LAB 1 - BACTERIAL STRUCTURE, GENETICS, AND METABOLISM

Bacterium

is a unicellular prokaryotic microorganism — it has no membranebound nucleus and no membrane-bound organelles, carries its genome as circular DNA, and reproduces by binary fission.

Mycoplasma and Ureaplasma

The only bacteria without cell wall

Mycoplasma

is also the sole bacterial exception to the “no sterols in the cell membrane” rule.

Eukaryotes

The nuclear body is an enclosed in a membrane

Archaea and Eubacteria

The nuclear body is nucleoid

Archaea and Eubacteria

The cell division is binary fission

Eukaryotes

The cell division is Mitosis

Arachaea

It has a cell wall but it lack peptidoglycanE

Eubacteria

It has a cell wall except for Mycoplasma and Ureaplasma

Eukaryotes

It has no cell wall, except in plants and fungi

Archaea, Eubacteria, and Eukaryotes

It has a cytoplasmic membrane

Arachaea and Eubacteria

It has no cell organelles

Eukaryotes

It has cell organelles

Bacteria

prokaryotic and unicellular; cell wall of peptidoglycan.

Protozoans

eukaryotic and unicellular.

Metazoans

eukaryotic and multicellular.

Fungi

eukaryotic; possess a cell wall like plants and bacteria. The fungal cell wall is made of chitin.

Viruses

acellular; nucleic acid.

Prions

acellular; protein.

Cocci

round-shaped.

Bacilli

rod-shaped.

Spirilla

– spiral or helical

Cell envelope

makes up the outer cell structure of the bacterium.

Outer membrane

found in gram negative bacteria only

Periplasm / periplasmic space

found in gram-negative bacteria only.

crystal violet and safranin O.

two primary dyes of gram staining

Gram-positive

a bacterium whose thick, rigid peptidoglycan layer retains the crystal violet–iodine complex through decolorization; stains purple.

Gram-negative

a bacterium with only a few layers of peptidoglycan and an outer membrane; loses the primary stain on decolorization and takes up the counterstain; stains red.

Crystal violet

Primary stain

Iodine

Mordant

Ethanol

Decolorizer

Safranin

Counterstain

lipopolysaccharide (LPS) and PORIN channels

cell envelope of a bacterial cell is composed of what? and contains what channels?

PORIN channels

control the passage of nutrients and solutes such as antibiotics.

O Antigen

● Responsible for serotypes. Restricts antibiotics and toxins from entering the bacterial cell.

● Absent in some gram-negative bacteria → these have lipooligosaccharide (LOS) instead.

● LOS organisms: Haemophilus influenzae, Neisseria gonorrhoeae, and Bordetella pertussis.

Core Polysaccharide

Built from monosaccharides arranged in linear and branched structures.

• Two regions:

  • Outer core – proximal to the O antigen (sugars).

  • Inner core – proximal to Lipid A.

Outer core

proximal to the O antigen (sugars)

Inner core

proximal to Lipid A

Lipid A

a.k.a. ENDOTOXIN – released after the bacterium dies.

● Causes fever and shock; found to initiate disseminated intravascular coagulopathy (DIC).

● High variability among bacterial genera.

Cell Wall / Murein Layer

A rigid structure that maintains the shape of the cell.

● Prevents bursting of the cell from the high osmotic pressure inside it.

● Made up of peptidoglycan:

• Alternating series of glycans — N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) — that carry short peptides.

• Cross-links with each other to form a mesh or lattice.

Cell Envelope — Outer Membrane

Found only in gram-negative bacteria.

● A bi-layer structure composed of lipopolysaccharide (LPS).

Peptidoglycan

• NAG–NAM are bound via β-(1,4) linkages.

• NAM carries the oligopeptides.

• The oligopeptide cross-links the NAM of adjacent peptidoglycan chains.

Gram-Positive Cell Wall

● Rich in teichoic acids.

Teichoic acids

• runs perpendicular to the peptidoglycan sheets.

• Building blocks – linear polymers of polyglycerol or polyribitol with phosphates and a few amino acids and sugars.

• Function – regulate the movement of cations into and out of the cell.

• Antigenicity – provide antigenic specificity (surface antigens).

• Virulence – can induce septic shock, similar to the gram-negative Lipid A.

