Bacterial Classification, Anatomy, Nutrition, Growth, Metabolism and Genetics

Classification Systems in the Prokaryotes1. Macroscopic morphology

• Colony appearance & color• Texture & size

2. Microscopic morphology• Cell shape, size • Staining

3. Physiological / biochemical characteristics• Enzymes

4. Chemical analysis• Chemical compound of cell wall

5. Serological analysis

1. Ag/ Ab binding

6. Genetic and molecular analysis• G + C base composition• Nucleic acid sequencing and rRNA analysis

G + C base composition

Low G+C Gram-Positive BacteriaClostridiaMycoplasmas

High G+C Gram-Positive BacteriaCorynebacteriumMycobacterium

Bacterial Taxonomy Based on Bergey’s Manual Bergey’s Manual of Determinative

Bacteriology – five volume resource covering all known procaryotesclassification based on genetic information –

phylogenetic two domains: Archaea and Bacteria five major subgroups with 25 different phyla

Major Taxonomic Groups of Bacteria

Vol 1A: Domain Archaea primitive, adapted to extreme habitats and modes of

nutrition Vol 1B: Domain Bacteria Vol 2-5:

2 - Phylum Proteobacteria – Gram-negative cell walls

3 - Phylum Firmicutes – mainly Gram-positive with low G + C content

4 - Phylum Actinobacteria – Gram-positive with high G + C content

5 – Loose assemblage of phyla – All gram negative

Species and Subspecies Species

bacterial cells which share overall similar pattern of traits Subspecies

Strain or variety culture derived from a single parent that differs in

structure or metabolism from other cultures of that species

E. coli O157:H7 Type

subspecies that can show differences

Bacterial Shapes, Arrangements, and Sizes

Typically described by one of three basic shapes: coccus

Spherical

bacillus Rod

coccobacillus vibrio

spirillum Helical, twisted rod,

Spirochete

Bacterial Shapes, Arrangements, and Sizes

Arrangement of cells dependent on pattern of division and how cells remain attached after division: cocci:

singles diplococci tetrads chains irregular clusters cubical packets

bacilli: chains palisades

Cocci Bacilli

Bacterial anatomy

Generalized structure of a prokaryotic cell

Appendages: Cell Extensions Flagella 3 parts

filament long, thin, helical structure

composed of proteins Hook

curved sheath basal body

stack of rings firmly anchored in cell wall

rotates 360o

1-2 or many distributed over entire cell

Flagellar Arrangements monotrichous

single flagellum at one end lophotrichous

small bunches arising from one end of cell

amphitrichous flagella at both ends of

cell peritrichous

flagella dispersed over surface of cell, slowest

Fig. 4.4

Movement by flagella

Polar Rotates counterclockwise Cell swims forward in

runs Reverse will stop it

Peritrichous All flagella sweep

towards one end

Chemotaxis

Internal Flagella Axial Filaments

aka Periplasmic Endoflagella

Spirochetes

enclosed between cell wall and cell membrane of spirochetes

Appendages for Attachment Fimbrae

fine hairlike bristles from the cell surface

function in adhesion to other cells and surfaces

Appendages for Mating Pili

rigid tubular structure made of pilin protein found only in Gram

negative cells Functions

joins bacterial cells for DNA transfer (conjugation)

Adhesion to form biofilms and

microcolonies

The Cell Envelope External covering outside the cytoplasm Composed of few basic layers:

glycocalyx cell wall cell membrane

Maintains cell integrity

fluid layer of phospholipid and protein phospholipid molecules are arranged in a bilayer Hydrophobic fatty acid chains in the phospholipids form a

permeability barrier

The Cell Membrane

The Bacterial Surface Coating Glycocalyx Coating of molecules

external to the cell wall Made of sugars and/or

proteins functions

attachment inhibits killing by white

blood cells receptor

The Bacterial Surface Coating Glycocalyx 2 types:

