chapter 19 - environmental microbiology

Question 1

Inoculum

Answer 1

samples where microorganisms will be isolated

Question 2

Enrichment cultures

Answer 2

used to isolate bacteria from mixed cultures (feces/soil)

Question 3

What do enrichment cultures do?

Answer 3

favor growth of target organisms while inhibiting growth of non-target organisms

Question 4

Enrichment bias

Answer 4

microorganisms are often minor components of microbial ecosystem, quantity in lab is bigger than nature.

Question 5

What are the techniques to isolate microorganisms ?

Answer 5

1. Most probable-number technique
   - serial dilution x10
   - estimate # of microorganisms
2. Pure (axenic) cultures need to be verified
   - microscopy cell shape/size
   - test living conditions
3. Laser Tweezers
   - laser beam that moves through a lens
   - focus on cell and seperates it
   - isolates slow growing bacteria
4. flow cytometry: counting + examining method
   - suspended stream pass through electronic detector
   - separate based on shape, size, fluorescent properties.

Question 6

How can you cultivate uncultured microorganisms?

Answer 6

High-throughput Screening (HTS)
- separate one cell per well in microtitre plate.
- each cell can grow without any competition (used for slow growing species that thrive in nutrient poor environments)
- microfluidic platforms are used for cultivation

Question 7

How can microscopic analyses of microbial communities be done in culture-independent bacteria?

Answer 7

using non specific fluorescent dyes under UV light

Question 8

What dyes are used and what color do they have?

Answer 8

DAPI - blue
Acridine orange - orange
SYBR green - green

Question 9

How are viability stains done?

Answer 9

green dye used to enter all cells
red dye used to enter dead cells
- provides info on abundance and viability of cells

Question 10

What are fluorescent protein receptors used for?

Answer 10

• gene encodes fluorescent proteins controlled by the promoter.
• introduced into bacteria to then track live bacteria + bacterial processes (infections)

Question 11

How can molecular analysis of microbial communities be done in culture-independent bacteria?

Answer 11

through nucleic acid hybridization
- probe will have reporter molecules that will bind with complementary nucleic acids. This will form a duplex formation, which will reveal the unknown.

Question 12

Situ

Answer 12

in place; used to see microorgansims in communities.

Question 13

How does FISH study phylogenetic of microbial populations?

Answer 13

they label oligonucleotides complementary to rRNA, as it is highly specific to species. It can be modified to measure gene expression or translational activity.

Question 14

What is the PCR method of microbial community analysis?

Answer 14

1 - isolate DNA from environment samples using Polymerase Chains Reaction, amplification of specific genes.

Question 15

How can you analyze amplified genes?

Answer 15

1. molecular cloning
2. electrophoresis
3. restriction enzyme digestion
4. sequencing

Question 16

Phylogenetic analysis

Answer 16

massively parallel DNA sequencing

Question 17

What are the results of phylogenetic analysis?

Answer 17

1. rRNA sequences differ from lab ones
2. new phylogenetic distinct prokaryotes
3. fewer than 0.1% bacteria have been cultured (enrichment bias)

Question 18

Geochip

Answer 18

funcitonal gene microarray
- FGA (functional gene array) target genes in different functional processes
- valuable for assessing functional composition and structure of microbial communities

Question 19

Environmental multi-omics

Answer 19

provides integrated perspective to power discovery across multiple levels of biology

Question 20

How does environmental genomics occur? (metagenomics)

Answer 20

1- DNA is extracted
2- it is sequenced directly, without cloning
3- assembled and then annotated (partial genomes are assembled)

Question 21

What are the outcomes of environmental genomics?

Answer 21

1 - identification of gene categories
2- discovery of new genes
3- linking of genes to phylotypes

Question 22

How does metatranscriptomics happen?

Answer 22

1- DNA is extracted
2- Single gene is amplified
3- remove rRNA before sequencing (>90% of RNA is rRNA)

Question 23

What are the outcomes of metatranscriptomics?

Answer 23

1- reveals genes in community
2- reveals level of gene expression

Question 24

Metaproteomics

Answer 24

measures diversity and abundance of different proteins

Question 25

Metabolomics

Answer 25

comprehensive analysis of cellular and extracellular metabolites

Question 26

Microsensors

Answer 26

measure wide range of activity (pH, oxygen)

Question 27

Stable isotopes

Answer 27

element can have multiple nonradioactive stable isotopes

Question 28

Isotopic fractionation

Answer 28

biological reactions prefer lighter isotopes

Question 29

Examples of isotopic geochemistry

Answer 29

Petroleum: displays similar isotopic fractionation as plants

Question 30

Stable isotope probing

Answer 30

• feed microorganisms with substrate labelled with heavy isotope.
• heavier DNA separates by ultracentrifugation