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
Microorganisms are everywhere Source:www.microbiomesupport.eu
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
Categories of Extremophiles Environmental factor Category Definition Major microbial habitat Temperature Hyperthermophile, Opt. growth at > 80 ° C Hot springs and vents, sub-surface. Thermophile < 15 ° C ice, deep-ocean, arctic Psychrophile Salinity Halophile 2-5M NaCl. Salt lakes, solar salterns, brines. Pressure Peizophile (Barophile) <1000atm Deep sea eg. Mariana Trench, sub- surface pH Low Acidophile pH < 2 acidic hot springs High Alkaliphile pH > 10 soda lakes, deserts Oxygen No Anaerobe (Anoxiphile) cannot tolerate O 2 sediments, sub-surface High high O 2 tention? sub-glacial lakes. Radiation Radioresistant Soil contaminated areas Toxic heavy metals Metallophiles tolerate heavy metals Contaminated areas Low nutrition Oligotrophs Lakes Inert substrates CH 4 oxidizers, hydrocarbons etc. Soil, water etc.
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
Sequencing technologies • 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
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
Evolution of Sequencing • 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
Cost of sequencing technologies over the years
I have enough of sequencing data ..Whats next ?
Strategies for Microbial Diversity Analysis Sample Community Direct collectio DNA cloning n Transformation Direct Sequencing using NGS Platform PCR Amplification Sequencin Metagenomic DNA g library Phylogenetic Trees DGG Structural and E Functional analysis Isolation of culturable Microbial Diversity Estimation microorganisms
Microbial Community Structure and their survival strategies Sample Date Humidity Overhead Pressure Temperature Total UVA UVB Wind ID ozone radiation Radiation radiation Speed (%) (hPa) (ºC) (DU) (MJ m -2 ) (MJ m -2 ) (MJ m -2 ) (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
Real time PCR based estimation of bacterial biomass Distribution of bacterial communities under the UVB radiation
Functional study: abundance and distribution of genes
Marisediminicola senii sp. nov. isolated from Queen Maud Land, Antarctica Scanning electron micrograph of strain SM7_A14 T . Strain SM7_A14T, isolated from the glacier fed sediment sample collected the Queen Maud Land, Antarctica (70 0 45’28” S, 11 0 37’36” E)
Marisediminicola senii SM7_A14 T (MT084553) 68 Marisediminicola antarctica ZS314 T (GQ496083) Glaciihabitans tibetensis MP203 T (KC256953) Glaciihabitans arcticus RP-3-7 T (SISG01000001) 91 Parafrigoribacterium mesophilum MSL-08 T (EF466126) Galbitalea soli KIS82-1 T (JX876866) 57 Yonghaparkia alkaliphila KSL-113 T (DQ256087) Lysinibacter cavernae CC5-806 T (KP411613) Frigoribacterium faeni 801 T (Y18807) Frigoribacterium endophyticum EGI 6500707 T (KM114212) 96 Frigoribacterium salinisoli LAM9155 T (KX094417) 100 Compostimonas suwonensis SMC46 T (JN000316) 64 Aurantimicrobium minutum KNC T (AP017457) Cryobacterium mesophilum MSL-15 T (EF466127) Reconstruction of phylogenetic tree based on 16S Diaminobutyricibacter tongyongensis KIS66-7 T (JX876865) Labedella endophytica EGI 6500705 T (KM095501) rRNA gene sequences using neighbour-joining 53 Cryobacterium zongtaii TMN-42 T (JX949938) 100 Cryobacterium arcticum SK-1 T (GQ406814) algorithm, depicting the position of strain Cryobacterium psychrotolerans CGMCC 1.5382 T (jgi.1076200) 60 SM7_A14 T with closest species belonging to the Cryobacterium psychrotolerans CGMCC 1.5382 T (jgi.1076200) 78 Frondihabitans australicus DSM 17894 T (RBKS01000001) 51 genera members of the family Frondihabitans peucedani RS-15 T (FM998017) 83 Frondihabitans sucicola GRS42 T (JX876867) Microbacteriaceae . Bootstrap values (expressed Frondihabitans cladoniiphilus CafT13 T (FN666417) 62 Subtercola lobariae 9583b T (KM924549) as percentages of 1000 replications) of above Subtercola frigoramans K265 T (AF224723) Subtercola vilae DB165 T (MF276890) 96 50% are shown at the branch points. Planctomonas deserti 13S1-3 T (MH287062) Clavibacter sepedonicus ATCC 33113 T (AM849034) Clavibacter capsici PF008 T (CP012573) Clavibacter michiganensis subsp. michiganensi s VKM Ac-1403 T (jgi.1118350) 100 Clavibacter tessellarius ATCC 33566 T (MZMQ01000001) 53 Clavibacter insidiosus LMG 3663 T (MZMO01000001) Clavibacter nebraskensis NCPPB 2581 T (HE614873) Clavibacter michiganensis subsp . phaseoli LPPA 982 T (HE608962) Clavibacter michiganensis subsp . chilensis ZUM3936 T (KF663872) Clavibacter michiganensis subsp . californiensis C55 T (KF663871) Mycetocola tolaasinivorans CM-05 T (AB012646) 100 Mycetocola saprophilus NRRL B-24119 T (JOEC01000010) 74 Mycetocola reblochoni JCM 30549 T (RCUW01000025) Rathayibacter tritici DSM 7486 T (X77438) Rathayibacter festucae DSM 15932 T (CP028137) 100 Rathayibacter rathayi VKM Ac-1601 T (OCNL01000027) Rathayibacter iranicus VKM Ac-1602 T (jgi.1118354) 69 Leucobacter komagatae JCM 9414 T (D45063) 0.005
Genome wide phylogeny constructed based on whole genome sequences depicting the distinct positioning of SM7_A14 T strain with members of the family Microbacteriaceae . Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at the branch points.
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