CH 1 - Microbial Life

Ubiquitous

present, appearing, or found everywhere.

Ex: Extremophiles

How microbial activity impacts our lives

> Human microbiome (Ex: gut bacteria)

> Environmental activity (Ex: Nitrogen triangle/cycle)

> Infectious disease (Ex: COVID-19)

> Biotechnology (Ex: Biofuels production)

> Scientific discovery (Ex: bacterial plasmids)

What is a Microbe?

• Requires a microscope to be observed

• Typically cellular entities, not multicellular

  > cellular properties: a genome, metabolize; respond to change; evolve; reproduce

  > Such cells can live singly, or form filaments, chains, or clusters

20-100 μm (Average size range of microbial types)

eukaryotic microbes

1-10 μm (Average size range of microbial types)

Prokaryotes

0.02-0.9 μm (20-900 nm) (Average size range of microbial types)

Viruses

Super-sized cells (Macroscopic/Microscopic: Contradictions to the Microbe Definition)

sulfur–oxidizing Thiomargarita magnifica (10 mm); also some algal species and fungal species

Microbial communities (Macroscopic/Microscopic: Contradictions to the Microbe Definition)

• multicellular assemblages: biofilms

• cells comprising tissues: are not microbes

• Bacterial plate culture

Multicellular “micro-animals” (Macroscopic/Microscopic: Contradictions to the Microbe Definition)

are not microbes (Ex: water bear)

Microbes can be divided into two main categories

Cellular and Acellular

Cellular Microbes

• Fungi (yeasts, molds)

• Protista (algae, protozoans)

• Bacteria (E. coli)

• Archaea (cyanobacteria)

Acellular Microbes

• Viruses - composed of proteins, nucleic acids

• Viroids - composed of RNA

• Prions - composed of protein

Prokaryotes characteristics (bacteria and archaea)

• Lack a nucleus & organelles; single circular chromosome; asexual reproduction; cell wall is common

• Diverse metabolisms: autotrophs, heterotrophs, phototrophs

main difference between bacteria and archaea

archaea have cell walls without peptidoglycan, while bacterial cell walls contain peptidoglycan

Eukaryotes characteristics (fungi, protists)

• larger, possess nucleus, organelles, reproduce asexually or sexually; multiple, linear chromosomes

Archaea characteristics

primarily “extremophiles” (prokaryotes)

Thermophiles (Archaea)

• Grow optimally at 50-80°C

• Hyperthermophiles (>80°C)

• Aerobic/anaerobic

• Sulfur metabolizers

• Acidophiles (thrive in highly acidic conditions)

Methanogens (Archaea)

• Produce methane

• Anaerobic

• Thermophilic types

Halophiles (Archaea)

• Require 12-30% NaCl for growth

• Non-chlorophyll based, photosynthetic types

• Aerobic

Ammonia-Oxidizing Archaea (Archaea)

• Oxidize ammonia (NH4 +) to nitrite (NO2 - )

• Key role in the nitrogen cycle

• Compete with ammonia oxidizing bacteria

Viruses (characteristics)

RNA- or DNA-containing non-cellular particles; that requires a host cell to replicate

Microbial Genomes - What do they reveal?

• Microbial genomes reveal the functional capabilities of the microbe in terms of growth, interactions with other species, and relatedness to each other.

  > Genomes can be sequenced relatively rapidly

  > Prokaryote genomes average 500 kb to 5 million bp in size (Ex: 2 million bp - 1700 genes)

  > Genome analysis: core genes shared by all organisms

Metagenomes

Total genome sequencing & analysis of environmental samples

Most microbes are unable to be cultured - what do you do?

Using metagenomes - Allows for collecting information regarding the physiology/genetics of uncultureable microbes

> Less than 1% of microbes are able to be cultured

Metagenomics Process

allows one to identify microbes without culturing them. Involves:

1. Extract all genomic DNA in the sample

2. Digest DNA into fragments and clone into vector

3. Produce library of all the fragments

4. Can express DNA and/or sequence DNA

Application in:  microbial ecology  pharmaceutical industry  biotechnology

Robert Hooke (late 1600’s)

first microscope (20-50X power); viewed macroscopic life (e.g., insects, molds) published drawings in Micrographia (1665)

Antonie van Leeuwenhoek

first to observe single-celled microbes (“animalcules”)

> used 300 X power microscope

Spontaneous generation

life from non-life; abiogenesis

• Require: organic matter, heat, & a vital force - "pneuma" (meaning "breath“; air)

What did Redi do?

disproved abiogenesis for macroscopic organisms (i.e. maggots)

