Understanding Life in Extreme Environments; from a single colony to - - PowerPoint PPT Presentation

Understanding Life in Extreme Environments; from a single colony to million sequences

Avinash Sharma (PhD) avinash.nccs@gmail.com Wellcome Trust-DBT India Alliance Fellow

Source:www.microbiomesupport.eu

Microorganisms are everywhere

What are they? Microbes living where nothing else can Why are they are interesting? Medicine, Environment, Human Gut, Agriculture, Food etc

Why we need to study Extreme Environments

• Microorganisms represent the most important and

diverse group of organisms

• Widely distributed in many environmental habitats
• Important for ecosystems functioning
• Diversity and structure of complex microbial

communities still poorly understood

• Great challenge in microbial ecology to evaluate

microbial diversity in complex environments

Woese and Fox, 1977

Introduction to Extremophiles

What are they? Microbes living where nothing else can How do they survive? Why are they are interesting? Extremophiles are well know for their enzymes Why enzymes from extremophiles…? Stabilty even at extreme conditions

Life in Extreme Environments

• Many organisms adapt to extreme environments

– Thermophiles (liking heat) – Acidophiles (liking acidic environments) – Psychrophiles (liking cold) – Halophiles (liking salty environments)

• Demonstrates that life flourishes even in the

harshest of locations

Environmental factor Category Definition Major microbial habitat Temperature Hyperthermophile, Thermophile Psychrophile

• Opt. growth at > 80°C

< 15°C Hot springs and vents, sub-surface. ice, deep-ocean, arctic Salinity Halophile 2-5M NaCl. Salt lakes, solar salterns, brines. Pressure Peizophile (Barophile) <1000atm Deep sea eg. Mariana Trench, sub- surface pH Low High Acidophile Alkaliphile pH < 2 pH > 10 acidic hot springs soda lakes, deserts Oxygen No High Anaerobe (Anoxiphile) cannot tolerate O2 high O2 tention? sediments, sub-surface sub-glacial lakes. Radiation Radioresistant Soil contaminated areas Toxic heavy metals Metallophiles tolerate heavy metals Contaminated areas Low nutrition Oligotrophs Lakes Inert substrates CH4 oxidizers, hydrocarbons etc. Soil, water etc.

Categories of Extremophiles

Microbial Identification Methods

• Morphological and microscopic features

– Colony morphology, cell shape and size, staining etc.

• Biochemical features

– Catalase, Oxidase, Indole, Citrate, Urease, Sugar fermentation, etc.

• Molecular features

– Nucleic acids (DNA and RNA), fatty acids, proteins, etc.

16S rRNA Gene Sequencing

• Most common housekeeping genetic marker

used for a number of reasons

– Its presence in almost all bacteria – Large enough for informatics purposes ( ̴1500 bp) – No change in the function

• 1980 in the Approved Lists, 1,791 valid

names

• Today, this number has ballooned to >16000

Unknown microbial diversity

The Great Plate Count Anomaly

The Great Plate Count Anomaly

• First generation:
• Maxam- Gilbert method
• Sanger’s Dideoxy method
• Next Generation:
• Roche 454
• SOLiD by ABI
• Genome Analyzer/ Hiseq by Illumina
• Compact PGM Sequencer
• Ion Torrent
• Miseq by Illumina
• Third Generation:
• SMRT by Pacific Bioscience
• Nanopore by University of ILLINOIS

Sequencing technologies

DNA sequencing technologies ideally should be 1. Fast 2. Accurate 3. Easy-to-operate 4. Cost effective

   

DNA sequencing: Importance

• Basic blueprint for life; Aesthetics.
• Gene and protein

– Function – Structure – Evolution

• Genome-based diseases- “inborn errors of metabolism”

– Genetic disorders – Genetic predispositions to infection – Diagnostics – Therapies

• Remarkable improvement in sequencing

efficiency since inception

• The amount of sequencing that one person can

perform has increased dramatically – 1980: 0.1– 1 kb per year – 1985: 2–10 kb per year – 1990: 25–50 kb per year – 1996: 100–200 kb per year – 2000: 500–1,000 kb per year

• -2020: ~ 300-1000 Gb per day

Evolution of Sequencing

Cost of sequencing technologies over the years

I have enough of sequencing data ..Whats next ?