Lipoteichoic acid

the teichoic acid species anchored into the cell membrane; teichoic acid proper is anchored in the peptidoglycan.

Gram-Negative Cell Wall

● Few layers of peptidoglycan. Relies on the outer membrane for further protection.

● Structural stack (outside → in): LPS / outer membrane → lipoprotein → peptidoglycan → cell membrane. Surface proteins are embedded in the outer membrane.

● Gram-positive stack (outside → in): teichoic acid, surface protein, lipoteichoic acid traversing a thick peptidoglycan → cell membrane (phospholipid).

acid-fast bacterium

is one whose cell wall contains high concentrations of mycolic acids, which makes it “resistant” to the Gram stain.

Acid-Fast Cell Wall

• mycolic acid in the cell wall

• the waxy, lipidrich mycolic acid layer makes the wall impermeable to the Gram-stain dyes; the organism does not stain reliably by Gram stain.

Cell Envelope — Periplasmic Space

● Exclusive to gram-negative bacteria.

● Bound to the peptidoglycan layer.

Contains the periplasm: a gel-like fluid consisting of detoxifying enzymes and transport proteins.

periplasm

gel-like fluid consisting of detoxifying enzymes and transport proteins.

Glycocalyx

a viscous, gelatinous layer external to the bacterial cell wall, composed of either polysaccharide, polypeptide, or both.

Glycocalyx

• Most often seen as a CAPSULE.

• Sometimes as a SLIME LAYER or as a BIOFILM.

• More often made inside the cell, then secreted outside

Capsule

In glycocalyx it -

● Determined via negative staining. ● Antiphagocytic. ● Barrier to hydrophobic compounds. ● Removed by boiling.

Slime Layer

In glycocalyx it is Unorganized and not firmly attached to the cell wall. ● Inhibits phagocytosis or aids in adherence to host tissue or synthetic implants.

Biofilm

In glycocalyx it Aggregates of microorganisms housed in a complex polysaccharide matrix. ● Cohesive and adheres to surfaces.

Cell Membrane

● Site of energy production.

Components: phospholipid, proteins.

● No sterols, except for Mycoplasma.

● Chromatophores: Infoldings of the plasma membrane.

○ Site for pigment and enzymes involved in photosynthesis.

Cytoplasm

● Site of protein synthesis. Has a granular appearance due to the presence of polysomes. ● A gel-like matrix composed of water, enzymes, nutrients, wastes, and gases. ● Location of the other interior structures of the bacterium.

Nucleoid

Contains a single long, continuous, circularly arranged thread of dsDNA → the bacterial chromosome. ● Not surrounded by a membrane and does not include histones. ● ● Attached to the plasma membrane.

Ribosomes

● Attached to the cytosol. ● Principal site for protein synthesis. ● Density: 70 Svedberg units (70S)

50S subunit

: 70 Svedberg units (70S).

point of attachment of tRNA

30S subunit

: 70 Svedberg units (70S).

point of attachment of mRNA.

Endospores

Dormant form, highly resistant to most common disinfection and sterilization methods.

● Achieved via sporulation. Occurs when the environment is not favorable.

● Induces structural changes to the cytosol and the cell envelope.

● Seen in gram-positive bacteria: Bacillus and Clostridium.

Flagella

Responsible for locomotion.

● Best observed at 25°C. Carries the H antigen; the protein is flagellin.

● Three parts:

○ Filament

○ Hook

○ Basal body

Visualized using: Leifson stain, Gray stain, Fisher and Conn stain.

Atrichous

No flagella at all.

Monotrichous

A single flagellum at one pole.

Amphitrichous

A flagellum at each of the two opposite poles (one on each end).

Lophotrichous

A tuft (cluster) of several flagella at one pole.

Cephalotrichous

Flagella arising from the head/anterior end of the cell.

Peritrichous

Flagella distributed all over the entire cell surface.

Genome

the entirety of the organism’s genes organized into chromosomes.

Gene

a segment of DNA that acts as the functional and physical unit of inheritance

Genotype

the nucleotide sequence carried by the organism

phenotype

– the expressed characteristic.

Mobilome

the extrachromosomal, mobile genetic elements of a cell.

Replicon

a DNA unit capable of replicating; transposons must physically integrate with a bacterial replicon to propagate.

Competent bacteria

microorganisms able to take up free DNA from the environment.

