from a single colony to million sequences - FMIPA UNJ

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

Avinash Sharma (PhD) [email protected]

Wellcome Trust-DBT India Alliance Fellow

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Source:www.microbiomesupport.eu

Microorganisms are everywhere

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What are they?

Microbes living where nothing else can

Why are they are interesting?

Medicine, Environment, Human Gut, Agriculture, Food etc

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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

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Woese and Fox, 1977

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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

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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

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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 O

₂

high O

₂

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 CH

₄

oxidizers, hydrocarbons etc. Soil, water etc.

Categories of Extremophiles

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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.

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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

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Unknown microbial diversity

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The Great Plate Count Anomaly

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• 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

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DNA sequencing technologies ideally should be 1. Fast

2. Accurate

3. Easy-to-operate 4. Cost effective



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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

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• 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

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Cost of sequencing technologies over the years

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I have enough of sequencing data ..Whats next ?

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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

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Microbial Community Structure and their survival strategies

Sample

ID Date Humidity

(%)

Overhead ozone

(DU)

Pressure (hPa)

Temperature (ºC)

Total radiation

(MJ m ^-2 )

Radiation UVA

(MJ m ^-2 )

radiation UVB

(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

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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

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Distribution of bacterial communities under the UVB radiation

Real time PCR based estimation of bacterial

biomass

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Functional study: abundance and distribution of genes

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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)

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Marisediminicola senii SM7_A14^T(MT084553) Marisediminicola antarcticaZS314^T(GQ496083) Glaciihabitans tibetensisMP203^T(KC256953)

Glaciihabitans arcticusRP-3-7^T(SISG01000001) Parafrigoribacterium mesophilumMSL-08^T(EF466126) Galbitalea soliKIS82-1^T(JX876866)

Yonghaparkia alkaliphilaKSL-113^T(DQ256087) Lysinibacter cavernaeCC5-806^T(KP411613)

Frigoribacterium faeni801^T(Y18807)

Frigoribacterium endophyticumEGI 6500707^T(KM114212) Frigoribacterium salinisoliLAM9155^T(KX094417) Compostimonas suwonensisSMC46^T(JN000316)

Aurantimicrobium minutumKNC^T(AP017457) Cryobacterium mesophilumMSL-15^T(EF466127)

Diaminobutyricibacter tongyongensisKIS66-7^T(JX876865) Labedella endophyticaEGI 6500705^T(KM095501)

Cryobacterium zongtaiiTMN-42^T(JX949938) Cryobacterium arcticumSK-1^T(GQ406814)

Cryobacterium psychrotoleransCGMCC 1.5382^T(jgi.1076200) Cryobacterium psychrotoleransCGMCC 1.5382^T(jgi.1076200) Frondihabitans australicusDSM 17894^T(RBKS01000001)

Frondihabitans peucedaniRS-15^T(FM998017) Frondihabitans sucicolaGRS42^T(JX876867) Frondihabitans cladoniiphilusCafT13^T(FN666417)

Subtercola lobariae9583b^T(KM924549) Subtercola frigoramansK265^T(AF224723) Subtercola vilaeDB165^T(MF276890) Planctomonas deserti13S1-3^T(MH287062)

Clavibacter sepedonicusATCC 33113^T(AM849034) Clavibacter capsiciPF008^T(CP012573)

Clavibacter michiganensissubsp.michiganensis VKM Ac-1403^T(jgi.1118350) Clavibacter tessellariusATCC 33566^T(MZMQ01000001)

Clavibacter insidiosusLMG 3663^T(MZMO01000001) Clavibacter nebraskensisNCPPB 2581^T(HE614873) Clavibacter michiganensissubsp. phaseoliLPPA 982^T(HE608962) Clavibacter michiganensissubsp. chilensisZUM3936^T(KF663872) Clavibacter michiganensissubsp. californiensisC55^T(KF663871) Mycetocola tolaasinivoransCM-05^T(AB012646)

Mycetocola saprophilusNRRL B-24119^T(JOEC01000010) Mycetocola reblochoniJCM 30549^T(RCUW01000025) Rathayibacter triticiDSM 7486^T(X77438)

Rathayibacter festucaeDSM 15932^T(CP028137) Rathayibacter rathayiVKM Ac-1601^T(OCNL01000027)

Rathayibacter iranicusVKM Ac-1602^T(jgi.1118354) Leucobacter komagataeJCM 9414^T(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 of strain SM7_A14 ^T with closest species belonging to the

genera 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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Genome wide phylogeny constructed based on whole genome sequences depicting the distinct positioning of strain SM7_A14 ^T 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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Source of Hot Water 3rd sampling site

2

^nd

Sampling site

4th sampling site

5

^th

sampling site 1

^st

sampling

site 92±1°C

88±1°C 90±1°C

90±1°C

Microbial Ecology Soldhar Hot Spring

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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

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17 isolates with 2 Genera

6 Genera in DGGE, 22 species in library construction

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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).

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Results:

Bacterial richness at different depths: Phyla distribution:

Beta diversity analysis Cultivable analysis

Phylogenetic analysis

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• 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 of marine habitats in relation to biogeochemical cycles.

• Strain SD111 ^T represents a novel species of the genus Domibacillus for which the name Domibacillus indicus sp.

nov. was proposed.

Conclusion

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