exguiobacterium cold adaption

8/9/2019 Exguiobacterium cold adaption

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Debora Frigi Rodrigues

Mentor: Dr. James Tiedje


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Outline

Importance of studying cold-adaptedmicroorganisms

Distribution and diversity of two cold-adaptedgenera on our planet

Characterization and identification of species

from a cold-adapted genus

Architecture of thermal adaptation in cold-adapted species


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Why study cold adapted organisms?

Ecology: nutrient cycling, greenhouse gasproduction and consumption

Biotechnology: industry, foods,bioremediation (new enzymes)

Evolution: microbial life a million years ago

Astrobiology: strategies for life on

cryoplanets; biomarkers for life detection


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Russia &Canada

55%

Cold temperatures on Earth

Alaska 85%

China 20%

l80% of Earths

biosphere is

permanently

cold

l20% of Earths

land is

permafrost

lPermafrost:

Soil, sand,sediment at or

below 0C for 2

or more years

Map provided by NSDIC


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Low temperature environments

~80% ofthe Earths

surface!

Deep Sea T95% of ocean

volume

Glaciers T= 0 to - 40C10% of land surfaceJLM Visuals

Seasonal Ice and Snow

3% of Earths surface 2005 AECOM

Permafrost T= 0 to -14C20% of land surface

Sea Ice T= - 2 to - 35C13% of Earths surface
http://www.noaa.gov/


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Kolyma Lowland, Siberia


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Why study the Siberian permafrost?

Cold temperatures, but stable

-10 to -12C

Low water activity

aw = 0.90

Low nutrients

frozen matrix

Radiation

0.03 Mrad/ year for 3 M years

Because of lots of environmental

stresses, such as

Depth

below

ground

Dr. Rivkina


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natural freezer

samples remained frozen

during transport

cores were stored at -20oC

in the labslow rotary drill

no solutions ordrilling mud

permafrost cores remain

frozen and uncontaminated

surface of extracted cores

was trimmed away

cores were divided into

sections and placed in pre-sterilized aluminum cans

Kolyma Lowland, Northeast Eurasia

Cold arid arctic climate

Mean annual air temperature of -14oC

Upper soil layers thaw and freeze

each year

Sampling soil in the permafrost


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Bacterial colonies grown from permafrost

Dr. Hinsa


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Isolates characterized from Arctic permafrost

Soil

oldest Growth

# of isolates age -2.5C

Actinobacteria

Arthrobacter (17) 3m 15:17

Microbacteriaceae (5) 3m 5:5

FirmicutesExiguobacterium (4) 3m 4:4

Planococcus (5) 30k 3:5

Bacteroidetes

Flavobacterium (4) 30k 1:4

Proteobacteria

- Psychrobacter (4) 30k 3:4 - Sphingomonas (2) 3m 2:2

Blue = Gram positive; Red = Gram negative; m= million

years; k= thousand years


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Psychrobacter arcticusPsychrobacter arcticus 273-4273-4

Psychrobactergenus

Gram negative

Gamma Proteobacteria

Predominantly isolated from

cold or salty environments

Shallow and deep marine

water

Sea ice

Salty Foods

Intestine of fish

Grow at temperatures down to

-10oC

Dr. Ponder


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Exiguobacterium genus

Gram-positive

Found in a variety of

environments:

Antarctica, Siberianpermafrost

Alkaline environments

Food processing effluents

Hot springs Defining characteristics of

genus

Peritrichous flagella

Facultatively anaerobic

Exiguobacterium sibiricumExiguobacterium sibiricum 255-15255-15


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Work design

Identification of genes related to

cold and heat acclimation in

Exiguobacterium 255-15

-Genome analysis-Transcriptome analysis

Characterization and Identification

ofExiguobacterium isolates from

Siberian permafrost

- Polyphasic approach

- 54 soil/sediment DNA

extraction from: Michigan, Iowa,Brazil, Puerto Rico, Hawaii,

Siberia and Antarctica-Q-PCR and 16S rRNA clone

libraries-Computational analyses of

Biodiversity

Biogeography, diversity and

abundance ofExiguobacterium

and Psychrobactergenus


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Siberia

Antarctica

Brazil

Puerto RicoHawaii

Iowa and Michigan

Samples for my study


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Quantitative

real-time PCR

with SYBRgreen

The more DNA target, the

more fluorescence is

seen

SYBR green dye attaches

only in double strandDNA and fluoresce.