1. slime layer - loosely organized and attached

2. capsule - highly organized, tightly attached

Cell Wall

Four Groups Based on Cell Wall Composition:1. Gram positive cells

2. Gram negative cells

3. Bacteria without cell walls

4. Bacteria with chemically unique cell walls

Structure of the Cell Wall Peptidoglycan

macromolecule composed of a repeating framework of long glycan chains cross-linked by short

peptide fragments provides strong,

flexible support keep bacteria from

bursting or collapsing because of changes in osmotic pressure

Gram Positive Cell Wall (1)

Consists of a thick, homogenous

sheath of peptidoglycan tightly bound acidic

polysaccharides teichoic acid and

lipoteichoic acid

Periplasmic space cell membrane

Gram Negative Cell Wall (2) Consists of

an outer membrane containing lipopolysaccharide (LPS)

periplasmic space thin shell of peptidoglycan periplasmic space cell membrane

Protective structure while providing some flexibility and sensitivity to lysis

Gram Negative Cell Wall

LPS endotoxin that may

become toxic when released during infections

may function as receptors and blocking immune response

contains porin proteins in upper layer

Regulates molecules entering and leaving cell

The Gram Stain Important basis of bacterial

classification and identification Practical aid in diagnosing infection

and guiding drug treatment Differential stain

Gram-negative lose crystal violet and stain red from

safranin counterstain Gram-positive

retain crystal violet and stain purple

Atypical Cell Walls

Some bacterial groups lack typical cell wall structure Mycobacterium and Nocardia Gram-positive cell wall structure with lipid mycolic acid

pathogenicity high degree of resistance to certain chemicals and dyes basis for acid-fast stain

Some have no cell wall Mycoplasma cell wall is stabilized by sterols pleomorphic

Chromosome single, circular, double-

stranded DNA molecule contains all the genetic

information required by a cell DNA is tightly coiled around

a protein dense area called the nucleoid central subcompartment in the

cytoplasm where DNA aggregates

Plasmids

small circular, double-stranded DNA

stable extrachromosomal DNA elements that carry nonessential genetic information

duplicated and passed on to offspring replicate independently from the

chromosome

Plasmids may encode antibiotic

resistance, tolerance to toxic metals, enzymes & toxins

used in genetic engineering readily manipulated &

transferred from cell to cell F plasmids allow genetic

material to be transferred from a donor cell to a recipient

R plasmids carry genes for resistance to antibiotics

Storage Bodies Inclusions & Granules

intracellular storage bodies

vary in size, number & content

Examples: Glycogen poly--hydroxybutyrate gas vesicles for floating sulfur

Endospores resting, dormant cells produced by some G+ genera

Clostridium, Bacillus & Sporosarcina resistance linked to high levels of

calcium & certain acids longevity verges on immortality

25 to 250 million years pressurized steam at 120oC for

20-30 minutes will destroy

Endospores have a 2-phase life cycle

vegetative cell endospore

sporulation formation of endospores

Germination return to vegetative growth

withstand extremes in heat, drying, freezing, radiation & chemicals

Endospores• stressed cell

• undergoes asymmetrical cell division• creating small prespore and larger

mother cell• prespore contains:

Cytoplasm DNA dipicolinic acid

• mother cell matures the prespore into an endospore

• then disintegrates• environmental conditions are again

favorable• protective layers break down • spore germinates into a vegetative

cell

Microbial nutrition, growth, and metabolism

Obtaining Carbon

Heterotroph organism that obtains carbon in an organic form

made by other living organisms proteins, carbohydrates, lipids and nucleic acids

Autotroph an organism that uses CO2 (an inorganic gas) as

its carbon sourcenot dependent on other living things

Growth Factors organic compounds

that cannot be synthesized by an organism & must be provided as a nutrient essential amino acids,

vitamins

Nutritional types Chemo-

Chemical compounds Photo-

light

Carbon source

Energy source

photoautotrophs CO2 sunlight

chemoautotrophs CO2 Simple inorganic chemicals

photoheterotrophs organic sunlight

chemoheterotrophs organic Metabolizing organic cmpds

Types of Heterotrophs

Saprobes Parasites / pathogens

Obligate

Nutritional Movement

Osmosis Facilitated diffusion Active transport Endocytosis

PhagocytosisPinocytosis

Extracellular Digestion

digestion of complex nutrient material into simple, absorbable nutrients

accomplished through the secretion of enzymes (exoenzymes) into the extracellular environment

