Microbiology Exam 2

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Diversity in bacteria can be generated via

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Diversity in bacteria can be generated via

Mutation

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Mutation

Modification in the sequence of DNA in a gene often resulting in an alteration in the protein encoded by the gene

Spontaneous

Induced

Essential for understanding genetics

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Spontaneous Mutation

Mutations are stable inheritable changes in the base sequence of DNA

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Vertical Gene Transfer

Mutation passed onto progeny; inherited

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Reversion

Change in a cell’s genotype and phenotype to its original state through a change in the mutated gene

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Spontaneous mutations can occur as results of

Base substitutions

Removal or addition of nucleotides

Transposable elements

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Base substitutions

Most common type of mutation

Results from mistakes during DNA synthesis

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Incorrect base is incorporated into DNA

Point Mutations

Missense Mutation

Nonsense Mutation

Null or knockout mutation

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Point mutations

Occur when one base pair is changed

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Missense mutation

Mutation resulting from amino acid substitution

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Nonsense mutation

Mutation that changes an amino acid codon to a stop codon

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Null or knockout mutation

Mutation that inactivates a gene

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Removal and addition of nucleotides

Shifts the translational reading frame

Shifts the codons

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Frameshift Mutation

Affects all amino acids downstream from addition or deletion

Mutations frequently result in premature stop codons

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Induced Mutations

Mutations can be intentionally produced to demonstrate function of particular gene or set of genes

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Mutations can be induced via

Chemical mutagens

Transposition

Radiation

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Chemical mutagens

Chemical modification of purines and pyrimidines

Increase frequency of mutations as DNA replicates (base substitutions)

Nitrous acid

Alkylating agents

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Base analogs

Chemicals that are structurally similar to the nitrogenous bases but have slightly altered base pairing properties (base substitutions)

May result in pairing with wrong base as complementary strand is being syn.

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Intercalating agents

Chemical Mutagen

Molecules that insert themselves between adjacent bases

Increase the frequency of frameshift mutations

Create space between bases

»Extra base is often added to fill space

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Ethidium Bromide

Common Intercalating Agent

Potential carcinogen

Used to stain DNA in gel electrophoresis

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Transposition

Common procedure used to induce mutation in laboratory

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Transposon (transposable element)

Genes that move from one replicon to another site in the same replicon, or to another replicon on the same cell

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Insertion Mutation

Gene that receives the transposon will undergo a knockout mutation

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Transposable elements

Special segments of DNA that move spontaneously from gene to gene

Elements called transposons

Transposons disrupt proper function of gene

Gene or gene product generally non-functional

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Two types of Radiation

Ultraviolet Light and X rays

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Ultraviolet light

Causes covalent bonding between adjacent thymine bases

»Forms thymine dimers which distorts DNA

»Prevents replication past the dimer (SOS repair system results in the incorporation of the wrong bases)

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X rays

Causes single and double stranded breaks in DNA

»Breaks that occur on both strands are often lethal

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Major problem in induced mutation

identifying bacteria with desired mutation

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Direct selection

Involves inoculating population of bacteria on medium on which only mutants will grow

Used to select antimicrobial resistant or auxotrophic mutants reverted to prototrophic organisms

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Indirect selection

Required to isolate organisms that require growth factor that parent strain does not have (auxotrophic mutants)

Replica plating

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Testing for cancer causing chemicals (carcinogens)

Many mutagens are also carcinogens

Microbes used to test potential carcinogenic activity

Tests are based on effect chemical has on microbial DNA

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Ames test

common chemical carcinogen test

Test assumes that the frequency of reversions is increased by mutagens and that most carcinogens are mutagens

Tests rate of reversion of Salmonella auxotroph

Also tests potential lethality

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Horizontal gene transfer

Genes transferred from one cell to another

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Genes are naturally transferred between bacteria using three mechanisms

DNA-mediated transformation

Transduction

Conjugation

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bacterial virus

bacteriophage

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DNA-mediated transformation

DNA transferred as “naked DNA”

Involves the transfer of naked DNA from the environment to the recipient cell

Cells rupture during the stationary and death phase

The chromosome breaks into small pieces and explodes through the ruptured cell wall