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1 2 3 4 5 6 7 1 2 3 4 5 61 2 3 4 5 6 7 1 2 3 4 5 6 7

8

1 2 3 4 5 6 7 8 9101121314151618192021222324

Puerto Rico Brazil Michigan Antarctica Siberian permafrostIowa

H

awaii

Samples


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Why patchy distribution?

Environmental factors? pH, salinity, organicmatter, micro and macronutrients

Principal component analysis (PCA):

Look at relationships between variables

Group large data set into small artificial variables

(PC) that will account for most variance in thelarge data set

Goal: Reduce redundancy in those variables =

correlate with one another


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Principal component analysis of the

different samples

C l ti bi l t f h i


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Correlation bi-plot of physico-

chemical and Q-PCR resultsVariables (axes F1 and F2: 76.60 %)

Salinity

Moisture

OM pH

CuCEC

Ca

Mg

K

Exiguobacterium

Psychrobacter

-1

-0.75

-0.5

-0.25

0

0.25

0.5

0.75

1

-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1

F1 (60.81 %)

F2(15.8

0%


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1 2 3 4 5 6 7 1 2 3 4 5 61 2 3 4 5 6 7 1 2 3 4 5 6 7

8

1 2 3 4 5 6 7 8 9101121314151618192021222324

Puerto Rico Brazil Michigan Antarctica Siberian permafrostIowa

H

awaii

Samples


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Samples for clone librariesCave system

LasCarmelitas

Mangrove LasCabezas de San

Juan

KBS Mid-successionalcommunity

Comandante Ferraz marine sediment

Botany point marinesediment

MSU BakerWoodlot

Sites studied forPsychrobacter spp.

Sites studied forExiguobacterium spp.

Cl lib t ti d l i


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Clone library construction and analysisSequence each cloneto identify OTUs(OperationalTaxonomic Unit,i.e.species)

7 OTUs

4 OTUs

2 OTUs

Measure Diversity

Bacterialcommunity

DNA extractionGene of interestamplified by PCR withspecific primer

PCR product

Insert PCR productinto plasmids

Clone the plasmidinto E.colicells

Select cloneswith plasmids

Richness and evenness

Find closest

relative


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Diversity concepts

Richness: number of

species per sample

Evenness: the frequency

of different speciesoccurrence

Community 1 Community 2

4 species

or OTUs

2 species

or OTUs

Community 1 is richerthan community 2

A community dominated by 1 or 2 species is considered to be less diversethan one in which several different species have a similar abundance

Community 1 Community 2

= 1 individual

= 9 individuals= 6 individuals= 3 individuals

= 5 individuals

= 5 individuals

Community 1 has less evenness than

community 2

E.aurantiacum


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Exiguobacterium spp.

clone library results

70%

80%

90%

100%

bu

ndance

C4

D2

D5

G4

C7

B3

H6

E. indicum K22-26

Michigan A04

Exiguobacterium sp. 8N

E.homiense

E.aestuarii strain TF-16

E.lactigenes

Exiguobacterium sp. A1

E.taiwanense

Exiguobacterium sp. India orange

Exiguobacterium sp. M37Exiguobacterium sp. AT4

Exiguobacterium sp. AT1b

Antarctica C04

E.marinum strain TF-80

Antarctica E12

Antarctica D10

Antarctica B08

Antarctica C05a

Antarctica D02

Antarctica B12Antarctica D11

Antarctica G05

E.acetylicum

E.sibiricum strain 255-15

Exiguobacterium sp. strain 5138

Exiguobacterium sp. India Stream

E.sibiricum strain 7-3

Antarctica D05

Puerto Rico E07

Puerto Rico B12E.oxidotolerans

Puerto Rico H06

Puerto Rico G06

E.undae

E.undae strain 190-11

Antarctica G04

Michigan C07

E.antarcticum

Michigan B03

B.subtilis

8090

81

96

56

70

98

72

47

66

91

65

7899

52

51

66

52

64

54

45

57

0.005

P. fozii


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Psychrobacter

spp. clonelibrary results

40%

50%

60%

70%

80%

90%

100%

n

ofOTU

sabun

dance

e

rcentage)

