Environmental microbiology Flashcards
what must microbes interact with
-each other and the environment
-Never get a microbe on its own, always present in a community
-We do not fully understand the nature of the interactions
Quorum sensing – just one type of interaction
what is interaction crucial for
microbial cell survival
where is most research taken upon
pure culture
what do individual cells reproduce to form
clonal populations of individual species
-Clonal populations mix and exchange genetic information with members of the same species and members of different species.
Transformation
Transduction
Conjugation
what do species interact to form
communities
what do communities assemble to form
ecosystems
what is the equation for ecosystems
organisms + environment + activities
what diseases can parasitism cause
plant, animal and microbe-microbe diseases
what does predation cause
Nematophagous fungi and Amoebae
what does mutualism cause
-N-fixation in legumes by Rhizobia
-Fungus-plant mycorrhizae
-Human gut microbiome
-Lichen (fungus-alga) symbiosis
what are examples of mixed cultures in nature
-GI tract
-Soil
-Plant roots (mycorrhizae)
-Teeth
what is involved in the traditional strategy for examining bacteria
-Enrichment
-Isolation in pure culture
-Identification= Biochemical tests, Microscopy and DNA sequencing
Relatively slow process
what is involved in the enrichment strategy
- samples include water, soil, human gut contents
-Enrichment medium formulated to support the growth of the desired organisms AND TO ALLOW SMALL NUMBERS TO BE AMPLIFIED
-Isolation medium formulated to support the growth of the desired organisms
what are the limitations to the enrichment strategy
-Many organisms cannot be cultivated so will not appear on the plates yet they may have an important role in the community
-Choice of growth conditions will influence results (bias) e.g. if the plates are incubated aerobically any obligate anaerobes will not be able to grow.
-the selection strategy will always exclude some organisms
what is a disadvantages to cultivation
bias
what does fluorescent staining use (DAPI)
DAPI- 4’,6-diamidino-2-phenylindole
What is the pros of fluorescent staining
-Stains every cell
-Stains dead cells= Excluded by viable cells
-Easy to see
-Every cell has DNA
what does FISH stand for
FISH= Fluorescent In Situ Hybridisation- This technique gives information on population distribution and community organisation
what does fish do
Permeabilize cells so that the DNA probe can enter Allow it to find its matching sequence on rRNA
- short DNA sequence
-complementary to rRNA
-specific sequence (eg. to genus)
-Florescent tag attached
what can we see in a microscope using FISH
-View cells (in situ) under fluorescent microscope, and see what cells fluoresce, showing they have bound the probe
-Fluorescent DNA probe will bind to rRNA gene in the cells only if it exactly matches complementary sequence of rRNA target region
-Many different coloured fluorophores, so can do simultaneous probes for different genera, families….
What are the strengths of FISH
-Information on physical structure of community
-Flexible format= Change probe to change overall analysis
what are the weaknesses of FISH
-Can be subjective interpretation= Same for all microscopy
-No high throughput
what can we detect when we detect DNA sequences
we can detect the species- 16S rRNA sequences most commonly used- Other targets possible
-This strategy can be applied along with some of our more traditional approaches
what is included in enrichment strategy and DNA sequencing (subculture is selected…)
-sample= water, soil and human gut contents
-Enrichment medium formulated to support the growth of the desired organisms AND TO ALLOW SMALL NUMBERS TO BE AMPLIFIED
-Isolation medium formulated to support the growth of the desired organisms
-Subculture the selected organism to obtain a pure culture.
Extract DNA
Sequence 16S rRNA gene
how do we obtain a DNA sequence (GEAPSD)
-Grow a pure culture, extract DNA, amplify the specific gene of interest (PCR) and sequence the amplicon
-Compare sequences with Database to find match
what is a limitation with the enrichment strategy and DNA sequencing
it is still biased towards culturable organisms
what does no cultivation mean
reduced bias but metagenomics
What is metagenomics (direct extraction…)
-Extract sequence data from microbial communities as they exist in nature
-Direct extraction of DNA from an environment
what does metagenomics bypass
Bypass the need for culture techniques
-Sequence all the DNA in sample
OR
-Select DNA of interest
Universal target sequences (16S rRNA)
Specific target sequences (huge variety possible)
what is the greatest challenge for studying metagenomes
-Metagenomes are big
-Soil has as many as 40,000 individual microbial species
-Soil metagenome orders of magnitude bigger than human genome
how are metagenomes analysed
-Screens for metabolic properties
-Phylogenetic studies
-Sequencing uncultivated organisms
-Studying metagenome under different conditions
what are the problems with metagenomics (purifcation complexity sequencing)
-Problems with DNA purification (Easy from sea/fresh water, Difficult from soil/solid matter)
-Sample complexity (Human gut procaryote metagenome, Human gut eucaryotic metagenome???)
-Issues with sequencing (Immensity of metagenome (gigabases), Errors in assembly due to inter-species similarities, Difficulties in sequencing less well-represented genomes)
what are the strengths of metagenomic sequencing strategies
-Technology has advanced making the process cheap, widely available and reliable
-Allows access to resources from uncultivated organisms- No cultivation bias
what are the weaknesses of metagenomic sequencing strategies
-Bioinformatic analysis has not kept pace with sequencing technology
-Have more data than we can currently process.
what is enrichment bias
Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias.
explain the marine metagenomics- case study
- J Craig Venter- 2 year voyage to sample oceans for metagenomic analaysis
-Bacterial discovery ing the Sargasso sea
1,800 species
148 new species
1.2 x 106 new genes
-Unexpected links between genetics & environment
Different rhodopsin proteins in open ocean vs coast line
