phenotypic classification is based upon

overall similarity.

Morphological, physiological, biochemical,

serological features are compared.

Is based on supposition that overall

similarity reflects well enough the

relatedness of organisms.

The method was developed in 1960-s, and was prevalent up

to 1980-s

Features or attributes or

descriptors

- enable to describe and classify the

individuals and taxons (taxa).

Quantitative

can be measured

or counted

Qualitative

shape, colour etc.

some feature

present or missing

Morphology in

microscope

Morphology of colony

Biochemical activity

Antibiogram

Serotyping

Phagotyping etc.

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2

Taxonomy in practical

medicine

Microorganisms are grouped by

simple features:

� main form (rod, cocci, screw-like)

� Gram stain reaction (positive,

negative)

� intra- or extracellular

� respiration type (aerobic, anaerobic)

Microorganisms�

groups in practicalmedicine

Bacteria� �common� bacteria � Spirochaetes� Chlamydiae� Rickettsiae�Mycoplasma

Viruses� Prions

FungiProtozoa

Phenogram: a branching diagram (tree) that links taxaby estimates of overall similarity (phenotypicclustering)

Genotypic classification (cladistics)

is based upon evolutionary

relationships i.e. upon

common ancestry.

Most precise method for

classifying microorganisms.

Started with development of

molecular methods and genetic

studies

Genetic taxonomy �nucleotide /

nucleocide compositions are used

to determine similarity between

species

GC-index

DNA hybridization

Nucleic acidsequence

analysis

Plasmid analysis

Ribotyping

Analysis of chromosomal DNA

fragments

Cladogram is a tree diagram which depicts a hypothesised evolutionary history.

Phylogram is a tree which indicates by branch length the degree of change believed to have occurred along each lineage

3

Phylogenesis of

microorgansisms

Ribosomal nucleic acids

� 16S and 23 S rRNA

Appearance of oxygen 700-800

million years ago

� anaerobic bacteria are older than

aerobic

Analytic classification

is based upon detection

of structural

components and

metabolic products

using chemical

methods.

� cell wall fatty acids

� whole cell lipids

� whole cell proteins

� enzymes

Chromatography

Mass spectrometry

Multilocus enzyme

electrophoresis

+ High objectivity

+ Rapid

- Needs special

equipment (only in

referent labs)

Two Kingdom System: Plantae/Animalia

� bacteria, fungi, and algae were classified with

plants; protozoa were classified as animals

Five Kingdom System (Whittaker,1969)

� Animalia, Plantae, Fungi, Protista, Procaryotae

� Bacteria were separated into the Kingdom

Prokaryotae

Three Domain System (Woese, 1978)

� based on comparison of sequences of rRNA

Classification systems Three Domain System

Eukarya - all eukaryotic organisms

� Kingdoms:

�Animalia

�Plantae

�Fungi

�Protista

Bacteria

Archaea - "ancient" bacteria which live in extreme environments methanogens

extreme halophiles

extreme thermophiles

From an evolutionary point of view, Archaea more closely related to Eukarya than Bacteria

Classification of Viruses

Viruses are not placed in a kingdom.

They are not composed of cells and

cannot grow without a host cell.

A viral species is a population of

viruses with similar characteristics that

occupies a particular ecological niche.

Species Genus Familia Ordo Classis Regnum

Liik Perekond Sugukond Selts Klass Riik

Species Genus Family Order Class Phylum (Division) Kingdom

Similar species are grouped into a genus; similar genera are grouped into a family; families into an order; orders into a class; classes into a division or phylum; and phyla into a kingdom.

4

Species as the main taxonhundreds of definitions for species!

Similar organisms are designated as

species.

Closely related individuals constitute a

species.

Species � the matter that is considered

species by a good systematizer (!)

...

The groups of individuals of the same

origin in nature is reality, but naming

them species is a model created by

human.

Alpha-taxonomy � description of

species

Beta-taxonomy � joining of similar

species into genus, similar genera to

family ...

Taxa below of species

Subspecies

Type or variant � differs from the othermicroorganisms of this species by a certainfeature� morphotype, biotype, phagotype, serotype

Pure culture � mass of microorganisms, originating from a single microbial colony grownon arteficial medium

Isolate � pure culture that has been identified

Strain � isolates that are identical (98%) bygenotyping

Clone � microbial culture that originates fromone microbial cell, all cells are identical.

