Genomics Technology to Genomics Technology to Assessing Microbial Activity in Assessing Microbial Activity in

the Environmentthe Environment

MICRO ARRAY TECHNOLOGYMICRO ARRAY TECHNOLOGY

• Array technology and how it works

• How it is likely to be applied and the implications

• What WILL be gained from its application

• Advantages and cost benefits

• Array technology and how it works

• How it is likely to be applied and the implications

• What WILL be gained from its application

• Advantages and cost benefits

Diverse Chemicals and Microorganisms

Diverse Chemicals and Microorganisms

• 10 x 106 Chemicals

– 8 x 106 Xenobiotic

– 1 x 106 Recalcitrant

• 0.4 x 106 traded at over 50 tonnes per year

• Toxicological/ biodegradative data on only around 5000-6000

• MICROORGANISMS IN COMPLEX COMMUNITIES IN THE ENVIRONMENT– Difficult to assess their effectiveness– Some cannot be cultured

• 10 x 106 Chemicals

– 8 x 106 Xenobiotic

– 1 x 106 Recalcitrant

• 0.4 x 106 traded at over 50 tonnes per year

• Toxicological/ biodegradative data on only around 5000-6000

• MICROORGANISMS IN COMPLEX COMMUNITIES IN THE ENVIRONMENT– Difficult to assess their effectiveness– Some cannot be cultured

Some soil numbers…

Mean particle Per gram

Soil type Diameter (mm) Volume (mm3) Number of Particles

Surface area (cm2)

Very coarse sand

2.00-1.00 4.18 x 101 9.0 x 101 1.1 x 101

Coarse sand 1.00-0.50 5.24 x 10-1 7.20 x 102 2.3 x 101

Medium sand 0.50-0.25 6.55 x 10-2 5.70 x 103 4.5 x 101

Fine sand 0.25-0.10 8.18 x 10-3 4.60 x 104 9.1 x 101

Very fine sand 0.10-0.05 5.24 x 10-4 7.22 x 105 2.27 x 102

Silt 0.05-0.002 6.50 x 10-5 5.78 x 106 4.54 x 102

Clay <0.002 4.20 x 10-9 9.03 x 1010 8.0 x 106

The ‘tree of life’rRNA sequence-basedphylogenetic tree

You are here…

Most of the biomass,and most of the diversity is here – there are many missing branches…

Assessing microbial diversity

• These are all nucleic acid (principally rRNA or rDNA) based methods. More direct, analytical methods (ie. FAME analysis / MS fingerprinting) can also be used to estimate diversity.• These address only diversity (“Who’s there?”) and not metabolic potential or activity – unless metabolic genes or their transcripts are targeted by gene probes etc.

Pure cultures

Amplified DNA

Nucleicacids

WholeCells

FISH /microarray

Molecularfingerprinting

Gene probe

DNAsequencing

Sequencedatabase

‘Evenness’(relative abundance

of each taxon)

‘Richness’(number of unique

taxa)

Microbial community

Clonelibrary

Overview of array technologyfor environmental samples

Overview of array technologyfor environmental samples

Slide or membrane base

Robotic arraymaker makesmany multigene arrays.

Environmentalsample: Extract totalDNA or RNA.

Use PCR to amplifygene copies (convert RNA to DNA with reversetranscriptase) and applyfluorescent dye label

Hybridise tocomplimentarysequence in array

Use laser fluorescencereader to scan slide.Two dyes can be used. One to target genes that identify the bacteria andthe other to detect activebiodegradation genes.

The genes present hybridse to their counterparts on the array and can be detected.

Preparation of a DNA MicroarrayPreparation of a DNA Microarray

DNA samples may be1. Oligonucleotides2. Clones3. Total DNA4. PCR products

Robotic armcompresses micro samplesonto slide

Silylated Slides have reactive aldehyde groups and covalently bind tissue, cells or single or double-strandedDNA directly to the glass surface of a high quality microscope slide via the Schiff base aldehyde-amine chemistry(lysine residues of proteins, primary amines of DNA bases, or via synthetic DNA bases bearing amino-modifications).

