Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis Milano May 2004 Latest from Essex Strain

Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth,

Ken Timmis

Milano May 2004

Latest from Essex

•Strain database

•Quick recap and new information about isolates

- All isolates

- Bacillales

- Interface consortium

•Life in MgCl2

•Publication plans

http://www2.geo.unimib.it/BioDeep/Project.html

Database of Strains from BIODEEP

Some information missing from database

•% 16S rRNA similarity to closest cultivated relative

•Next relative with standing in nomenclature +

•Isolation medium

•Link to maintenance medium

Location of diff erent phylogenetic/physiological groups in the DHABs

Phylogenetic group AI AB AS BI BB BS UI UB US DI DB DS SW ? TotalArchaea - Halobacteriales 1 0 1 0 0 0 0 0 0 0 0 0 0 0 2Archaea - Methanosarcinales 0 0 4 0 0 0 0 0 0 0 0 0 0 0 4Firmicutes - Bacillales 2 0 42 1 0 42 0 2 29 7 1 31 1 0 158Firmicutes - Clostridiales 0 0 17 0 0 6 0 0 1 0 0 0 0 0 24Firmicutes - Lactobacillales 0 0 0 0 0 1 1 0 0 0 0 0 0 0 2 Proteobacteria 1 0 1 2 0 1 0 0 0 0 0 0 0 0 5 Proteo - unknown 0 0 0 0 0 1 1 0 0 0 0 0 0 0 2 Proteo - Alteromonadaceae 9 0 0 21 0 0 3 0 5 4 1 1 0 1 45 Proteo - Chromatiaceae 0 0 0 3 0 0 14 0 0 0 0 0 0 0 17 Proteo - Enterobacteriales 0 0 0 2 0 0 1 0 0 0 0 0 0 0 3 Proteo - Halomonadaceae 2 0 2 4 0 7 1 4 3 7 0 1 0 0 31 Proteo - Idiomarina 1 0 0 3 0 0 4 0 0 0 0 0 0 1 9 Proteo - Marinobacter-Alcanivorax 4 1 3 40 1 1 2 1 1 0 0 0 4 5 63 Proteo - Pseudomonadaceae 1 0 0 1 0 6 1 0 0 0 0 0 0 0 9 Proteo - Vibrionales 3 0 0 2 0 0 0 0 0 0 0 0 0 0 5 Proteobacteria 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1Actinobacteria 0 0 2 0 0 2 0 0 0 0 0 0 0 0 4Bacteroidetes 0 0 0 2 0 0 0 0 0 0 0 0 0 0 2Halanaerobiales 5 0 6 2 0 0 3 3 0 0 1 1 0 0 21Total 29 1 78 84 1 67 31 10 39 18 3 34 5 7 407

B - hypersaline brine: 15S - sediment: 218I - seawater-brine interface: 162

Paper 1Isolates



ASM Library

AS22US13

AN-BS10BAS2 and AS3

AN-BS5A

BS18Virgibacillus panthothenicus

AN-BS1BAN-AS13

Bacillus halodenitrificans

Gracilibacillus halotoleransBS16

BS17BS14

Halobacillus trueperiHalobacillus litoralis

LC16US16

Halobacillus halophilus

Bacillus halophilusAS28

LC39

Bacillus niacini

Bacillus jeotgaliBS29 (similar: US7)DS5

Bacillus sp. MB-5Bacillus benzoevoransBacillus firmus

US1

AN-BS8Bacillus cohniiBacillus horikoshii

„Bacillus aquaemaris“

Bacillus licheniformisBacillus subtilis

Bacillus pumilus

DS1US2 (=US4)

BS3

Bacillus marinusJeotgalibacillus alimentarius

„Bacillus aminovorans“

AS13

DS10

DS9

BS19

Bacillus sp. MK03

Bacillus simplex

AS12BS12

AS7

Bacillus pseudofirmus

Bacillus sp. OS-5Bacillus baekryungensis

AS5

DS16

Bacillus barbaricus

Bacillus alcalophilus

Bacillus sp. NRRLB-14851

BS25

BS6 (similar: AS6)