Recipient “competent” cell picks up piece of the naked DNA

The naked DNA is integrated onto the recipient  chromosome

replaces the homologous gene on the chromosome of the recipient cell

Natural transformation occurs when bacterial cells are “competent

bacterial cells are capable of taking up and integrating larger fragments of DNA

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Natural transformation occurs in four stages

Entry of the DNA

Integration of the donor DNA

Mismatch repair

Cell multiplication

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Entry of the DNA

Only single strands enter, double strands are degraded

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Integration of the donor DNA

Donor DNA is integrated by hydrogen bonding

Enzymes cleave recipient DNA

Donor DNA is put in place

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Mismatch repair

Repair mechanism removes either donor or recipient DNA that doesn’t match

Repairs with correct nucleotides

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Cell multiplication

Transformed cells multiply under selective conditions in which non-transformed cells will not grow

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Other methods of transformation

chemically induced transformation and the use of biolistic transformation (gene guns)

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Transduction

Bacterial DNA that is transferred from donor to recipient via a bacterial virus (bacteriophage)

Two types of transduction

Generalized

Any gene of donor can be transferred

Specialized

Only specific genes can be transferred

is a mis-packaging of DNA during viral replication

The mis-packaged  phage infects a new bacterial cell and inserts the donor DNA into the recipient cell

The donor DNA is integrated into the cell by homologous recombination

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Conjugation

only form of gene exchange in which donor survives

Conjugation is frequently mediated by a plasmid

Plasmid is self replicating extrachromosomal piece of DNA

Can code for traits that give bacteria advantage

Conjugation requires direct contact between cells

Cells must be of opposite mating types

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R plasmids

Group of plasmids that confer resistance to many antimicrobial agents

Self-transmissible

Carry all of the genetic information they need for transfer

Mobilizable

Encode some but not all of the information needed for transfer

Example

F (fertility) plasmid

Self-transmissible plasmid

During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient

After, plasmid is transferred, F- cell becomes F+

Some cells can be cured

Spontaneously lose their plasmid

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Basic components of molecular biologist’s “toolkit” include

Restriction enzymes

Gel electrophoresis

DNA probes

primers

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Restriction enzymes

Naturally occurring enzymes that cut DNA into fragments

Cut in predictable and controllable manner

Generate pieces of DNA called restriction fragments

These fragments can be joined to new fragments

Enzymes produce jagged cuts called sticky ends or blunt cuts called blunt ends

»Ends anneal together to form new strand

DNA ligase covalently joins fragments

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Gel electrophoresis

Used to separate DNA fragments according to size

DNA is put into wells in gel

Gel subjected to current (negative to positive)

DNA moves through the gel towards positive electrode

Fragments are separated according to size

»Large fragments remain high in the gel

»Small fragments migrate lower

Gel must be stained to view DNA

Oftentimes stained with ethidium bromide solution

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DNA probes

Used to locate nucleotide sequences in DNA or RNA

Probe is single-stranded piece of DNA tagged with detectable marker

Location can be easily determined

Probe will hybridize to complementary fragment of interest

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technologies that employ DNA probes

Colony blotting

Southern blotting

Fluorescence in situ hybridization (FISH)

DNA/RNA microarrays

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Colony blotting

Used to detect specific DNA sequences in colonies grown in agar plates

Colonies are transferred in place on nylon membrane

Colony blots are used to determine which cells contain gene of interest

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Southern blotting

Uses probes to detect DNA sequences in restriction fragments separated using gel electrophoresis

Application of Southern blotting is locating DNA sequences similar to ones being studied

Northern Blot – RNA

Western Blot - Protein

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Fluorescence in situ hybridization (FISH)

Uses fluorescently labeled probe to detect certain nucleotide sequences

Detects sequences inside intact cell

Specimen is viewed using fluorescence microscope

FISH can be used to identify specific properties of bacteria

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DNA microarray technologies

Used to study gene expression under certain conditions

DNA arrays are solid supports with fixed patterns of different single-stranded DNA fragments attached

