Microbial Diversity: Acellular and Procaryotic Microbes

Categories of Microorganisms

• Microbes are divided into cellular (bacteria, archaea, algae, protozoa, fungi) and acellular (viruses, viroids, prions) categories.

• Cellular microbes are divided into procaryotic (bacteria, archaea) and eucaryotic (algae, protozoa, fungi) categories.

• Viruses, viroids, and prions are acellular microbes or infectious particles.

Acellular Microbes: Viruses

• Complete virus particles are called virions.

• Most viruses range from 10 to 300 nm in diameter.

• Viruses can infect humans, animals, plants, fungi, protozoa, algae, and bacteria.

• Oncogenic viruses or oncoviruses can cause specific types of cancer.

• A typical virion consists of a genome of either DNA or RNA, surrounded by a capsid (protein coat).

• The capsid is composed of protein units called capsomeres.

• Some viruses (enveloped viruses) have an outer envelope composed of lipids and polysaccharides.

Viral Components

• Capsomeres form the capsid around the nucleic acid in helical and icosahedral viruses.

• Enveloped viruses contain a nucleic acid, capsid, envelope, and proteins within the envelope; these can also be helical or icosahedral.

Distinguishing Properties of Viruses

• Viruses possess either DNA or RNA, whereas living cells possess both.

• Viruses cannot replicate on their own; they require a host cell.

• Viruses do not divide by binary fission, mitosis, or meiosis.

• Viruses lack the genes and enzymes necessary for energy production.

• Viruses depend on the host cell's ribosomes, enzymes, and metabolites for protein and nucleic acid production.

Virus Classification

• Viruses are classified by:

  • Type of genetic material (DNA or RNA)

  • Shape and size of capsid

  • Number of capsomeres

  • Presence or absence of an envelope

  • Type of host it infects

  • Disease it produces

  • Target cell(s)

  • Immunologic/antigenic properties

Viral Nucleic Acid Categories

• Four categories of viruses based on nucleic acid type:

  • Double-stranded DNA viruses

  • Single-stranded RNA viruses

  • Single-stranded DNA viruses

  • Double-stranded RNA viruses

• Viral genomes can be circular or linear.

Capsid Shape and Symmetry

• Capsids can be:

  • Polyhedral (many-sided, often icosahedral with 20 facets)

  • Helical (coiled tubes)

  • Bullet-shaped

  • Spherical

  • Complex combinations

Origins of Viruses Theories

• Coevolution theory: Viruses originated in the primordial soup and coevolved with bacteria and archaea.

• Retrograde evolution theory: Viruses evolved from free-living prokaryotes that evaded other living organisms and lost functions provided by the host cell.

• Escaped gene theory: Viruses are pieces of host cell RNA or DNA that escaped from living cells and are no longer under cellular control.

Virus Envelopment and Release

• Viruses can acquire an envelope by budding from the host cell membrane.

• Viral glycoproteins are synthesized and transported to the host cell membrane.

• The cytoplasmic domains of membrane proteins bind nucleocapsids.

• The host cell membrane provides the viral envelope during budding.

Simplified HIV Life Cycle

• Virus attaches to the cell surface.

• The viral core enters the cell, and its RNA is converted to DNA.

• Viral DNA enters the nucleus and combines with host cell DNA.

• New viral RNA copies are made and leave the nucleus.

• New viral components congregate at the cell surface.

• New virus particle buds from the cell.

Herpesviruses

• Herpesviruses exit the nucleus of the host cell, enveloping themselves in membranes.

Comparative Sizes

• Virions, nucleic acids, and bacteria vary in size.

• Examples include bacteriophages, poliovirus, adenovirus, tobacco mosaic virus, and Escherichia coli.

Acellular Microbes: Bacteriophages

• Viruses that infect bacteria are known as bacteriophages or phages.

Bacteriophage Categories

• Virulent bacteriophages: Cause the lytic cycle, resulting in the destruction of the bacterial cell.

• Temperate bacteriophages.

Lytic Cycle Steps

• Attachment

• Penetration

• Biosynthesis

• Assembly

• Release

Bacteriophage T4

• Bacteriophage T4 is an assembly of protein components.

• Viral DNA enters the cell through the core.

• The structure includes 20 facets filled with DNA.

Lytic Cycle Summary

1. Attachment of phage to cell surface receptors.

2. Injection of phage DNA into the cell.

3. Synthesis of new phage DNA and phage proteins.

4. Assembly of new phage particles.

5. Lysis of the cell and release of progeny phage.

Animal Viruses

• Animal viruses escape from their host cells by lysis or budding.

