-obligate parasites

-contain genome surrounded by protective protein coat that allows genome to be transmitted from one host to another; host can be eukaryotic or prokaryotic

-exists in 2 states: extracellular & intracellular

-can contain DNA or RNA in circular or linear form, or go through DNA &/or RNA intermediates during life cycle

extracellular: nucleic acid surrounded by proteins; termed 'virion' or 'virus particle'

intracellular: where virus genome is introduced into host cell (infection) & replicates itself

-destroy 40% of bacteria in oceans every day

-imp. source of genetic diversity

-imp. agents for gene exchange b/w microorganisms

-cause human diseases (flu, HIV, small pox, polio, rabies, Ebola...)

-cause disease to plants & animals on which we depend for food

-can kill bacterial human pathogens & used for food fermentation

-useful for immortalizing animal cell lines & potential for gene therapy

-powerful model organisms; more available to be analyzed for rare genetic events

-much of nature of genetic code was discovered using bacteriophages

-biotechnology (ex reverse transcriptase)

-functions of host cells learn from studying viral disease

-inner core of nucleic acid: single or double stranded, single or multiple segments, circular or linear, DNA or RNA

-capsid: protective protein coat surrounding nucleic acid

-proteins assemble in capsomers

-capsomers aggregate into protein coat

-entire capsid + nucleic acid = nucleocapsid

mostly in eukaryotes; nucleocapsid's often icosahedral, surrounded by host-derived membrane

-membrane functions like cloaking device

overlapping genes--diff. genes are encoded in diff. reading frames

-highly efficient use of limited genetic material

-early mRNA depends on host cell factors or enzyme cargo of phage

-phage proteins direct phage replication & expression of late genes & hijacking of host cell

-phage self-assembles & lyses host cell

-latent period: time phage takes to take over & lyse host cell

lysogeny: phage integrates into host genome (is replicated w/ host genome)

-can lyse host when host is sick (bail from sinking ship!)

lysogen: host cell contains phage integrated into host genome

-cloudy centres for plagues are lysogens, which grow resistant to lysis, can themselves become lytic

animal viruses: consequences of infection

causing cancer:
Hep B & C --> liver cancer

Papilloma virus --> cervical cancer

causing lysis:

poliovirus

Ebola virus

causing persistent infections:

herpes simplex virus

varicella-zoster virus

HIV

CMV

-positive-strand RNA viruses w/ dsDNA intermediate

-possess reverse transcriptase that copies RNA into DNA

-DNA form has props. similar to transposable elements

-1st viruses demonstrated to cause cancer

-HIV-1, causing AIDS

-integrate into host genome where they can exist in latent state for many years

-terminal repeats: essential for viral replication

-gag gene encodes nucleocapsid proteins

-pol gene encodes protease, reverse transcriptase, & integrase

-env gene encodes envelope glycoproteins

-some retroviruses carry 4th gene after env, associated w/ cancer

1) entry by fusion of envelope w/ cell membrane

2) uncoating

3) reverse transcriptase

4) dsDNA travels to nucleus & integrates into host DNA

5) transcription of viral DNA to make RNA & mRNA

6) translation & polyprotein processing

7) encapsidation

8) budding

9) release

-in some, like HIV, translation of several proteins occurs as single polyprotein

-specific viral protease cuts long polypeptide into individual proteins

-protease inhibitors are drugs developed to prevent this critical step

where there is no growth advantage for mutation & each mutant is examined for desired phenotype (ex each mutant colony is examined for loss of pigmentation)

auxotroph: mutant phenotype where nutritional supplement is required for mutant growth that's not required for wild-type growth

prototroph: strain not requiring nutritional supplement

-4.6 million base pairs
1 kilo base pair

1 Mega base pair

-slightly more than 300 amino acids & gene size of 1 kpb

-4,288 open reading frames (ORFs)

-operon: diff. genes co-transcribed as single mRNA (30% of genes co-transcribed)

