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Microbiology Final Prep!!! > Lecture 14 - 15 > Flashcards

Lecture 14 - 15 Flashcards

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1
Q

PCR-based methods of microbial community analysis

A

Major techniques include:

1) Polymerase chain reaction (PCR) or targeted genes (16S rRNA or metabolic marker genes)
2) DNA fragment analysis by gel electrophoresis (e.g. DGGE or T-RFLP)
3) DNA sequencing and analysis

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2
Q

Importance of 16S rRNA gene

A

-Phylogenetically informative and the techniques for their analysis are well developed.
Other reasons:
1) 16S rRNA genes are universally distributed among all cellular organisms
2) They contain regions of high sequence conservation, hence PCR primers can be designed that will amplify the gene from “all organisms”

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3
Q

PCR methods useful indicators for:

A

1) The presence of a microorganism

2) The metabolic potential, if an unknown sequence can be associated to a cultured bacteria of known physiology

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4
Q

PCR methods limitations

A
  • PCR amplification can be biased towards specific microorganisms
  • not all organisms with identical 16S rRNA gene sequences possess similar metabolic activities
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5
Q

Denaturing gradient gel electrophoresis (DGGE)

A

-PCR-based community fin

An approach that separates DNA fragments based on their base sequences (e.g. GC content)

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6
Q

Metagenomics

A

Cultivation-independent analysis of the collective genomes of microbial assemblages obtained directly from the environment.

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7
Q

Steps of the phylogenetic anchor approach:

A

1) Isolate DNA from an environmental sample
2) Clone large fragments of DNA (40 kb) into a suitable vector
3) Transform vector into a host bacterium (e.g. E. coli)
4) Screen the transformants for phylogenetic markers (e.g. 16S rRNA or recA) by PCR or hybridization
5) Completely sequence the DNA fragment linked to the phylogenetic marker

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8
Q

Bacteriorhodopsin

A

Light driven proton pump that halophilic archaea use to generate energy through ATP synthesis

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9
Q

Whole genome shotgun (WGS)

A

1) Isolate DNA from an environmental sample
2) Randomly sequences the DNA
3) Assemble the short clones into overlapping DNA contigs
4) Analyze the function of the genes to understand metabolic properties of the community

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10
Q

Sargasso Sea

A
  • Complex ecosystem
  • Intriguing observations:
    1) Genes for the use of phophonates in this extremely phosphate limited system
    2) Identification of a high diversity of rhodopsin genes, that could be linked to taxa that were previously not known to have light-harvesting functions
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11
Q

Microbial activity

A

What microbes are actually doing in their environment

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12
Q

Technique to measure microbial activities can:

A
  • Can provide bulk estimates of the physiological reactions occurring in the entire microbial community
  • Can assess the metabolic or physiological activities of specific, targeted populations
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13
Q

Examples of techniques to measure microbial activities

A

1) Fluorescent in situ hybridization-microautoradiography (FISH-MAR)
2) Stable Isotope probing (SIP)
3) Halogen in situ hybridization- secondary ion mass spectrometry (HISH-SIMS)

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14
Q

Radioactive isotopes

A

Unstable isotopes that breakdown due to radioactive decay. Useful for: measuring turnover rates of a compound of interest or tracking an element as it is metabolized by a community.

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15
Q

FISH - Microautoradiography (MAR)

A

Cells from a community are exposed to a radioisotope, then affixed to a microscope slide, dipped in a photographic emulsion, and exposed by precipitation of silver grains on film.

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16
Q

Stable isotopes

A

Not radioactive, but are metabolized differently by microorganisms and can be used to study microbial processes in nature.

  • Can yield information on microbial activities:
    1) Isotopic fractionation
    2) Stable isotope probing
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17
Q

Stable Isotope fractionation

A
  • Organisms exhibit this because enzymes typically favour the lighter isotope.
  • Since it’s typically the result of biological activity, can be used to measure whether or not a particular transformation was catalyzed by a microorganism.
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18
Q

Stable isotope probing (SIP)

A

Can be used to experimentally identify the active fraction of the community utilizing the supplied labeled substrate.

-Limitation: The substrate must be incorporated into biomass, and DNA specifically, for the metabolic activity to be detected.

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19
Q

Ecosystem

A

Dynamic complex of organismal (microbial, plant, animal) communities and their abiotic surroundings, all of which interact as a functional unit.

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20
Q

Habitat

A

Part of the ecosystem best suited to one or a few populations. Hence, an ecosystem can be comprised of many different habitats.

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21
Q

Species richness

A

Total number of different species present.

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22
Q

Species abundance

A

Proportion of each species in an ecosystem.

23
Q

Populations can be categorized as:

A
  • Metabolically related populations are referred to as Guilds
  • Sets of guilds interact in Microbial Community
  • Sets of interacting communities are defined as an Ecosystem
24
Q

Biogeochemical cycle

A

Defines the transformations of an element that are catalyzed by either biological or chemical means.

25
Q

Surfaces

A

Important microbial habitats:

1) Offer greater access to nutrients
2) Protect from predation and physicochemical disturbances
3) Cells can remain in a favourable habitat

26
Q

Biofilms

A

Assemblages of bacterial cells attached to a surface and enclosed in an adhesive matrix excreted by the cells (matrix = mixture of polysaccharides, proteins and nucleic acids)

27
Q

How do biofilms form?

A
  • Initiated by attachment of a cell to a surface followed by expression of biofilm-specific genes (e.g. polysaccharide secretion), and repression of genes for motility
  • Switch from planktonic to biofilm growth is triggered by production of cyclic di-guanosine GMP (c-di-GMP)
  • Intraspecies communications (quorum sensing) is critical in the development and maintenance of a biofilm (increased c-di-GMP levels)
  • Major intracellular signalling molecules: acyl homoserine lactones (AHLs)
28
Q

Why form biofilms?

