Marine Actinomycetes Flashcards
What are actinobacteria?
Actinobacteria are a group of Gram-positive bacteria with high guanine and cytosine content in their DNA, which can be terrestrial or aquatic.
They are considered as the biotechnologically valuable bacteria that are exploited for its secondary metabolite production. Approximately, 10,000 bioactive metabolites are produced by Actinobacteria, which is 45% of all bioactive microbial metabolites discovered.
Whihc strains of actinobacteria are considered particulay valuble?
Especially Streptomyces striains produce industrially important microorganisms as they are a rich source of several useful bioactive natural products with potential applications.
- Account for almost half of all microbial bioactive compounds with nearly 80% of them produced by
- Streptomyces* strains.
Draw a flow chart of finding new natural products.
Dereplication technology needs to be important
First 4 steps give you a selection of novel actinobacteria – then the screening process can start
The current bottleneck is finding the new molecules – deep sea
Coupling between taxonomic diversity and chemical diversity. If you look for taxonomically unusual microbes to find the unusual chemicals
Why Focus on Actinobacterial Diversity from Extreme Habitats?
- Isolation of novel actinobacteria from extreme biomes rests on the premise that harsh environmental conditions give rise to unique taxa (species, genera) with novel chemistry.
- Evidence of a coupling between taxonomic and chemical diversity means that taxonomic diversity can be used as a surrogate for chemical diversity.
- Screening common actinobacteria from well studied habitats leads to the costly rediscovery of known bioactive compounds
What is high throughput screening?
High-throughput screening (HTS) is a method of scientific experimentation that comprises the screening of large compound libraries for activity against biological targets via the use of automation, miniaturized assays, and large-scale data analysis.
Using robotics, data processing/control software, liquid handling devices, and sensitive detectors, high-throughput screening allows a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the noninteraction or role of a particular location.
What taxonomic methods are required to find drugs focusing on rare and previously unknown taxa, including novel species of
Streptomyces.
Key taxonomic methods to meet these aims:
-
Choice of selective isolation procedures.
- Selection medium – get the bugs in colonies form the enviroment
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Selection of cultivation media
- Cultavation medium – to grow the bacterium (may be expansive medium)
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Comparative sequencing of conserved genes / proteins, notably 16S rRNA genes.
- Production medium has to be cost effective – so a cheaper food source
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Choice of production media.
- Deep sea bacteria – only grow the bacterias that can survive at surface pressure / get the gene for a protien and grow it in ecol
What is selective isoation media?
Growing ytarget organisms on a speciifc slelevctive agent, which contains the best food source for that bacterium alongside other selective agents which disadvantages other competeing strains , such as adding an antibiotic that the strain is resistant too.
As an example of a selective medium, what are the medium an dselective media used to grow Streptomyces?
Media
Starch-casein agar
Selective media
High carbon to nitrogen ratio
Target strains
Streptomyces spp.
Wha are some methods used in Dereplication?
Chemical: fatty acid analyses, pyrolysis and MALDI-TOF mass spectrometry (type of anaysis of protiens).
Molecular: rep-PCR, genus specific primers, 16S rRNA and whole-genome sequencing (becoming cheap to do).
Colour and morphology, similar strains will have siilar colours and apprearences.
Conclusions
Actinobacteria are a rich source of novel bioactive metabolites
They are difficult to grow – but targeting of unusual / unstudied habitats is a fruitful strategy
Dereplication is a critically important step
Novel drug targets being developed constantly
Recently being explored as a source of other biotechnologically useful molecules (not just antibiotics)
Actinomycetes for Marine Drug Discovery Isolated from Mangrove Soils and Plants in China
Study
Actinomycetes for Marine Drug Discovery Isolated from Mangrove Soils and Plants in China
(Hong et al, 2009)
Study
A study looking for actinomycetes in mangrove soil and plant samples from 8 sites around China. Within the 1000 actinomycetes isolates 20 % were found to inhibit the growth of Human Colon Tumor 116 cells. An average of 9.3 strains per sample were collected, with the highest numbers occurring in rhizome soil samples.
Actinomycetes for Marine Drug Discovery Isolated from Mangrove Soils and Plants in China
Results
Actinomycetes for Marine Drug Discovery Isolated from Mangrove Soils and Plants in China
(Hong et al, 2009)
Results
They have also been found to restrict the bacteria Candida albicans and Staphylococcus aureus and a protein related to diabetes.
Over 2,000 actinomycetes were isolated and of these approximately 20%, 5%, and 10% inhibited the growth of Human Colon Tumor 116 cells, Candida albicans and Staphylococcus aureus, respectively, while 3% inhibited protein tyrosine phosphatase 1B (PTP1B), a protein related to diabetes.
(actinomycetes isolated from mangrove habitats are a potentially rich source for the discovery of anti-infection and anti-tumour compounds, and of agents for treating neurodegenerative diseases and diabetes)
Mangrove forests may become an invaluable source for discovering novel actinomycetes hence new chemo diversity should salinity prove to be a driver of bacterial diversity as proposed by Lozupone and Knight
Selective isolation of actinomycetes from environmental samples
mangrove
Air dried soil samples were sieved to exclude large mineral and organic matter particles then ground in a pestle and mortar.
Selective pretreatments of the soil samples included dry heat (120 °C, 60 min)
Dilutions of soil and plant tissues were plated out onto one or more of 11 different selective isolation media.
The inoculated plates were incubated at 28 °C for one up to twelve weeks.
Colonies of streptomycete-like and non-streptomycete-like strains growing on the isolation plates were inoculated onto plates.
4.5. Identification of isolates
(recognition of novel taxa and dereplication)
(DNA - chemical structure?)
- The presumptive streptomycetes were inoculated onto oatmeal agar plates
- Extraction of DNA from pure cultures and PCR amplification
- Total genomic DNA samples from 243 representative strains were extracted with the Fast DNA®SPIN Kit used for extracting community DNA from soil