• Viruses that escape by budding become enveloped viruses.

Latent Viral Infections

• Viral infections where the virus hides from the host's immune system by entering cells and remaining dormant.

Examples

• Herpes viral infections (e.g., cold sores, genital herpes, chickenpox/shingles).

• Chickenpox may be followed by shingles years later, both caused by the same virus.

Herpes Simplex Virus Infection Process

• Adsorption

• Penetration (involves local digestion of viral and cellular membranes)

• Uncoating and digestion of the capsid

• Fusion of the two membranes and release of the nucleocapsid into the cytoplasmic matrix

• The nucleocapsid is digested, leaving a core containing DNA and protein.

Antiviral Agents

• Antibiotics are ineffective against viral infections.

• Antiviral agents are drugs used to treat viral infections.

• They interfere with virus-specific enzymes and virus production by disrupting critical phases in viral multiplication or inhibiting the synthesis of viral DNA, RNA, or proteins.

Oncogenic Viruses or Oncoviruses

• Viruses that cause cancer.

• Examples: Epstein-Barr virus, human papillomaviruses, and HTLV-1 (Human T Lymphotropic virus type 1).

• Human Immunodeficiency Virus (HIV).

  • The cause of acquired immunodeficiency syndrome (AIDS).

  • It is an enveloped, single-stranded RNA virus.

  • The primary targets for HIV are CD4+ cells.

HIV Structure

• HIV is an enveloped virus with two identical single-stranded RNA molecules.

• Each surface knob contains a glycoprotein (gp120) that binds to a CD4 receptor on certain host cells (e.g., T-helper cells).

• The stalk supporting the knob is a transmembrane glycoprotein (gp41), involved in attachment to host cells.

• Reverse transcriptase is an RNA-dependent DNA polymerase.

Important Human Viral Diseases

• AIDS, chickenpox, cold sores, the common cold, Ebola virus infections, genital herpes infections, German measles, Hantavirus pulmonary syndrome, infectious mononucleosis, influenza, measles, mumps, poliomyelitis, rabies, severe acute respiratory syndrome (SARS), and viral encephalitis.

• All human warts are caused by viruses.

Mimiviruses

• Extremely large double-stranded DNA viruses.

• Recovered from amebas.

• Named Mimivirus because it mimics bacteria.

• Large enough to be observed using a standard compound light microscope.

Plant Diseases

• More than 1,000 different viruses cause plant diseases, affecting citrus trees, cocoa trees, rice, barley, tobacco, turnips, cauliflower, potatoes, tomatoes, and many other fruits, vegetables, trees, and grains.

Mimivirus Structure

• Consists of a double-stranded DNA core, surrounded by two lipid membranes and a protein capsid.

• Numerous fibrils extend outward from the capsid surface.

Acellular Microbes: Viroids and Prions

• Smaller and less complex infectious particles than viruses.

Viroids

• Short, naked fragments of single-stranded RNA.

• Interfere with the metabolism of plant cells.

• Transmitted between plants in the same manner as viruses.

Examples of Plant Diseases Caused by Viroids

• Potato spindle tuber

• Citrus exocortis

Prions

• Small infectious proteins that cause fatal neurologic diseases in animals.

Examples

• Scrapie

• Bovine Spongiform Encephalopathy (“Mad Cow Disease”)

• Creutzfeldt-Jacob disease

• Of all pathogens, prions are the most resistant to disinfectants.

• The mechanism by which prions cause disease remains a mystery.

The Domain Bacteria

Characteristics

• Bacteria are divided into 3 major phenotypic categories:

  • Gram-negative with a cell wall

  • Gram-positive with a cell wall

  • Lacking a cell wall (Mycoplasma spp.)

Classification and Identification

• Characteristics used include:

  • Cell morphology

  • Staining reactions

  • Motility

  • Colony morphology

  • Atmospheric requirements

  • Nutritional requirements

  • Biochemical and metabolic activities

  • Enzymes produced

  • Pathogenicity

  • Genetic composition

Cell Morphology

• Three basic categories based on shape:

  • Cocci (round bacteria)

  • Bacilli (rod-shaped bacteria)

  • Curved and spiral-shaped bacteria

Cocci

• May be seen singly or in pairs (diplococci), chains (streptococci), clusters (staphylococci), packets of 4 (tetrads), or packets of 8 (octads).

• The average coccus is about $1 \mu m$ in diameter.