Environmental Influences on Microbial Growth

1. temperature 2. oxygen requirements 3. pH 4. Osmotic pressure 5. UV light 6. Barophiles

1. Temperatures Minimum temperature

lowest temperature that permits a microbe’s growth and metabolism

Maximum temperature highest temperature that

permits a microbe’s growth and metabolism

Optimum temperature promotes the fastest rate of

growth and metabolism

Temperature Adaptation Groups Psychrophiles

• optimum temperature 15oC• capable of growth at 0 - 20oC

Mesophiles • optimum temperature 40oC• Range 10o - 40oC (45)• most human pathogens

Thermophiles • optimum temperature 60oC• capable of growth at 40 - 70oC

Hyperthermophiles Archaea that grow optimally

above 80°C found in seafloor hot-water

vents

2. Oxygen Requirements Aerobe

requires oxygen

Obligate aerobe cannot grow without

oxygen

Anaerobe does not require oxygen

Obligate anaerobe Facultative anaerobe and aerobe

capable of growth in the absence OR presence of oxygen

Fluid thioglycollate media can be used to test an organism’s oxygen sensitivity

Gas chamber

3. pH

The pH Scale Ranges from 0 - 14 pH below 7 is acidic

[H+] > [OH-]

pH above 7 is alkaline [OH-] > [H+]

pH of 7 is neutral [H+] = [OH-]

3. pH

Acidophiles optimum pH is relatively to

highly acidic Neutrophiles

optimum pH ranges about pH 7 (plus or minus)

Alkaphiles optimum pH is relatively to

highly basic

4. Osmotic Pressure Bacteria 80% water

Require water to grow Sufficiently hypertonic media at concentrations

greater than those inside the cell cause water loss from the cell Osmosis Fluid leaves the bacteria causing the cell to contract

Causes the cell membrane to separate Plasmolysis

Cell shrinkage extreme or obligate halophiles

Adapted to and require high salt concentrations

5. UV Light

Great for killing bacteria Damages the DNA

(making little breaks) in sufficient quantity can kill

the organisms in a lower range causes

mutagenisis Endospores tend to be

resistant can survive much longer

exposures

6. Barophiles

Bacteria that grow at moderately high hydrostatic pressures Oceans membranes and enzymes

depend on pressure to maintain their three-dimensional, functional shape

Barotolerants Grows at pressures from 100-

500 Atm Barophilic

400-500 Extreme barophilic

Higher than 500

Microbial Associations Symbiotic

organisms live in close nutritional relationships; Mutualism

Obligatory Dependent Both members benefit

Commensalism One member benefits Other member not harmed

Parasitism Parasite is dependent and benefits Host is harmed

Microbial Associations

Non-symbiotic organisms are free-livingrelationships not required for survival

Synergism members cooperate and share nutrients

Antagonism some member are inhibited or destroyed by others

Microbial Associations

Biofilms Complex relationships

among numerous microorganisms

Develop an extracellular matrix

Adheres cells to one another

Allows attachment to a substrate

Sequesters nutrients May protect individuals in

the biofilm

Microbial Growth in Bacteria Binary fission:

Prokaryotes reproduce asexually

one cell becomes two basis for population growth

Process: parent cell enlarges duplicates its chromosome forms a central septum

divides the cell into two daughter cells

Population Growth

Generation / doubling time time required for a complete

fission cycle Length of the generation time

is a measure of the growth rate of an organism

Some populations can grow from a small number of cells to several million in only a few hours!!