> open container vs. guaze covered container

What did Spallanzani do?

disproved abiogenesis for microscopic organisms (i.e. bacteria)

> spallanzani experiment

> open flash —> growth in broth

> Sealed, sterilized flask —> no growth

Proponents of abiogenesis: lack of air (O2) prevented growth (NOT TRUE)

Louis Pasteur Contributions (Overview)

• Swan-necked flask experiments: refutes spontaneous generation

• Fermentation studies: microbes are catalysts for chemical transformations

• Silkworm disease investigation: links microbe to animal disease

• Pasteurization: gentle heat to reduce microbial numbers in food products

• Vaccines: weakend microbes stimulate immunity

• Aseptic technique

• Advocate for germ theory

Fermentation (Pasteur)

• Pasteur discovered that yeast carry out fermentation; catalysis in the absence of oxygen (anaerobic) to produce alcohol (ethanol)

  > Growth of yeast accompanied formation of alcohol

  > Bacterial contamination & growth accounted for acetic acid production

Pasteur’s germ theory of fermentation

fermentation is not a spontaneous, abiotic process, rather it is caused by the presence of living microorganisms (bacteria).

Virchow

biogenesis concept – life arises only from pre-existing life

Spontaneous Generation (Pasteur)

demonstrated that microbes in the air could contaminate sterile solutions; devised experiment to prove biogenesis.

> devised the swan neck flask: admitted air, but prevented travel of airborne contaminants in the broth.

> Growth appeared only when contaminants contacted the broth

John Tyndall

periodically found opposite results when duplicating Pasteur’s experiment with swan-necked flasks - DUE TO ENDOSPORES!

• Some broth types yielded microbes following sterilization by boiling

• Broths contaminated with heat-resistant endospore forming bacteria

• Endospores eliminated via repeated cycles of boiling & resting (Spore germination at ambient (resting) temperature; susceptible to boiling in this form)

Microbial Origins

• Microbial mats comprising stromatolites; date to 3.8 billion years ago

• Chemical simulations of early Earth’s environment:

• mixture of reduced compounds & electrical discharge & UV light —> biological compounds formed (amino acids, adenine)

• Transition from abiotic —> biotic world

Early archaea, bacteria:

phototrophic, lithotrophic metabolisms; oxidation of reduced inorganic compounds

Microbial Origins: Timelines of Microbial Evolution

Pasteur’s study of silkworm disease

• Microscopic examination of tissues and eggs of diseased worms to link protozoal parasite to the disease

  > showed a possible “chain of infection” (microbe to disease) before Koch (who is more important)

• This work, along with prior fermentation studies, led him to the concept/idea of the germ theory

Robert Koch

• proved the germ theory of disease by providing direct causal proof for specific human diseases; formulated Koch’s Postulates.

chain of infection (Koch)

link a specific microbe to a specific disease

Anthrax - Bacillus anthracis (Koch)

established etiology (cause) via rabbit model using anthrax-infected blood from cow

Tuberculosis - Mycobacterium tuberculosis

proved more difficult to establish etiology

> Difficult to distinguish M. tuberculosis from tissue and other bacteria in the body; required pure culture on solid agar

Koch’s Postulates

• Using solid growth media, Koch isolated pure culture of M. tuberculosis from diseased animals.

  > Allowed him to establish a causative link between the isolate and tuberculosis.

Exceptions of Koch’s Postulates

• Asymptomatic carriers

• One disease: one pathogen

• One pathogen: one disease

• Culturability of pathogen; lack of an animal host

Postulate 1

The microbe is found in all cases of the disease but is absent from healthy individuals (technically false)

Postulate 2

The microbe is isolated from the diseased host and grown in a pure culture

Postulate 3

When the microbe is introduced into a healthy, susceptible host, the same disease occurs

Postulate 4

The same strain of microbe is obtained from the newly diseased host

18th century (Medical Microbiology: Immunization)

• smallpox & variolation - inoculation of smallpox pustules

  > Edward Jenner: use of cowpox to vaccinate; less virulent form

  > Pasteur: attenuated (make virus weaker by leaving outside host) vaccines for fowl cholera & rabies

Vaccination

• use less virulent form of pathogen

  > attenuate vaccine via heat/chemical treatment; “aging” of pathogen.

  > molecular components of the pathogen (antigens) stimulates the body’s immune system providing immunity (immunization).