Arrangements of Cocci

• Diplococci: Cocci in pairs (e.g., Neisseria gonorrhoeae, causing gonorrhea).

• Streptococci: Cocci in chains (e.g., Streptococcus pyogenes, causing strep throat).

• Staphylococci: Cocci in clusters (e.g., Staphylococcus aureus, causing boils).

• Tetrad: A packet of 4 cocci (e.g., Micrococcus luteus, rarely pathogenic).

• Octad: A packet of 8 cocci (e.g., Sarcina ventriculi, rarely pathogenic).

Bacilli

• Referred to as rods; may be short or long, thick or thin, and pointed or with curved or blunt ends.

• May occur singly, in pairs (diplobacilli), in chains (streptobacilli), in long filaments, or branched.

• An average sized bacillus is $1 x 3 \mu m$.

• Extremely short bacilli are called coccobacilli.

Medically Important Bacilli Examples

• Escherichia, Klebsiella, and Proteus spp.

• Pseudomonas, Haemophilus, and Bacillus spp.

Curved and Spiral-Shaped Bacteria

Curved Bacteria Examples

• Vibrio spp.

• Campylobacter spp.

• Helicobacter spp.

Spiral Shaped Bacteria Examples

• Treponema spp.

• Borrelia spp.

Staining Procedures

• Three Major Categories of Staining Procedures

  1. Simple stains

  2. Structural staining procedures

     • Capsule stains

     • Spore stains

     • Flagella stains

  3. Differential staining procedures

     • Gram and acid-fast staining procedures

Bacterial Smears

• Bacterial smears must be fixed prior to staining.

• The fixation process serves to kill organisms, preserve their morphology, and anchors the smear to the slide.

• Heat-fixation: Not a standardized technique; excess heat will distort bacterial morphology.

• Methanol-fixation: A standardized technique; the preferred method.

Simple Bacterial Staining

1. Smear loopful of microbes onto a slide.

2. Air-dry.

3. Drip methanol onto the specimen to fix it.

4. Flood slide with stain.

5. Rinse with water and blot dry.

6. Examine with x100 objective (oil immersion).

The Gram Staining Procedure

• Divides bacteria into 2 major groups:

  • Gram-positive (bacteria are blue-to-purple)

  • Gram-negative (bacteria are pink-to-red)

Gram Reaction Basis

• Depends upon the organism’s cell wall structure.

Gram-Positive Bacteria

• Have a thick layer of peptidoglycan, making it difficult to remove the crystal violet-iodine complex.

Gram-Negative Bacteria

• Have a thin layer of peptidoglycan, making it easier to remove the crystal violet; the cells are subsequently stained with safranin.

Gram Staining Steps

1. Methanol-fix the specimen to the slide.

2. Flood the slide with crystal violet solution; allow to act for 1 minute.

3. Rinse the slide, then flood with iodine solution; allow iodine to act for 1 minute.

4. Rinse off excess iodine. Decolorize with ethanol, approximately 5 seconds

5. Apply safranin counterstain for 30 seconds.

6. Wash in water, blot, and dry in air.

Gram-Positive vs. Gram-Negative

Gram-Variable Bacteria

• Some bacteria are neither consistently purple nor pink after Gram staining; they are known as Gram-variable bacteria; example, Mycobacterium spp.

Acid-Fast Stain

• Mycobacterium spp. are often identified using the acid-fast stain.

  • Carbol fuchsin is the red dye that is driven through the bacterial cell wall.

  • Heat is used to soften the waxes in the cell wall.

  • Because mycobacteria are not decolorized by the acid-alcohol mixture, they are said to be acid-fast.

Bacterial Motility

• If a bacterium is able to “swim,” it is said to be motile.

• Bacterial motility is most often associated with flagella; less often with axial filaments.

• Most spiral-shaped bacteria and about 50% of bacilli are motile; cocci are generally nonmotile.

• Motility can be demonstrated by stabbing the bacteria into a tube of semisolid medium or by using the hanging-drop technique.

Methods to Determine Motility

• Semisolid Agar Method for Determining Motility

• Hanging-Drop Prep for Study of Living Bacteria

Colony Morphology

• A bacterial colony contains millions of organisms.

• Colony morphology (appearance of the colony) varies from one species to another.

Colony Morphology Characteristics

• Size

• Color

• Overall shape

• Elevation

• Appearance of the edge or margin of the colony

• Results of enzymatic activity on various types of media

• Colony morphology is an important “clue” to the identification of bacteria.

Atmospheric Requirements

• Bacteria can be classified based on their atmospheric requirements, including their relationship to $O<em>2$ and $CO</em>2$.