-NotI = 8 bp restriction endonuclease

self-replicating accessory coding DNA, not essential for viability

MAJOR SOURCE OF ANTIBIOTIC RESISTANCE

selection: mutants are selected for mutant phenotype; other mutants fail to grow

screening: must screen (sort) through mutants to find mutant phenotype of interest; most mutants grow

missense: point mutation changes in amino acids

nonsense: point mutations resulting in stop codon

silent: point mutations resulting in diff. codon for same amino acid; doesn't change amino acid sequence!

employment of chemical, physical, & biological agents to generate mutations

chemical mutagens include base analogs, chemicals that react w/ DNA, chemicals that intercalate w/ DNA

biological mutagens include transposons that hop into DNA

physical mutagens include ultraviolet & ionizing radiation

point mutations

1) same site reversion: goes back to wild-type genome

2) second site reversion: corrects defect of 1st mutation (restores reading frame of frame-shift mutation)

mutant that suppresses mutant phenotype

1) nonsense suppressor: tRNA anticodon mutated to read stop codon

insertion sequence: minimal mobile genetic element

transposon: contains antibiotic resistance genes

transposase: enzyme that catalyzes transposition b/w recognition sequences that are often inverted repeats

*2 IS2 elements & transpose DNA b/w each & can evolve  into transposon that hops as unit*

-although bacteria reproduce asexually, gene exchange w/in lineages & across lineages occurs

-transformation: uptake of free DNA into cell

-transduction: phage-mediated DNA transfer

-conjugation: direct cell-to-cell transfer requiring cell-to-cell contact

-homologous recombination to recombine w/ genome

-site-specific recombination to insert into genome

-transposition hop into genome

-replicate independently of genome (ex plasmids)

exchange of DNA segments from 1 DNA molecule to another

-exchange of DNA b/w 2 homologous DNA sequences

1) nick 1 of DNA molecules (cut off 1 strand of double helix)

2) SSB--nicked strand detaches from other w/ help of proteins, especially single-stranded binding protein (SSB)

3) strand displacement--nicked strand displaces homologous region of recipient DNA

4) crossed-strand exchange--formation of crossed-strand structure

5) resolution--cutting of DNA strands to yield new DNA w/ heteroduplex regions--segments originating from diff. DNA molecules

TRANSFORMATION

competence (2 types)

competence: DNA that enters cells can be double-stranded, or be degraded to single-stranded as it enters cell

1) natural competence: only certain microbes have natural ability to take up foreign DNA; many others can be made to uptake DNA using artificial means (Haemophilus, Streptococcus, Bacillus)

2) artificially-induced competence: many bacteria can be induced to uptake plasmids by exposing them to certain growth conditions followed by chemical treatments; alternately, exposure to electric currents results in competence & uptake of plasmid DNA

TRANSDUCTION

2 types

1) generalized: random pieces of chromosomal DNA are transferred by bacterial virus particle

2) specialized: chromosomal segments adjacent to integrated phage DNA are transferred; only occurs @ certain sites in chromosomes

ex

-in E. coli, F (fertility) plasmid is involved in conjugation

-cells having F plasmid are designated F+

-F plasmid encodes pilus & other genes

-F+ cells donate DNA through sex pilus to F- cells

-F plasmid can exist in 2 states:
1) as separate plasmid

2) incorporated into cell genome

F plasmid is conjugative plasmid--codes for genes necessary for conjugation

high frequency of recombination

-strains have conjugative integration in genome, can transfer chromosomal genes & used for mapping

untranslated region: DNA transcribed to mRNA, but not translated (before START & after STOP codons)

-transcription & translation are coupled in bacteria & archaea, unlike eukaryotes that export mRNA out of nucleus for translation

regulation of enzyme activity: allosteric inhibition/feedback inhibition

define 'effector'

What does allosteric inhibition mean?

-common in biosynthetic enzymes

-effector: molecule having effect of regulation

-allosteric effector diff. than substrate & binds to diff. site, triggering conformational change that causes enzymatic inhibition

-provides means for cell to express functions appropriate for given environment

1) control activities of gene products--rapid alteration of pre-existing products (feedback inhibition)

2) control amounts of gene products--slower synthesis of new products & breakdown of old products @ transcriptional & translational levels

leucine zipper (prokaryotes)

zinc-finger proteins (eukaryotes)

ex glucose

uptake of glucose by E. coli requires 5 enzymes that sequentially transfer phosphate from phosphoenolpyruvate to transporter, which transports & phosphorylates glucose

REGULATION

some facts to know...