Entire DNA specimen to be studied is labeled

Enable researchers to screen sample for numerous sequences simultaneously

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Applications for
DNA Sequencing

Knowing DNA sequence of particular cell helps identify genetic alterations

Alterations that may result in disease

Sickle cell anemia

Due to single base-pair change in gene

Cystic fibrosis

Caused by three base-pair deletion

DNA sequence analysis assists in studying evolutionary relatedness

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Dideoxychain termination

Elements for termination reaction include

Single stranded DNA template

Primer that anneal to template

DNA polymerase

Each of the nucleotide bases

One of these bases is labeled with marker for detection

Dideoxynucleotides

Like deoxynucleotide counterparts but lack 3’ OH

»Incorporation causes chain termination

Polyacrylamide gel electrophoresis used to separate DNA fragments by size

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Automated DNA sequencing

Most automated systems use fluorescent dyes to detect newly synthesized DNA

Gel electrophoresis used to separate fragments into colored bands

Laser used to detect color differences

Order of color reflects nucleotide sequence

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Primers

Single-stranded DNA fragments bind the sequences of DNA

Used in in vitro DNA synthesis

Primer serves as fragment for addition of DNA nucleotides (PCR)

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Polymerase Chain Reaction

Creates millions of copies of given region of DNA in matter of hours

Technique exploits specificity of primers

Allows for selective replication of chosen regions

Termed target DNA

Large amounts of DNA can be produced from very small sample

Starting with double-stranded DNA molecule, process involves number of amplification cycles

DNA is amplified exponentially

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PCR requires three step amplification cycle

Step 1: double-stranded DNA denatured by heat

Step 2: primers anneal to complementary sequence of target DNA and DNA synthesis occurs with heat stable DNA polymerase

Step 3: duplication of target DNA

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DNA cloning

Process of producing copies of DNA

Cloned DNA generally combined with carrier molecule called cloning vector

Insures replication of target DNA

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Researching gene function and regulation

Gene expression, regulation and function can be studied by gene fusion

Joining gene being studied to reporter gene

Reporter gene encodes observable trait

»Trait makes it possible to determine the conditions that affect gene activity

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Why are living organisms divided into groups

To better understand relationships among species

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Taxonomy

The science that studies organisms to order and arrange them

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Taxonomy can be viewed in three areas

Identification

Process of characterizing in order to group them

Classification

Arranging organisms into similar or related groups

Nomenclature

System of assigning names

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True or False

Phenotype can be used in the process of identification of bacteria

True

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Methods used to identify prokaryotes

Microscopic morphology

Metabolic capabilities

Serology

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Strategies Used to Classify Prokaryotes

Understanding organisms’ phylogeny assists in classification

Allows for organized classification of newly recognized organisms

Development of molecular techniques for classification and identification make genetic relatedness possible

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Taxonomic hierarchies

Classification categories arranged in hierarchical order

Species – group of related isolates or strains

Most basic unit

Genus – group of related species

Family – collection of similar genera

Order – collection of similar families

Class – collection of similar orders

Phylum – collection of similar classes

Kingdom – collection of similar phyla

Domain – collection of similar kingdoms

New taxonomic category

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Microscopic morphology

Important initial step in identification

Can be used to determine size, shape and staining characteristics

Size and shape can readily be determined microscopically

Gram stain differentiate Gram (+) from Gram (–)

Narrows possible identities of organism

Special stains

Identifies unique characteristics of organisms

Acid fast stain

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Metabolic capabilities

Identification relies heavily on analysis of metabolic capabilities

Culture characteristics

Colony morphology can give clues to identity

Green pigment of Pseudomonas aeruginosa

-b-hemolytic colonies of Streptococcus pyogenes

  (beta=“complete” hemolysis)

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Biochemical tests

More conclusive identification

Most tests rely on pH indicators or chemical reaction that results in a color change when a compound is degraded

pH change can be acidic (i.e. fermentation of sugars), alkaline (production of CO2 which can raise the pH) or no change at all (could be due to the bacteria not growing or utilizing the specific nutrient source)

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Catalase test

Bacteria that produce catalase break down hydrogen peroxide to release oxygen gas causing bubbling

Also done on plates

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Sugar Fermentation

Fermentation of sugars results in acid production causing pH indicator to turn yellow (inverted tube w/n larger tube traps any gas produced)

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Urease test

Breakdown of urea by urease enzyme releases ammonia and CO2 leads to alkaline environment within tube as indicated by pink color

Breakdown of urea by urease enzyme releases ammonia and CO2

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MACCONKEY AGAR

identification of lactose fermenting, Gram-negative enteric pathogens (differential)

inhibiting growth of Gram-positive organisms (selective).