Sample Preparation and hybridisation Sample Preparation and hybridisation

Complex community of microorganisms

Extract total DNAOR RNA

Reverse Transcriptaseto give DNA

PCR to amplifytarget gene copies

Hybridise to complimentary DNA on the array and wash offexcess

Reading a DNA Microarray after hybridisation

Reading a DNA Microarray after hybridisation

Dual labelled fluorescencethat can be quantified

OR other chromogeniclabelling method

SLIDE PLACED INAUTOMATED LASERREADER

DATA LOGGED ON COMPUTER

INVOLVES APPLICATION OF ADVANCED DNA ARRAY TECHNOLOGY

• A ‘DNA microarray’

• 20,000 features, printed at 170µm pitch. Spot diameter approx 145µm.

• A different DNA ‘probe’ can be printed on each spot

ribosome

Bacterial SSU rRNA variability map; red = highly conserved

Primary structure(sequence) and secondary structure (folding, loops etc) dictate tertiary (3D) structure of ribosome

16S rDNA gene

• ~1500bp gene - encodes 16S (small subunit) rRNA molecule

• Parts of the sequence are conserved among all living things

• Central metabolism – inherited from the ‘last common ancestor’

• 40000+ sequences in the ‘Ribosomal Database Project’ DB

• Conserved ‘blocks’ can be exploited to ‘amplify’ the gene in vitro

• Variable regions are used for taxonomy – ‘phylogenetics’

27f primer

519r primer

Highly conserved sequence

Variable sequence

V1 V2 V3 V5V4 V7V6

How it is likely to be applied and the implications.

How it is likely to be applied and the implications.

• Large amount of gene sequence data – Estimated 10 years worth now added in 10 weeks!

• Gene expression studies– e.g. New drug targets sought

• Rapid detection of pathogenic bacteria and viruses from complex samples– Organisms detected AND their active genes

• Rapid detection of biodegrading bacteria in the environment• “NO SINGLE AREA OF BIOSCIENCES WITH REMAIN

UNAFFECTED BY THIS TECHNOLOGY” (Recent US government report)

• Large amount of gene sequence data – Estimated 10 years worth now added in 10 weeks!

• Gene expression studies– e.g. New drug targets sought

• Rapid detection of pathogenic bacteria and viruses from complex samples– Organisms detected AND their active genes

• Rapid detection of biodegrading bacteria in the environment• “NO SINGLE AREA OF BIOSCIENCES WITH REMAIN

UNAFFECTED BY THIS TECHNOLOGY” (Recent US government report)

What will be gained from its application?

What will be gained from its application?

• For the first time it will be possible to analyse a complex microbial population and its relative performance in a SINGLE step.

• The implications are enormous!• This will become the standard diagnostic tool• Legislation will follow on from this development• THE DESIGN AND SUITABILITY OF THE

ARRAYS WILL BE THE MAIN LIMITING STEP IN APPLICATIONS

• For the first time it will be possible to analyse a complex microbial population and its relative performance in a SINGLE step.

• The implications are enormous!• This will become the standard diagnostic tool• Legislation will follow on from this development• THE DESIGN AND SUITABILITY OF THE

ARRAYS WILL BE THE MAIN LIMITING STEP IN APPLICATIONS

Variability in dioxygenase genes in the Rhodococci (unpublished data)

STRAIN edoA edoB edoC edoD bphC-RHA1

Rhodococcus sp. Acr33 ++ (95%) ++ (99%) - ++ (98%) + (97%)

Rhodococcus sp. A1 + (90%) ++ (97%) - - + (96%)

Rhodococcus sp. B1 - ++ (92%) - - -

Rhodococcus sp. C1 - ++ (92%) - - + (97%)

Rhodococcus sp. E0 + (100%) ++ (98%) ++ (100%) + (97%)

Rhodococcus sp. E3 - ++ (98%) ++ (92%) + (97%)

Rhodococcus sp. I1 + (95%) ++ (100%) ++ (100%) ++ (100%) + (97%)

Rhodococcus sp. P5 - + (89%) - - -

Advantages and cost benefitsAdvantages and cost benefits• Revolutionises environmental analysis• Analysis of MANY organisms / genes handled in

single step. • DNA/RNA extracted on site can remain stable• High throughput back at the laboratory within one

day• Fluorescence methodology is sensitive and

quantitative• We can take advantage of our existing databases

and expertise

• Revolutionises environmental analysis• Analysis of MANY organisms / genes handled in

single step. • DNA/RNA extracted on site can remain stable• High throughput back at the laboratory within one

day• Fluorescence methodology is sensitive and

quantitative• We can take advantage of our existing databases

and expertise