Bergey�s Manual of

Systematic

Bacteriology= the standard taxonomic

reference on bacteria;

best consensus at the time

& most widely accepted and used system

Manual of Determinative Bacteriology� 1923, � , 1994

� not based on phylogeny!

� based on cell wall type (differential staining), oxygen

requirements, morphology, motility, biochemical testing

Manual of Systematic Bacteriology� 1984, 2001, 2005

� based on phylogeny - requires molecular testing

used in practical medicine

2. Identification

- practical side of taxonomy: process of determining the specific identity of an isolate;

- placement of a new strain into a previously described group.

Identification is the routine work in

microbiology laboratory:

� usually starts from getting of pure culture of the

new microorganism

� investigation of properties follows (mostly

morphological, biochemical and/or serological, sometimes molecular)

Colonies of bacteria on solid medium in Petri dish

5

Investigation of thebiochemicalpropertis of microorganismsusing different test media in tubes

Methods of Classifying and Identifying

Microorganisms1. Bergey's Manual of Determinative Bacteriology is the standard reference for

laboratory identification of bacteria.

2. Morphological characteristics are useful in the identification of microorganisms,

especially when aided by differential staining techniques.

3. The possession of various enzymes as determined by biochemical tests is used in the

identification of microorganisms.

4. Serological tests, involving the reactions of microorganisms with specific antibodies,

are useful in determining the identity of strains and species as well as relationships

among organisms. ELISA (enzyme-linked immunosorbent assay) and Western blot are

examples of serological tests.

5. Phage typing is the identification of bacterial species and strains by the determination of

their susceptibility to various phages.

6. The sequences of amino acids in proteins of related organisms are similar. Related

organisms have identical proteins; this characteristic can be ascertained by PAGE

(polyacrylamide gel electrophoresis) "fingerprints."

7. Flow cytometry measures physical and chemical characteristics of cells.

8. The percentage of G-C pairs in the nucleic acid of cells can be used in the classification

of organisms.

9. The number and sizes of DNA fragments produced by restriction enzymes are used

to determine genetic similarities.

10. The sequence of bases in rRNA can be used in the classification of organisms.

11. PCR (polymerase chain reaction) can be used to amplify small amounts of microbial

DNA in a sample.

12. Single strands of DNA, or of DNA and RNA, from related organisms will hydrogen-bond

to form a double-stranded molecule; this bonding is called nucleic acid hybridization.

Southern blotting and DNA probes are examples of hybridization techniques.

13. Dichotomous keys are used for identification of organisms.

14. Cladograms show phylogenetic relationships between organisms.

3. Nomenclature

- branch of taxonomy concerned with

assignment of names to taxonomic groups

- the naming of (micro)organisms

Binomial nomenclature

Carl Linné, 1753

Genus name + species name

Capitalize genus

Underline or italicize both the genus and species

Generally Latinized (genus may be Greek)

Staphylococcus aureus

Treponema pallidum

Chlamydia trachomatis

Family -aceae Chlamydiaceae

Order -ales Chlamydiales

Rules for the assignment of names to

bacteria are established by the

International Committee on

Systematic Bacteriology.

Rules for naming fungi and algae are

published in the International Code for

Botanical Nomenclature.

Rules for naming protozoa are found in

the International Code of Zoological

Nomenclature.

Principles of nomenclature

1) distinct organisms are designated as species

2) latin / latinized binomial nomenclature

3) naming congruent with international code

4) law of priority

5) designation of categories (classification)

6) criteria used for formation and publication of

new names

6

Structure of

bacterial cell

One bacterial cell is an

independent organism

feeds and respires

multiplies, inhabits certain

environment

is able to survive in wide

variety of environmental

conditions

Structure of bacterial cell

cell wall

flagellum

spore

capsule

and pili

Prokaryotic and eukaryotic cell

Feature Eukaryote Prokaryote

Size Nuclear membrane Genome Endoplasmatic network Golgi complex Mitochondria Ribosomes Plasmides Cytoplasmatic membrane Cell wall