H3a

H2

G10

H10

C9

H11

A12

F7

B4

E12

B3

Michigan H11

P. okhotskensis

P. luti LMG 21276

Puerto Rico G03

Michigan H02

Puerto Rico H02

P. arcticus 273-4

Puerto Rico B03Puerto Rico A07

P. glacialis DD43

P. cryohalolentis K5

Antarctica G10

Michigan E07

Antarctica C07

Antarctica H02

Antarctica E07P. cibarius JG-220

Michigan C02

P. immobilis DSM 7229T

Antarctica H10

Michigan A12

Puerto Rico E12

Michigan F07

Antarctica H03a

Antarctica H03

P. faecalis OS-38

P. pulmonis CECT 5989T

Psychrobacter sp. ikaite

Puerto Rico D03

Michigan C09

Puerto Rico B04

Moraxella atlantae

89

87

98

49

73

74

68

87

76

52

6087

6769

53

46

49

0.01


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Diversity analysis Measuring diversity within a community

Shannon index (H)Shannon index (H)

Accounts for both richness (number of species) and evenness(proportion) of the species. The index increases either byhaving more unique species, or by having a greater speciesevenness. More sensitive for impact ofrare species. Samplingsensitive.

Simpson index (D)Simpson index (D)Accounts for both richness and evenness of the species. D variesfrom 0 to 1. When (D) is close to 0, the diversity is high. Moresensitive for impact ofcommon/abundant species, less for rarespecies. Sampling insensitive.

Chao1 estimatorChao1 estimator

Chao1 index, a nonparametric estimator suitable for microbialdiversity analysis. It estimate the richness of a community.


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Results Diversity

Samples Genus studied Total No ofsequences

No. OfOTUs

ShannonIndex

Chao 1estimator

SimpsonIndex(D)

Ant. Psychrobacter 129 21 2.60 21.43 0.09MI Psychrobacter 150 14 2.22 17.00 0.13

PR Psychrobacter 137 11 1.84 11.50 0.21

Ant. Exiguobacterium 149 7 1.53 7 0.26

MI Exiguobacterium 109 4 0.34 5 0.84

PR Exiguobacterium 135 1 0 1 1

Ant.= Antarctica; MI= Michigan; PR= Puerto Rico


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Conclusions

Exiguobacterium is more widely distributed onour planet, but less diverse thanPsychrobacter

NeitherPsychrobacternorExiguobacteriumare cosmopolitan due to environmentalfactors: salinity, pH and Cu

Both genera seems to preferentially inhabitpolar regions


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Work design







Siberian permafrost






Biodiversity




E i b t iE i b t i t i f Sib it i f Sib i


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ExiguobacteriumExiguobacterium strains from Siberianstrains from Siberian

permafrostpermafrost

E. sibiricum 7-3

pleiromorphic

rods. Borehole

6Kh-Yu (1989),

8 m

E. undae 190-11

rods. Borehole

5/94 (1994), 5.5 m

E. sibiricum 255-15,

motile short rods.

Borehole 2/94 (1994),

43.6 m

~250miles

Qu

ate

rna

ry

T

ert

iary

Ho

locen

e

Pleisto

ce

ne

P

liocen

e

20-30 tds

200-600 tds

2-3Mi

7-3

190-11

255-15


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Polyphasic approach

* Phenotypic analyses: Chemotaxonomic markers

Metabolic characteristics

FAME Cell morphology at different temperatures

* Genotypic analyses:

rep- PCR DNA- DNA percent similarity

Multi-loci sequence typing


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Summary of phenotypic resultsStrains Peptidoglycan Quinones Polar Lipids Carbon

source

FAMES

255-15

7-3

190-11

E. undae

E. antarcticum

E. acetylicum

E. aurantiacum

A3 L-Lys-Gly MK7 majorMajor:PG,DPG ,PE

The same symbol means very similar/identical traits


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Summary of genotypic clusters