A

1) Self-defence against phagocytosis by protists and immune cells
2) Biofilms resist physical forces that sweep away unattached cells, and penetration of toxins (e.g. antibiotics)
3) Allow cells to remain in a favourable niche (nutrient rich environment)
4) Allows bacterial cells to live in close association with one another, facilitates cell-cell communication and survival
* ** Important in industrial and medical settings and therefore receives a lot of research attention

29
Q

Microbial mats

A

Among the most visible microbial communities - can be considered as thicc biofilms

  • constructed by phototrophic and/or chemolithotrophic bacteria
  • Change dramatically within a 24 h period (diel cycle) : consequence of changing light intensity
30
Q

Phototrophic mats

A

Contain filamentous cyanobacteria

31
Q

Chemolithotrophic mats

A

Contain sulfur-oxidizing bacteria (SOBs)
-Grown of marine sediments at interface of O2 (electron acceptor) and H2S (electron donor) (produced by sulfate reducing anaerobes in the sediments)

32
Q

Soils

A

The loose outer material of Earth’s surface, distinct from the underlying bedrock
-Soil divided into two broad groups:
Mineral soils & Organic soils

33
Q

Mineral soils

A

Derived from rock weathering and other inorganic materials

34
Q

Organic soils

A

Derived from sedimentation in bogs and marshes

35
Q

Vegetated soils composition

A
  • Inorganic mineral matter (~40%)
  • Organic matter (~5%)
  • Air and water (~50%)
  • Living organisms (~5%)
36
Q

Freshwater environments

A
  • Highly variable in the resources and conditions available for microbial growth
  • Balance between photosynthesis and respiration controls the oxygen and carbon cycles
37
Q

Planktonic species

A

Suspended freely in water; include algae and cyanobacteria

38
Q

Benthic species

A

Attached to the bottom or sides of a lake or stream

39
Q

Biochemical Oxygen Demand (BOD)

A

Microbial oxygen-consuming capacity of a body of water

40
Q

Marine environment

A
  • Saline (around 35 PSU)
  • Low in nutrients (especially nitrogen, phosphorus, iron)
  • Cooler (deep ocean)
  • 70% of planet: Microbial activities taking place = major factors in Earth’s biogeochemical cycles
41
Q

Major phototrophs in the ocean

A

1) Prochlorococcus (single cell cyanobacterium)
- >40% of the biomass of marine phototrophs
- ~50% of the net primary production

2) Trichodesmium (planktonic filamentous cyanobacterium)
- Abundant phototroph in tropical and subtropical oceans (as nitrogen-fixing bacterium: critical role in nitrogen cycle)

3) Ostreococcus (small phototrophic eukaryotes)
- Inhabit coastal and marine waters

4) Aerobic anoxygenic phototrophs: Another class of marine microbes that use light energy but they DON’T fix carbon dioxide
- photoheterotroph
- light is used for ATP synthesis via photophosphorylation

42
Q

Pelagibacter

A

The most abundant marine heterotroph

  • One of the smallest bacteria
  • Example of oligotroph: organism that grows best at very low nutrient concentrations
  • They (and other marine heterotrophs) contain proteorhodopsin, a light driven proton pump that allows cells to use light energy to drive ATP synthesis: Light-mediated ATP production compensates for the low level of organic carbon
43
Q

Marine viruses

A
  • Most abundant biological entities in the oceans

- Through cell lysis: play major role in controlling prokaryotic populations in the ocean

44
Q

Oligotroph

A

Organism that grows best at very low nutrient concentrations

45
Q

Proteorhodopsin

A

Light driven proton pump that allows cells to use light energy to drive ATP synthesis

46
Q

Oxygen minimum zones (OMZs)

A
  • Unique microbial habitat in the sea
  • Regions within the oceans characterized by permanent low dissolved oxygen levels (< 20 uM)
  • High primary production in surface waters = transport of OM into deeper regions of water column
  • OM: Subjected to aerobic mineralization: depletes the O2 from water column
47
Q

Organisms that inhabit deep sea must deal with:

A
  • Low temperature
  • High pressure
  • Low nutrients levels
  • Absence of light energy
48
Q

Deep-sea microbes are:

A
  • Psychrophilic (cold-loving) or psychrotolerant

- Piezophilic (pressure-loving) or piezotolerant

49
Q

Hydrothermal vents

A
  • Thriving animal and microbial communities associated with these vents
  • Two types of vents:
    1) Warm diffuse vents
    2) Very hot vents
50
Q

Carbon reservoirs

A

Atmosphere, land, oceans, sediments, rocks, and biomass

51
Q

Carbon cycle

A
  • CO2 in atmosphere rapidly transferred to carbon reservoir
  • CO2 is fixed by photosynthetic land plants and marine microbes
  • CO2 is returned to the atmosphere by respiration as well as anthropogenic activities
  • Microbial decomposition is the largest source of CO2 released to the atmosphere
52
Q

Methane hydrate

A
  • Forms when high levels of methane are under high pressure and low temperature
  • Huge amount trapped underground
  • Can absorb and release methane
  • Fuels deep-sea vent ecosystems
53
Q

Photosynthesis

A
  • Drives light driven primary production
  • Oxygenic photosynthetic organisms use light to “split” or oxidize water

2H2O + light –> 4H+ + 4e- + O2

54
Q

Light reaction

A

The electrons are used to generate energy (ATP) and reducing power (NADPH), as well as a useful byproduct O2.

Microbiology Final Prep!!! (6 decks)

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