>10 µm Yes DNA chains Yes Yes Yes Yes (80S) No Sterols No (except fungi)

0.3-20 µm No DNA ring No No No Yes (70S) Yes (frequently) No sterols Yes

Genome of bacteria

Genome � substance having hereditary

information - DNA

Genome of bacteria � single round

(ring) molecule of double-strandedDNA

Nucleoid - DNA + proteins

Plasmid - separate small DNA ring,

additional information, facultative

Escherichia coli: 5 million nucleotide pairs 2000-3000 genes

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Bacteria multiply by dividing

Coli-bacteria divideafter every 20 minutes

DNA replication in bacteria

Bacteria multiply by dividing

(no mitose, no nuclear membrane)

DNA is associated with a coiled cytoplasmatic

membrane = mesosome

� Mesosome acts as an anchor to bind and pull apart

daughter chromosomes during cell division

Replication of bacterial DNA differs from eukaryote

cell:

� DNA chains separate (cleave) and both chains produce a

new copy

DNA replication has 3 stages:

� initiation

� elongation

� termination

DNA replicationof E. coli takes 40 minutes at 37°C

Cytoplasm

Colloidal

� of dry weight: 40% protein, 35% RNA

� vacuoles appear in old cell � honeycomb structure

Ribosomes � for protein synthesis

� ~5000 in one cell

� small round

� 60% RNA, 40% protein

� attach to cell membrane

� join to polysomes

Dissolved enzymes

Inclusions � reserve nutrients

Cell cover

1. Cytoplasm membrane

or cell membrane � 50% lipids, 50% proteins

� phospholipid double layer

� peripheric and integral proteins

� assistant-lipids

2. Cell wall

different in Gram(+) and Gram(-)

bacteria

3. Capsule or glycocalyx or slime

missing in some bacteria

Functions of cell membrane

Selective permeability

� permeases

Secretion of enzymes

� Gram(+) bacteria: to environment

� Gram(-) bacteria: to periplasmatic space

In aerobic bacteria: electron transport

and oxidative phosphorylation

� cytochromes, dehydrogenases, ATPase

� Hence, the cell membrane performs also the

function of missing mitochondria �

respiration and production of energy.

8

2. Cell wall

Between cell membrane and capsule

Rigid

Missing in eukaryote cell

Different in Gram(+) and Gram(-)

bacteria

� Gram(+) thick, one layer

� Gram(-) thin but several layers

1880 Christian Gram: staining method

Gram(+) Gram(-)

Gram stain method

Cell membrane + cell wallCell wall:

peptido-

glycane

or murein

network-like polymere

surrounds the cell like net bag

many layers

very rigid

Gram(+): lysozyme may break protoplast

Gram(-): penicillin may break spheroplast

L-form

Cell wall Gram(-) cell wall

thin peptidoglycane

no teichoic acids

outer membrane - �molecular

screen�

� integral proteins

� porins

� permeases,

� translocation proteins

� protein receptors

lipopolysaccharide LPS =

endotoxin (negative charge,

hydrophobic, anti-phagocytotic,

toxic)

� lipid A

� core polysaccharides

� O-antigen

Gram(+) cell

wall

thick

peptidoglycane

teichoic acid,

lipoteichoic acid

polysaccharides

9

3. Capsule

Glycocalyx

Slime

Capsule -� extracellular mucous polymere �

polysaccharide, tightly surrounds the cell

� tasks:� defends

� helps to attach

Glycocalyx� the same, but as network of unfixed (loose)

fibrils

Slime� some bacteria release lot of mucous

polysaccharides into environment

Other components of

bacterial cell

Pili, fimbria ø 3-8 nm, pilin

Flagella ø 15-20 nm, flagellin

Sporescell wall

flagellum

spore

capsule

and pili

Pili

- for attachment;- for changing of DNA between bacteria (sexof bacteria).

Urethra:Bacteria Bacteria without pili with pili.

Flagella

- for motility.long spiral proteinchains � flagellin

10

Position

of

flagella

ecoli[1].mov

Movement of bacteria (E. coli)

http://www.cellsalive.com/animabug.htm

Spores

- for survival of bacteria in badenvironmentalconditions.