Strategies for Microbial Diversity Analysis

Sample collectio n Community DNA Direct cloning Transformation Metagenomic DNA library

Structural and Functional analysis

PCR Amplification

Phylogenetic Trees

Sequencin g DGG E Direct Sequencing using NGS Platform

Isolation of culturable microorganisms

Microbial Diversity Estimation

Microbial Community Structure and their survival strategies

Sample ID Date Humidity (%) Overhead

• zone

(DU) Pressure (hPa) Temperature (ºC) Total radiation

(MJ m-2)

UVA Radiation

(MJ m-2)

UVB radiation

(MJ m-2)

Wind Speed (m s-1) ST01 8-Jan-19 78.32 271.26 978.28 0.44 0.14 0.011 78.16 17.88 ST02 10-Jan-19 49.7 272.44 985.69 3.09 0.18 0.014 78.08 12.43 ST03 12-Jan-19 41.42 276.86 982.01 1.57 0.18 0.013 78.09 13.33 ST04 14-Jan-19 48.71 277.53 986.73 0.98 0.19 0.013 78.07 8.58 ST05 16-Jan-19 44.97 307.67 980.71 1.6 0.19 0.013 77.64 16.79 ST06 18-Jan-19 46.38 306.21 981.19 0.46 0.19 0.013 78.14 10.17 ST07 20-Jan-19 66.48 295.83 982.98 0.05 0.11 0.009 78.17 10.06 ST08 22-Jan-19 47.4 299.81 971.88 0.85 0.17 0.011 78.13 10.71 ST09 24-Jan-19 54.5 305.79 978.32

• 2.53

0.12 0.009 78.15 7.8 ST10 26-Jan-19 72.97 304.04 977.76

• 0.78

0.06 0.006 78.07 7.98

Assessment of physical parameters under temporal variation of UV radiation

Assessment of physical parameters under temporal variation of UV radiation

Sample ID Chao1 Observed ASVs Shannon ST01 1863 1863 7.28 ST02 1151 1151 6.76 ST03 1550 1550 7.13 ST04 1431 1431 6.89 ST05 1629 1629 7.06 ST06 1746 1746 7.19 ST07 1448 1448 6.97 ST08 1240 1240 6.90 ST09 1584 1584 7.08 ST10 1431 1431 7.03

Estimates of alpha diversity parameters

Distribution of bacterial communities under the UVB radiation

Real time PCR based estimation of bacterial biomass

Functional study: abundance and distribution of genes

Marisediminicola senii sp. nov. isolated from Queen Maud Land, Antarctica

Scanning electron micrograph of strain SM7_A14T.

Strain SM7_A14T, isolated from the glacier fed sediment sample collected the Queen Maud Land, Antarctica (70045’28” S, 11037’36” E)

Marisediminicola senii SM7_A14T (MT084553)

Marisediminicola antarctica ZS314T (GQ496083) Glaciihabitans tibetensis MP203T (KC256953) Glaciihabitans arcticus RP-3-7T (SISG01000001) Parafrigoribacterium mesophilum MSL-08T (EF466126) Galbitalea soli KIS82-1T (JX876866) Yonghaparkia alkaliphila KSL-113T (DQ256087) Lysinibacter cavernae CC5-806T (KP411613) Frigoribacterium faeni 801T (Y18807) Frigoribacterium endophyticum EGI 6500707T (KM114212) Frigoribacterium salinisoli LAM9155T (KX094417) Compostimonas suwonensis SMC46T (JN000316) Aurantimicrobium minutum KNCT (AP017457) Cryobacterium mesophilum MSL-15T (EF466127) Diaminobutyricibacter tongyongensis KIS66-7T (JX876865) Labedella endophytica EGI 6500705T (KM095501) Cryobacterium zongtaii TMN-42T (JX949938) Cryobacterium arcticum SK-1T (GQ406814) Cryobacterium psychrotolerans CGMCC 1.5382T (jgi.1076200) Cryobacterium psychrotolerans CGMCC 1.5382T (jgi.1076200) Frondihabitans australicus DSM 17894T (RBKS01000001) Frondihabitans peucedani RS-15T (FM998017) Frondihabitans sucicola GRS42T (JX876867) Frondihabitans cladoniiphilus CafT13T (FN666417) Subtercola lobariae 9583bT (KM924549) Subtercola frigoramans K265T (AF224723) Subtercola vilae DB165T (MF276890) Planctomonas deserti 13S1-3T (MH287062) Clavibacter sepedonicus ATCC 33113T (AM849034) Clavibacter capsici PF008T (CP012573) Clavibacter michiganensis subsp. michiganensis VKM Ac-1403T (jgi.1118350) Clavibacter tessellarius ATCC 33566T (MZMQ01000001) Clavibacter insidiosus LMG 3663T (MZMO01000001) Clavibacter nebraskensis NCPPB 2581T (HE614873) Clavibacter michiganensis subsp. phaseoli LPPA 982T (HE608962) Clavibacter michiganensis subsp. chilensis ZUM3936T (KF663872) Clavibacter michiganensis subsp. californiensis C55T (KF663871) Mycetocola tolaasinivorans CM-05T (AB012646) Mycetocola saprophilus NRRL B-24119T (JOEC01000010) Mycetocola reblochoni JCM 30549T (RCUW01000025) Rathayibacter tritici DSM 7486T (X77438) Rathayibacter festucae DSM 15932T (CP028137) Rathayibacter rathayi VKM Ac-1601T (OCNL01000027) Rathayibacter iranicus VKM Ac-1602T (jgi.1118354) Leucobacter komagatae JCM 9414T (D45063)