What are features of high amts of glucose?

What is cyclic AMP?

What is catabolite expression?

How does the process of induction of lac work?

high glucose: inhibits aden. cvc.

cyclic AMP: regulatory nucleotide synthesized by adenylate cyclase

catabolite expression: genes are not expressed when preferred carbon source is available

induction of lac: allolactase inactivates lac1 receptor & glucose is consumed & CPR becomes active

recognize specific promoters, recruit RNA polymerase, activate transcription

-expression of diff. sigma factors changes which genes are expressed & often to great extent

2-component signal transduction system

-sensor kinase

sensor kinase senses signal & becomes phosphorylated, transducing signal by acting as kinase, transferring phosphate to response regulator that functions as regulatory output

-output binds to DNA in phosphorylated form & inhibits transcription

-phosphatase activity removes phosphate to reset response regulator

non-coding RNAs are not translated to protein

-common type of regulatory RNA is antisense RNA, which bind to sense mRNA of gene & affecting expression

-small regulatory RNAs can also enhance translation & mRNA stability

attenuation: express Trp biosynthetic genes when Trp is needed

-significance of Trp cxn level! (2)

1) @ low cxns of tryptophan, ribosomes pause/stall @ Trp codons & transcription continues most of time & full-length mRNAs are synthesized

2) @ high cxns of Trp, ribosomes don't pause @ Trp codons in leader sequence & transcription stops by factor-independent termination

1) preparation of DNA

2) ligation: join DNA to cloning vector (often plasmid)

3) transform into cells

4) use for applications, like sequencing

CLONING

restriction endonuclease

enzyme that cleaves DNA @ specific recognition sites

-can recognize diff. # of bases

-can make staggered/blunt cuts

-base pairing of sticky ends is used to clone DNA w/ complementary ends

-used to amplify DNA frag. of interest

-very powerful method to isolate, amplify, & clone specific piece of DNA

-thermocycler: machine that rapidly cycles temp.

-Taq polymerase: thermostable DNA polymerase from Thermus aquiticus, isolated from Yellowstone

1) melt--denature DNA (95 deg)

2) melt for 30s

3) anneal primers--temp. depends on melting temp. of primers

4) polymerize

5) go to #2, repeat 25x

agarose: gelling agent, more pure form of agar

agarose gel: wafer of solid agarose containing running buffer, so it conducts electricity

loading buffer: added to nucleic acid so it's more dense than water & sinks into wells

running buffer: fills gel rig so it conducts electricity buffered, EDTA to inhibit nucleases

run to red: negatively-charged nucleic acid runs toward + cathode & away from - anode

screen to determine which clones have inserts

1) digest vector & insert DNA are digested w/ same compatible restriction enzyme; enzyme is heat inactivated or removed prior to next step

2) ligate: vector & insert are joined by DNA ligase

3) transformation: DNA is transformed into other strain

-genes of interest may be found by screening w/ DNA probe

-proteins of interest may be found by screening w/ protein probe, often antibodies

-probes detected by radioactive, chemiluminescent, or colormetric methods

1) library of plasmids are grown & replica plated

2) cells are lysed onto filter

3) filter probed

4) positive colonies revealed by probe label

DNA polymerization of template strand initiated by primer & base terminators that terminate DNA polymerization

-only fraction of bases are terminators, so terminator can be incorporated @ each instance of base

-dye termination: each terminator is labeled w/ diff. fluor. so each can be detected in 1 sample

food additives, fuel, solvents, fine chemicals

metabolite produced during primary phase of growth, usually byproduct of metabolism

ex alcohol

metabolites produced after cell growth & non-essential to growh & metabolism of cell

ex penicillin

-unpredictable--formation not consistent among all members of species

-non-essential for growth

-highly dependent on growth conditions

-producing strains can be manipulated to overproduce secondary metabolites in large quantities