The Bacterial Chromosome

Single, supercoiled, circular dsDNA. Contains the information for cell growth and replication.

Housed in the nucleoid: no membrane, no histones, attached to the plasma membrane.

Binary Fission

1. The bacterial cell elongates and the chromosomal DNA is replicated → two replicated chromosomes.

2. The bacterial cell wall and plasma membrane begin to invaginate → a fission ring forms.

3. A cross-wall (septum) forms two distinct bacterial cells.

4. The bacterium separates into two bacterial cells; each daughter cell contains a single chromosome

Mobilomes

extrachromosomal elements; genetic elements that can move within a genome and/or between different genomes. They comprise transposons and plasmids.

Plasmids

● Non-chromosomal circular dsDNA.

● Autonomously replicating. May also be incorporated into the chromosome (recombinant technology / cloning).

● May code for virulence, toxins, and antimicrobial resistance (AMR) factors.

● Not necessary for the viability of the bacterium.

Transposons (“Jumping Genes”)

Segments of DNA that can incorporate between microorganisms even if no homology exists.

● Propagation depends on their physical integration with a bacterial replicon (chromosome or plasmid).

● Achieved via transposase.

Replication

Definition: Copying of the parental dsDNA to produce identical daughter DNA molecules, driven by DNA polymerase III and the replisome End-product: Two identical double-stranded DNA molecules (one per daughter cell)

Transcription

Definition: Synthesis of mRNA by RNA polymerase from a DNA template strand End-product: mRNA

Translation

Definition: Synthesis of polypeptides from mRNA; a codon becomes an amino acid End-product: Polypeptide / protein

Phase 1: Initiation

1. Identification of the Origin of Replication (ORI) — the site at which replication commences, recognized by an origin-recognising complex.

2. Formation of the replication fork — the parent strand is separated into two by helicase. Leading strand – synthesized from the ORI along the 3′→5′ template strand as a continuous strand. Lagging strand – synthesized from the ORI along the 5′→3′ template strand as Okazaki fragments.

3. Primase attaches to the DNA and synthesizes an RNA primer.

Phase 2: Elongation / Synthesis

Mainly driven by DNA polymerase III and the other components of the replisome. ● Replisome components

DNA gyrase

– relieves the supercoiling ahead of the advancing fork.

Helicase / primase

unwinds the parental duplex and lays down the RNA primers

SSB (single-strand binding protein)

– coats and stabilizes the exposed singlestranded template, preventing re-annealing.

β-subunit “sliding clamp”

encircles the DNA and tethers the polymerase to the template, conferring processivity

Trimeric replicative DNA polymerase (Pol III)

extends the newly synthesized leading strand and each Okazaki fragment of the lagging strand.

Leading strand template and lagging strand template

– copied continuously and discontinuously, respectively.

Phase 3: Excision and Termination

• Excision of the RNA primers by DNA polymerase I (5′→3′ exonuclease activity).

• The RNA primer is replaced with deoxyribonucleotide by DNA polymerase I.

• DNA ligase joins the ends of the DNA fragments together.

• Replication terminates when the two replication forks meet.

DNA polymerase III

Main synthetic enzyme; builds the leading strand and the Okazaki fragments Phase: Elongation

DNA polymerase I

5′→3′ exonuclease — excises the RNA primer and replaces it with deoxyribonucleotide Phase: Excision / Termination

DNA ligase

Joins the ends of the DNA fragments together Phase: Excision / Termination

Transcription

– synthesis of mRNA by RNA polymerase. ● Template strand = the 3′–5′ strand. ○ Also called the non-coding strand. ○ Also called the antisense strand.

Translation

– synthesis of polypeptides from mRNA. ● A codon becomes an amino acid. ○ Occurs on the 70S ribosome: mRNA binds the 30S subunit, tRNA binds the 50S subunit.

Recombination

Occurs when a segment of foreign DNA combines with a homologous portion of the original bacterial chromosome. ● Forms a partially hybrid chromosome with segments originating from both the donor and the recipient

Transformation

• DNA is released into the environment following cell lysis.

• The released DNA can be incorporated into another microorganism, followed by recombination.

• Microorganisms that take up the DNA are called COMPETENT BACTERIA:

• ○ Streptococcus pneumoniae ○ Neisseria gonorrhoeae ○ Haemophilus influenzae