The same symbols means very similar trait or clustered strains

Strains BOX-PCR DNA-DNAsimilarity

16SrRNA

rpoB recA hsp70 gyrB citC

255-15

7-3

190-11 E. undae

E. antarcticum

E. acetylicum

E. aurantiacum


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Conclusions

The permafrost isolate 190-11 (200 K) is

the same species as E. undae DSM 14481T

Type strain is isolated from warmer

environment (Germany)

The permafrost isolates 255-15 (2-3 Mi)

and 7-3 (20-40 K) are the same species

even though they have different cellmorphology


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Work design







Siberian permafrost






Biodiversity





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Genome analysis on

Exiguobacterium sibiricum 255-15

General features

2.9 Megabase genome

47.8 % GC content

2,978 candidate ORFs

138 Major contigs 65 Minor contigs


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Natalia Ivanova, Lawrence Berkeley National Labs


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E x i g u o b a c t e r i u mb ip h a s ic A r rh e n i

S t ra in 2 5 5 - 1 5

R2

= 0 . 9 8

R2

= 0 . 9 4

-5

-4

-3

-2

-1

0

1

2

3

3 .1 3 .2 3 .3 3 .4 3 .5 3 .6 3 .7

1 0 0 0 / T (oK )

LNg

en/h

r

Exiguobacterium sibiricum

biphasic arrhenius plot

-2.5oC39oC 28oC 10oC


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6 biological

replicates

0.1 O.D.

600 0.3

Experimental design

Dye swaps

TechnicalReplicates:

Indirect labelling

100mL for RNA

extraction

39oC 28oC 10oC -2.5oC

cy3 cy5 cy3 cy5 cy3 cy5 cy3 cy5

6 hybridizations for each comparison


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Direct comparisons of the samples

39oC 28oC

10oC -2.5oC

70 mer probes

Duplicate spots and negative controls: 10 Human and10 Arabidopsis 2931 gene-specific probes 6 group-specific probes for 25 ORFs Data analysis by limma within CarmaWeb environment

Differential expression when P-value < 0.01


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Conclusions

Exiguobacterium is adapted to coldtemperatures: not many differences ingene expression at 4oC, 10oC and 28oC.

Genes believed to be expressed underheat- and cold-shock conditions are also

expressed under continuous growth at thehigher extremity of the arrhenius profileand at subzero temperatures, respectively.


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Take home messages

Microorganisms successful in the poles arenot confined to the poles

Environmental factors affect presence anddiversity of microorganisms

Several molecular and physiologicaladaptations seem to be necessary forsurvival and growth at the higher extremity ofgrowth range as well as at subzero

temperatures

ACKNOWLEGEMENTS
http://images.google.com/imgres?imgurl=http://www.pha.jhu.edu/~france/PICTURES/NASA_logo.GIF&imgrefurl=http://www.pha.jhu.edu/~france/pubs.html&h=782&w=876&sz=73&tbnid=XXCZ_CUU_VofwM:&tbnh=129&tbnw=145&hl=en&start=8&prev=/images%3Fq%3DNASA%2Blogo%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3DN


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ACKNOWLEGEMENTS

My Committee members:

Dr.Tiedje

Dr.Thomashow

Dr. Kathariou

Dr.Bagdasarian

Dr.Britton

Especial thanks:

Dr. Goris Dr. Ramette Dr. Gilichinsky Dr. Pellizari

Dr. Ivannova Dr. Zhou Dr. He Dr. Gantner

Chia-Ju Lin Ederson Jesus

All tiedje lab My friends

Astrobiology group:

Dr.Ayala-del-Ro Dr. Bergholz

Dr. Bakermans

Dr. Hinsa
http://images.google.com/imgres?imgurl=http://www.pha.jhu.edu/~france/PICTURES/NASA_logo.GIF&imgrefurl=http://www.pha.jhu.edu/~france/pubs.html&h=782&w=876&sz=73&tbnid=XXCZ_CUU_VofwM:&tbnh=129&tbnw=145&hl=en&start=8&prev=/images%3Fq%3DNASA%2Blogo%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3DN

exguiobacterium cold adaption

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