Only in 2 genus:Bacillus sp.Clostridium sp.

Spores are dehydrated

Very strongcover(containsdipicolonicacid)

Sporulation

11

Physiology of

microorganisms

Main aims of

microorganisms:

To feed

To grow

To multiply

http://www.cellsalive.com/ecoli.htm

Metabolism

(intermediary metabolism):

Anabolism (assimilation)

feeding, building the cell up

Catabolism (dissimilation)

making nutrients suitable,

respiration, getting energy

Differences from metabolism

of macroorganism:

Microorganisms are able to use thesubstrates that cannot be used by

macroorganisms

Unique metabolic products

Single cell has independent

metabolism

Metabolic processes are more

quicker (up to 100 x)

Bacteria require for growth:

Sources of energy

�Organic" carbon (e.g. sugars and fatty acids)

Minerals: K, Ca, Mg, Fe

Other elements: N, P, S, H, B, Mb, Zn, Co, Ni �

Growth factors are needed by some bacteria

� Prototrophs (synthesize them)

� Auxotrophs (need them from environment)

Some bacteria need native proteins

= Fastidious bacteria

Optimal temperature, pH

Oxygen or oxygen-free environment

Bacteria can be divided by

source of energy:

Organotophs

� use organic compounds

Litotrophs

� use anorganic compounds

Phototrophs

� use photochemical reactions

Paratrophs

� use the cell of other organism

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Bacteria can be divided by

source of carbon:

Heterotrophs (most of bacteria)

� The source of carbon is organic compouns

(sugars, amino acids, vitamins, growths

factors)

Autotrophs

� Carbon dioxide or some other anorganic

compounds. All necessary organic

compounds are synthesised by bacteria.

Bacteria consist:

Mechanisms of nutrition

2000 chemical reactions

Transport of nutrients into cell

Accumulation of prekursors and

energy

Biosynthesis, enzymatic processes

Polymerisation

Montage of cell compounds and cell

Transport of nutrients into cell

Simple diffusion

� according to concentration gradient (water,

salts �)

Passive transport

� carrier-proteins, no need for additional energy

Active transport

� need for additional energy

Transport of metal ions� Bacteria secrete small molecules that bind iron

(siderophores, e.g. enterobactin, mycobactin).

Siderophores (together with bound iron) are then

internalized via receptors by the bacterial cell. The

human host also has iron transport proteins (e.g.

transferrin). Thus bacteria that ineffectively compete

with the host for iron are poor pathogens.

Passive. Active.Bacteria get energy:

from energetic metabolism =

catabolism.

At least one of the following processes is

necessary:

fermentation

oxidation or respiration

photosynthesis

The energy is bound to ATP.

13

Catabolism of proteins, polysaccharides, and lipids produces glucose, pyruvate, or intermediates ofthe tricarboxylicacid (TCA) cycleand, ultimately, energy in the form of adenosine triphosphate (ATP) or the reduced form of nicotinamide-adenine dinucleotide(NADH).

Fermentatsioon

During fermentation, substrate (like

glucose) is phosphorylated step-by-step

Different pathways are used for that.

All pathways have 3 general stages:

� Conversion of substrate to donor of phosphate group,

NAD+ NADH

� Phosphorylation of ADP with energy-rich phosphate

donor

� Reactions that balance the end products with substrate

(NADH NAD+)

End products are mainly acids and

alcohols, CO2 etc.

Only 1�4 moleculs of ATP is produced

Glycolysis or

Embden-Meyerhof-

Parnas [EMP]

pathway

Pentose phosphate

pathway

Entner-Doudoroff

pathway

Metabolic pathwaysEmbden-Meyerhof-Parnas (EMP) rada

EMP pathway

This is the most common pathway in bacteria for sugar catabolism (It is also found in most animal and plant cells).

A series of enzymatic processes result in conversion of sugars into pyruvate, generating ATP (adenosine triphosphate) and NADH (nicotinamide adenine dinucleotide).

Chemical energy needed for biosynthetic purposes is stored in the newly formed compounds (ATP and NADH)

Fermentation

of pyruvate

by different

micro-

organisms

results in

different end

products.