100 100 53 100 69 100 78 100 96 60 74 62 51 83 96 91 64 53 68 57 0.005

Reconstruction of phylogenetic tree based on 16S rRNA gene sequences using neighbour-joining algorithm, depicting the position

• f

strain SM7_A14T with closest species belonging to the genera members

• f

the family

• Microbacteriaceae. Bootstrap values (expressed

as percentages

• f 1000 replications) of above

50% are shown at the branch points.

Genome wide phylogeny constructed based on whole genome sequences depicting the distinct positioning of strain SM7_A14T with members

• f

the family Microbacteriaceae. Bootstrap values (expressed as percentages

• f

1000 replications) of above 50% are shown at the branch points.

Source of Hot Water 3rd sampling site 2nd Sampling site 4th sampling site 5th sampling site 1st sampling site 92±1°C 88±1°C 90±1°C 90±1°C 90±1°C

Microbial Ecology Soldhar Hot Spring

Geography of the Spring

• Longitude 79° 39’ 29”
• Latitude 39° 29’ 25”
• Altitude 1900m amsl
• Surrounding temperature during sampling

min -2° C, max 8° C

• Interesting hydrogeology as the entire region has an array of small hot springs

17 isolates with 2 Genera 6 Genera in DGGE, 22 species in library construction

Sampling:

Agatti Island (10° 52' 47.32"N, 72° 10'11.86"E) is surrounded by land on northern side which making it unique geographic location as it is distinguished from the extend of northern low temperature zones. Sediment samples from various depths viz 1 meter to 40 meter of the continental shelf were collected. Microbial community structure was analyzed by targeting V3 region of the 16S rRNA gene on Illumina MiSeq platform (2x150bp).

Results:

Bacterial richness at different depths: Phyla distribution: Beta diversity analysis Cultivable analysis Phylogenetic analysis

• The

continental shelf harbors a wide diversity. (Differences in UCS and LCS)

• Understanding the widespread bacterial diversity of the

marine environment, can serve as an elementary data to several future multi-omics studies aiming to understand the ecology

• f

marine habitats in relation to biogeochemical cycles.

• Strain SD111T represents a novel species of the genus

Domibacillus for which the name Domibacillus indicus sp.

• nov. was proposed.

Conclusion

Number of shared (50%) and unique genera obtained from samples processed onsite and laboratory.

Two Approaches

• 1. Onsite Cultivation
• 2. Cultivation in laboratory after

transportation of samples (after 2 days) Total 449 isolates were obtained

Kajale et al., 2020

Extremophiles-Research Group

Acknowledgements

• Director NCCS, Pune
• Dr. Yogesh Shouche
• Dr. Kunal Jani
• Mr. Swapnil Kajale
• Master Students
• DBT, ICMR and MoES

“All our dreams can come true, if we have the courage to pursue them”

Walt Disney

Thank you for attention