Oxygen Requirements

• Obligate aerobes: Require oxygen to grow (e.g., Mycobacterium tuberculosis, Pseudomonas aeruginosa).

• Microaerophilic aerobes: Require low levels of oxygen (e.g., Campylobacter jejuni).

• Facultative anaerobes: Can produce energy through aerobic respiration but switch to anaerobic respiration (e.g., E. coli, Group A Streptococcus, Staphylococcus aureus, Staphylococcus epidermidis).

• Aerotolerant anaerobes: Produce cellular ATP energy without oxygen (e.g., Lactobacilli and Streptococci).

• Obligate anaerobes: Live only in the absence of oxygen (e.g., Clostridium).

• Capnophilic organisms: Grow best in increased $CO_2$ concentrations (usually 5 to 10%).

Nutritional Requirements

• All bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur, phosphorus, and nitrogen for growth.

• Some bacteria require special elements (e.g., calcium, iron, or zinc).

• Organisms with especially demanding nutritional requirements are said to be fastidious (“fussy”).

• The nutritional needs of a particular organism are usually characteristic for that species and are sometimes important clues to its identity.

Biochemical and Metabolic Activities

• As bacteria grow, they produce many waste products and secretions, some of which are enzymes.

• Pathogenic strains of many bacteria, like staphylococci and streptococci, can be tentatively identified by the enzymes they secrete.

• In particular environments, some bacteria produce gases such as carbon dioxide or hydrogen sulfide.

• To identify bacteria in the lab, they are inoculated into various substrates (i.e., carbohydrates and amino acids) to determine whether they possess the enzymes necessary to break down those substrates.

Pathogenicity

• Many pathogens can cause disease because they possess capsules, pili, endotoxins, or secrete exotoxins and exoenzymes that damage cells and tissues.

• Frequently, pathogenicity is tested by injecting the organism into mice or cell cultures.

Examples of Pathogenic Bacteria

• Neisseria meningitidis

• Salmonella typhi

• Shigella spp.

• Vibrio cholerae

• Yersina pestis

• Treponema pallidum

Genetic Composition

• Laboratory identification of bacteria is moving toward analyzing the organism’s DNA or RNA – techniques collectively referred to as molecular diagnostic procedures.

• The composition of the genetic material (DNA) of an organism is unique to each species.

• DNA probes make it possible to identify an isolate without relying on phenotypic characteristics.

• Through the use of 16S rRNA sequencing, the degree of relatedness between 2 different bacteria can be determined.

Unique Bacteria

• Rickettsias, chlamydias, and mycoplasmas are bacteria but do not possess all the attributes of typical bacterial cells.

Rickettsias and Chlamydias

• Have a Gram-negative type of cell wall and are obligate intracellular pathogens (i.e., they must live within a host cell; they cannot grow on artificial culture media).

  • Rickettsias have “leaky membranes.”

  • Chlamydias are “energy parasites,” meaning they prefer to use ATP molecules produced by their host cell.

Typhus vs. Typhoid Fever

• Typhus fever is caused by the bacteria Rickettsia, which is transferred to humans through arthropods like lice, ticks, mites, or fleas.

• Typhoid fever is caused by the bacteria Salmonella typhi, which is related to the salmonella that causes food poisoning.

Mycoplasmas

• Smallest of the cellular microbes.

• Lack a cell wall and therefore assume many shapes (they are pleomorphic).

• In humans, pathogenic mycoplasmas cause primary atypical pneumonia and genitourinary infections.

• Because they have no cell wall, they are resistant to drugs like penicillin that attack cell walls.

• They produce tiny “fried egg” colonies on artificial media.

Photosynthetic Bacteria

• Include purple bacteria, green bacteria, and cyanobacteria; they all use light as an energy source, but not in the same way.

  • Purple and green bacteria do not produce oxygen, whereas cyanobacteria do.

  • Photosynthesis that produces oxygen is called oxygenic photosynthesis.

  • Photosynthesis that does not produce oxygen is called anoxygenic photosynthesis.

The Domain Archaea

• Archaea (meaning ancient) were discovered in 1977; they are procaryotic organisms.

• Genetically, archaea are more closely related to eucaryotes than they are to bacteria.

• Archaea vary widely in shape; some live in extreme environments, such as extremely acidic, extremely hot, or extremely salty environments.

• Archaea possess cell walls, but their cell walls do not contain peptidoglycan (in contrast, all bacterial cell walls contain peptidoglycan).