Jenner: why was cowpox effective as a vaccine even though it did not contain smallpox?

cross-reacting antigens

antisepsis vs. disinfection vs. sterilization

• antisepsis - chemical agent applied to skin (Semmelweis (1847))

• disinfection - chemical agent applied to objects (Lister (1865))

• sterilization - heat treatment (i.e. autoclaving)

Antibiotics

• produced naturally by microbes

  – Alexander Fleming (1929): penicillin

  – Antibiotic resistant bacteria

Antibiotic Resistant Bacteria

Winogradsky 1880’s (Environment and Ecology)

studied bacteria in natural environments; discovered unusual metabolic activities attributable to microbes in natural ecosystems (soil & wetlands)

Winogradsky column

Model of wetland ecosystem

• Combine mud (a source of wetland bacteria) mixed with

• shredded newsprint (a source of CARBON) and…

• calcium salts of sulfate and…

• carbonate (source of CO2)

• Expose to light for several weeks

Sulfate-reducing bacteria / Green sulfur bacteria / Purple sulfur bacteria

SO4—>H2S

H2S—>S₀

• Anaerobic

Purple and green nonsulfur bacteria

H₂—>2 H⁺

• Anaerobic/Microaerophilic

Cyanobacteria

H2O—>O₂

• Aerobic

Iron-oxidizing bacteria

Fe²⁺—>Fe³⁺

• Aerobic

Winogradski analyzed soil, sewage

Soil: H₂—>2 H⁺

Sewage: NH₄⁺—>NO₃^-

• Biotic activity in soil accounts for these conversions

what was used to discover lithotrophic bacteria?

enrichment culture!

• Use NH4 +, H2S, Fe2+ as energy sources

chemolithoautotrophs

use inorganic minerals (NH4 + , H2S, Fe2+) for growth

Enrichment medium for nitrifying bacteria

NH₄⁺—>NO₂^- —>NO₃^-

• what is the carbon source?

  Not organic, but inorganic CO2

Geochemical cycling by bacteria

interconversion of inorganic & organic forms of N, S, P, C, and other minerals; essential to all ecosystems

Fundamental metabolic types:

• heterotroph and autotrophs

  > Can further differentiate—> photo-, chemo-, heterotroph/autotroph

Decomposition

breakdown of dead organic material

Where are microbes found in this cycle?

EVERYWHERE - microbes fit all

Ecology of microbial communities

• Syntrophy

• Biofilms

• Interactions

Microbial Endosymbioses

intimate association between host and its endosymbiont(s) growing within it (i.e. ruminant metabolism, human microbiome, bacteria- plant endosymbiosis)

Species definition: eukaryotes v. prokaryotes; Bacteria/Archaea do not readily fit the species definition —> WHY?

They reproduce asexually - need to be able to sexually reproduce to be considered a species

So….How are they classified? (prokaryotes)

Taxonomic classification of microbes relies on genotypic, phenotypic, and chemical analyses.

Taxonomic history of microbes

‒ Pre-19th century: all life classified as either plant or animal (Linnaeus)

‒ Thereafter, microbial taxonomy changed over the centuries

Discovery of Archaebacteria

Carl Woese (1977): discovery of prokaryotes in extreme environments

• Used 16S ribosomal RNA gene sequences (16S rRNA gene) as a “molecular clock” to measure the time since the divergence of two species

16s rRNA

Eukaryotes: 18S rRNA (equivalent to 16S rRNA)

Revealed new prokaryotic group: archaea; differences to both bacteria and eukaryotes. Established the three-domain classification (Woese)

Archaea - similarities to bacteria

• Lack a nucleus

• Lack organelles

• Similarity in: gene structure, metabolism, environmental niches

Archaea - similarities to eukaryotes

• Components of transcription, protein synthesis

• intron-exon gene structure (mostly tRNA genes)

Three domain system

Two domain system?

refers to a classification of life into two domains, Bacteria and Archaea, with Eukarya (animals, plants, fungi) being incorporated into the Archaea domain. This system is supported by the discovery of the Asgard archaea, a group of microorganisms found to be the closest prokaryotic relatives to eukaryotes.

> a plausible theory

Endosymbiont Theory of Eukaryote Evolution

• Pre-eukaryote cell merged with bacteria to form a composite cell

• Termed intracellular endosymbiosis

• Endosymbiosis evolved to a single organism

  > respiring bacteria - mitochondria

  > phototrophic bacteria - chloroplasts

Evidence of Endosymbiont Theory of Eukaryote Evolution

mitochondria & chloroplasts possess:

(a) DNA w/homology to bacteria

(b) ribosomes & tRNAs

(c) organelles can duplicate

*Closest relatives to mitochondria:

Rickettsia Rhodospirillum