Prokaryotic Growth Bacterial Growth Curve

• lag phase• no cell division occurs while bacteria adapt to their new

environment• logarithmic (log) phase

• Exponential growth of the population occurs • Human disease symptoms usually develop

• stationary phase• When reproductive and death rates equalize

• decline (exponential death) phase• accumulation of waste products and scarcity of resources

Other Methods of Analyzing Population Growth

Turbidity Direct microscopic count Coulter counting

Turbidity

Direct Microscopic Count

Electronic Counting

Microbial genetics

Genomes

Prokaryotic Genomes

Prokaryotic chromosomes Main portion of DNA, along

with associated proteins and RNA

Prokaryotic cells are haploid (single chromosome copy)

Typical chromosome is circular molecule of DNA in nucleoid

DNA Replication in Prokaryotes

Genetic Recombination in Prokaryotes Genetic recombination

occurs when an organism acquires and expresses genes that originated in another organism

Genetic information in prokaryotes can be transferred vertically and horizontally Vertical gene transfer (VGT)

transfer of genetic material from parent cell to daughter cell

Horizontal gene transfer (HGT) transfer of DNA from a donor cell to

a recipient cell Three types

Bacterial conjugation Transformation Transduction

DNA Recombination Events

3 means for exogenous genetic recombination in bacteria:

1. Conjugation

2. Transformation

3. Transduction

Transmission of Exogenous Genetic Material in Bacteria

conjugation requires the attachment of two related species & formation of a bridge that can transport DNA

transformation transfer of naked DNA

transduction DNA transfer mediated by bacterial virus

1. Conjugation transfer of a plasmid or chromosomal fragment from

a donor cell to a recipient cell via direct connection Gram-negative

cell donor has a fertility plasmid (F plasmid, F′ factor) allows the synthesis of a conjugation (sex) pilus

recipient cell is a related species or genus without a fertility plasmid

donor transfers fertility plasmid to recipient through pilus

F+ and F-

Physical Conjugation

2. Transformation chromosome fragments from a

lysed cell are accepted by a recipient cell genetic code of DNA fragment is

acquired by recipient

Donor and recipient cells can be unrelated

Useful tool in recombinant DNA technology

Transformation of Insulin Gene human insulin gene isolated and cut from its

location on the human chromosome using a restriction enzyme

plasmid is cut using the same restriction enzyme desired DNA (insulin gene) and plasmid DNA can

be joined using DNA ligase plasmid now contains the genetic instructions on

how to produce the protein insulin Bacteria can be artificially induced to take up the

recombinant DNA plasmids and be transformed successfully transformed bacteria will contain

the desired insulin gene transformed bacteria containing the insulin gene

can be isolated and grown As transformed bacteria grow they will produce

the insulin proteins coded for the recombinant DNA Insulin harvested and used to treat diabetes

3. Transduction DNA is transferred from one

bacterium to another by a virus

Bacteriophages Virus that infects bacteria consist of an outer protein

capsid enclosing genetic material

serves as a carrier of DNA from a donor cell to a recipient cell

Other ways genetics can change:

Transposons Mutations

Transposons Special DNA segments that have the

capability of moving from one location in the genome to another “jumping genes”

Can move from one chromosome site to anotherr chromosome to a plasmid plasmid to a chromosome

May be beneficial or harmful Changes in traits Replacement of damaged DNA Transfer of drug resistance

Mutations Result of natural

processes or induced Spontaneous mutations

heritable changes to the base sequence in DNA

result from natural phenomena such as radiation or uncorrected errors in replication

UV light is a physical mutagen that creates a dimer that cannot be transcribed properly

Nitrous acid is a chemical mutagen that converts adenine bases to hypoxanthine Hypoxanthine base pairs with cytosine instead of thymine

Base analogs bear a close resemblance to nitrogenous bases and can cause replication errors

Result of spontaneous or induced mutations

affects just one base pair in a gene

Base-pair substitutions result in an incorrect base in

transcribed mRNA codons Base-pair deletion or

insertion results in an incorrect

number of bases

Point Mutation

Attempt to correct mistakes or damage in the DNA Mismatch repair involves DNA polymerase

“proofreading” the new strand removing mismatched nucleotides

Repair Mechanisms

Excision repair involves cutting out

damaged DNA replacing it with

correct nucleotides