1) air lift fermentors: air flow keeps culture mixed

2) solid state formation: growth w/o added water

3) fixed-bed reactor: microbes grow on porous solid surface

4) fluidized-bed reactor: microbes grow on surface of inert particles suspended in flowing growth medium

1) grow starter culture in liquid medium

2) sterilize solid growth substrate w/ steam

3) inoculate substrate

4) grow in uniform layers in sterilized trays

5) extract desired product from growth substrate

1) direct bacterial oxidation of copper

2) indirect oxidation of copper by chemical reaction w/ Fe⁺³ ions

-increased use of low grade copper ore contains <1% copper

-in most cases, copper exists as insoluble sulfides

-Cu⁺¹ compounds tend to be insoluble, whereas Cu⁺² are soluble

-insoluble forms converted to sulfate salts by leaching w/ dilute sulfuric acid

-if ore has low abundance of copper, then microbial leaching can increase yields

use of microorganisms for recovery of metal sulfides from ore

-also used for extraction of uranium & gold

-mercury is highly toxic--found in many man-made devices

-toxicity results from combination w/ methyl groups to make methyl- & dimethyl mercury & from combination w/ protein thiol groups

-some microbes have mechanisms of mercury resistance--often encoded on plasmid

mercuric ion reductase system--3 steps

1) mercury binds to MerP protein in periplasm

2) membrane-bound MerT protein transports mercury into cytoplasm

3) mercury is substrate for enzyme mercuric ion reductase & is reduced

synthetic chemical compound that doesn't naturally occur in nature

-in recent years, many microbes have been found that can degrade these compounds & produce product compounds that are less harmful

-complete degradation of xenobiotic can occur in single organism, or by combined metabolic activity of 2+ organisms

-complete conversion of xenobiotic compound into small, inorganic products is 'mineralization'

reductive:  halogenated organic compounds are used as terminal e- acceptors

-is of interest b/c most halogenated compounds are found in anoxic subsurface environments

oxidative: involves oxygen, often using it as growth substrate

-initiated by oxygenase enzyme followed by multistep degradation of compound

-individuals of same type multiply to form populations

-metabolically-related populations are guilds

-mixtures of guilds conducting complementary physiologically processes are communities

-microbial communities interact w/ macrorganisms & environment to constitute ecosystem

biological & chemical processes that result in interconversion of key elements

-life would cease to exist w/o microbial contribution to biogeochemical cycling

-microbial metabolism transforms nutrients

-biogeochemical cycles are interlinked & have global impacts

-bacteria & archaea are only organisms able to fix nitrogen

-fixation: brings N from atmosphere into living systems

-symbiotic bacteria: rhizobia & Frankia

-free-living: cyanobacteria, Azotobacteria

-anaerobic: Clostridia

-denitrification: takes N out of living systems into atmosphere

-another way nitrogen is lost to atmosphere

-carried out by Planctomycetes of bacteria // obligate anaerobes, lack peptidoglycan, contain membrane organelles

-50% of ammonia removed from marine sediments by anammox

where 1 species lives off byproducts of another species

ex fermentations yield E when hydrogen product is consumed

-primary producers: algae & cyanobacteria fix carbon dioxide (reduce it to sugar)

-detritus: byproduct of dead organisms

-anoxic sediments: metabolism based on most E yielding

-oligotrophic: carbon sparse

-eutrophic: carbon rich

lake stratification & turnover

-fall turnover: surface waters cool, becoming more dense & then mixing w/ bottom waters (when ice melts & lake mixes)

-anoxic zone: few metazoans

1) oxygen concentration decreases

2) respiring bacteria increase

3) ammonia, phosphate, then nitrate increase

4) algae & cyanobacteria bloom; some produce toxins in lakes, rivers, & coastal waters

5) oxygen & algae eventually return to close to pre-spill levels

grow @ high temperatures!

-piezotolerant: grow optimally @ 1atm, but still grows under pressure

-piezophile: grows optimally under pressure

-extreme piezophile: will ONLY grow under pressure