Fermentation of lactose turns the medium red/pink. (acidic environment from fermenting lactose).

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Biochemical typing

Biochemical tests can be used to identify species

They can also be used to identify strains by tracing specific biochemical characteristics called biovar or biotype

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Serological typing

Identification made based on differences in serological molecules (molecules that react with antibodies)

Serological characteristics are termed serovar or serotype

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Serology

Technique relying on specific interaction between antibodies and antigens

Serological tests are available for rapid detection of many organisms

E.coli O157:H7

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Using Genotype to
Identify Prokaryotes

Nucleic acid probes can locate unique nucleotide  sequence of a particular species

PCR

Used to amplify sequences that allow for detection of specific sequences for identification

Sequencing ribosomal RNA genes

There is little genetic variation in rRNA

16S rRNA is “gold standard for identifying unknown

    bacteria and determining evolutionary relationships (this is how “tree of life” was determined)

Advantage

Identification of organism that can’t be grown in culture

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Genomic typing

Restriction fragment length polymorphisms (RFLPs)

Uses restriction enzymes to digest DNA from each organism

Resolved using pulse-field gel electrophoresis

Variation in fragments b/w organisms are termed polymorphisms

National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet)

Catalogs RFLPs of certain foodborne pathogens

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Antibiograms

Identify organisms based on antibiotic susceptibility

Disc impregnated with antimicrobial placed on inoculated plate

Clear indicates microbial susceptibility

Different strains will have different susceptibility pattern

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True or False

Prokaryotes display an amazing degree of metabolic diversity

True

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True or false

Diversity allowed prokaryotes to emerge as first life-forms,

due to radically different environment on earth 4 billion years ago

True

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True or false

Prokaryotes continue to thrive in areas inhospitable to eukaryotic life,

e.g. thermal hotsprings, acidic, alkaline, salty environments, areas

with no oxygen, no sunlight, etc.

True

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True or false

Metabolic diversity of prokaryotes allows eukaryotes (us!) to live on

earth, some portions of e.g. nitrogen, sulfur cycles only provided

by prokaryotes, large portion of photosynthesis provided by prokaryotes

True

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True or false

Considering that mitochondria and chloroplasts are derived from

prokaryotes, all life on earth is due to metabolic diversity

of prokaryotes.

True

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Phototroph

harvests energy from sunlight

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Photoautotroph

obtains carbon from CO2

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Photoheterotroph

obtains carbon from organic compounds

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Restriction enzymes:

A. Recognize specific DNA sequences

B. Cleave DNA

C. Are used to clone DNA

D. All of the above

E. None of the above

D. All of the above

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DNA probes are used for all of the following EXCEPT:

A. Southern blot

B. Microarrays

C. Western blot

D. Colony blot

E. fluorescence in situ hybridization (FISH)

C. Western blot

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PCR requires all of the following EXCEPT:

A. DNA template

B. RNA primer

C. Thermostable DNA polymerase

D. deoxynucleotides

B. RNA primer

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Urease test is useful to identify:

A. Salmonella typhimurium

B. Helicobacter pylori

C. Neisseria gonorrhea

D. Psudomonas aeruginosa

B. Helicobacter pylori

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Anoxygenic phototroph

phototroph that does not produce O2

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Oxygenic phototroph

phototroph that produces O2

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Chemotroph

harvests energy by oxidizing chemicals

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Chemolithotroph

oxidizes inorganic chemicals

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Chemoorganotroph

oxidizes organic chemicals

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Aerobic respiration

Uses O2 as terminal electron acceptor

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