The clinical

laboratory

uses these

pathways and

end products

as a means of

distinguishing

different

bacteria

14

End products of different

microorganisms� metabolism

Pärmid Etanool, CO2

Streptokokk,laktobatsill

Laktaat

Enterobakter Etanool, atsetoiin,laktaat, CO2

Difteeriatekitaja Propionaat, atsetaat,suktsinaat, CO2

E. coli Laktaat, atsetaat,suktsinaat, etanool,H2, CO2

Oxidation (respiration)

In the presence of oxygen,

the pyruvate may be

completely oxidized (controlled

burning) to water and CO2 using thetricarboxylic acid (TCA) cycle (Krebs cycle).

Oxidation is a series of red-ox reactions

Transport of electrons to final acceptor and oxidation

of substrate take place

Oxidizer is mostly the molecular oxygen

This process is catalysed by several enzymes that are

on cytoplasmatic membrane

� quinones, cytochromes, terminal cytochrome oxidase

Oxidation: tricarboxylic acid

(TCA) cycle

TCA cycle:

1. Is the major mechanism

for the generation of ATP

2. Serves as the final

common pathway for the

complete oxidation of

amino acids, fatty acids

and carbohydrates

3. Supplies key

intermediates for the

ultimate synthesis of amino

acids, lipids, purines and

pyrimidines

The last two functions make the

TCA cycle amphibolic � it may

function in both anabolic and

catabolic functions of the cell

Shows sequential oxidation and energy-generating steps. Electron transfer is

accompanied by the flow of protons (H+) from NADH, through coenzyme Q (CoQ), and electrons through the

cytochromes (CYTO). Three ATPs are formed per molecule of NADH reoxidized, but only two ATPs are formed per

molecule of FADH2 reoxidized.

FMN, flavin mononucleotide.

Electrontransport chain

Aerobic glucosemetabolism oroxidation ismore effectivethanfermentation:

much more

energy is

released -

26�40 ATP

moleculs

Breathing types of bacteria

Bacteria are separated by their ability

� to survive in O2�containing environment

� to use O2 as terminal electron acceptor

Aerobic

Anaerobic

Facultative anaerobic

� most of pathogens

Microaerophilic

15

Obligate aerobes must grow in the presence of oxygen; they cannot carry out fermentation.

Obligate anaerobes do not carry out oxidative phosphorylation. Furthermore, they are killed by oxygen; they lack certain enzymes such as catalase [which breaks down hydrogen peroxide, H2O2, to water and oxygen], peroxidase[by which NADH + H2O2 are converted to NAD and O2] andsuperoxide dismutase [by which superoxide, O2

., is converted to H2O2]. These enzymes detoxify peroxide and oxygen free radicals produced during metabolism in the presence of oxygen.

Facultative anaerobes can perform both fermentation and aerobic respiration. In the presence of oxygen, anaerobic respiration is generally shut down and these organisms respire aerobically.

Microaerophilic bacteria grow well in low concentrations of oxygen, but are killed by higher concentrations.

Biosynthesis

Polymerization

DNA replication

Synthesis of RNA,

peptidoglycan,

teichoic acids,

phospholipids,

lipopolysaccharides

etc.

Series of biochemicalreactions

Material12 central precursors

C-containingsubstances, NADH, NADPH, ATP, NH3, S

Productsamino acids, nucleotides, sugars, fatty acids

Pro

tein

synt

hesi

s

Protein synthesis and

inhibition of itGrowth and multiplication

of bacteria

�Growth of bacteria� � what does it mean:

� growth of one bacterial cell

� growth of bacterial mass = multiplication of

bacteria

Bacteria multiply by cell division, two

equivalent daughter cells are produced

� 20-30 min.

Growth is limited:

� insufficiency of metabolites

� insufficiency of oxygen

� decrease of pH

� accumulation of toxic end products

16

Growth phases

Initial phase (lag-phase)� 1�2 h, cells enlarge

� metabolic activity increases

Exponential phase (log-phase)� rapid multiplication� bacteria are especially susceptible to antibiotics

Stationary phase� slow multiplication, some cells die � synthesis of DNA continues on some level

� smaller cells appear

Decline� decrease of the number of viable cells

due to lack of nutrients, decrease of pH, accumulation of toxic end products

Multiplication

of bacteria