Bacillus decolorationisBacillus halodurans

AN-BS10A

Phylogenetic tree of Bacillales based on 16S rRNA sequences

Strains from the DHABs are distributed throughout the order Bacillales

A S 2

0 : 0 0

1 : 0 0

2 : 0 0

3 : 0 0

4 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

A S 3

0 : 0 0

1 : 0 0

2 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

A S 5

0 : 0 0

1 : 0 0

2 : 0 0

3 : 0 0

4 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

A S 6

0 : 0 0

1 : 0 0

2 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

A S 7

0 : 0 0

1 : 0 0

2 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

A S 1 2

0 : 0 0

1 : 0 0

2 : 0 0

3 : 0 0

0 5 1 0 1 5 2 0

% N a C l

td(h)

•25 strains studied in detail•23/25 strains grow at 12 oC•7/25 strains grow at 20% NaCl•2/25 strains grow anaerobically (by fermentation)

Salt tolerance of selected L’Atalante Isolates

Can they grow in situ?

0

0.1

0.2

0.3

0.4

0.5

0 100 200 300 400

Time (h)

O.D

.Series1

Series2

Series3

Series4

Series5

Series6

Series7

Series8

5% w/v NaCl

10%

15%

20%

22%

25%

27%

30%

Strain US13 doesn’t grow below 5% w/v NaCl

US13 (and related strains) is phenotypically similar to AS2 (and related

strains), e.g.:

•use as sole source of carbon and energy of:

n-acetyl-glucosamine

glycerol

trehalose

AS2AS3US13AS6BS6AS7BS3US2US4AS28BS29US7DS10DS9US1BS17BS18AS12BS12DS16AS5US16BS25DS1DS5

UPGMA analysis showing phenotypic diversity of 25 Bacillales strains (based on 108 tests)

Jaccard's Coefficient

0.4 0.52 0.64 0.76 0.88 10.28

AS22US13, US14

AN-BS10BAS2, AS3, AS4

AN-BS5A

BS18Virgibacillus pantothenticus

AN-BS1BAN-AS13

Bacillus halodenitrificans

Gracilibacillus halotoleransBS16

BS17BS14

Halobacillus trueperiHalobacillus litoralis

LC16US16

Halobacillus halophilus

Bacillus halophilusLC39

Anaerobic growth by fermentation

Strain NO3- NO2

- SO3- S2O3

2- MnIV fumarate DMSO TMAO

AS2, AS3, AS4 – + + – +/ – – + –

US13, US14, AS22 – + – +/ – +/ – + – +

Anaerobic growth by respiration

•Bacillus-like strains predominatly from sediments

•Most unable to grow under in-situ conditions

•New halophilic facultatively anaerobic strains have the potential to be active in situ

Bacillales Summary

Paper 2 Bacillales diversity

Paper 3Taxonomy of halophilic, anaerobic Bacillales



AN-BI4

AN-BI3A

Desulfovibrio vulgaris DSM 644T

Wolinella succinogenes ATCC 29543T

Helicobacter pylori 181

Thiomicrospira denitrificans DSM 1251T

Arcobacter nitrofrigilis DSM 7299T

Campylobacter jejuni SSI 5384-98

Sulfurospirillum arcachonense DSM 9755T

Chlorobium limicola DSM 245T

Sulfurospirillum barnesii DSM 10660T

Sulfurospirillum deleyianum DSM 6946T

Sulfurospirillum sp. strain 18.1

Uncultured hydrothermal vent bacterium C1_B008Uncultured hydrothermal vent bacterium P. palm A/C 22

Cytophaga fermentans ATCC 19072T

Marinilabilia salmonicolor DSM 6480T

Anaerophaga thermohalophila DSM 12881T

Rikenella microfusus ATCC 29728T

Empedobacter brevis ATCC 14234Chryseobacterium gleum ATCC 35910T

Tenacibaculum maritimum IFO 15946T

Flavobacterium aquatile ATCC 11947T

Cytophaga marinoflava ATCC19326T

Flexibacter aggregans ssp. catalaticus IFO 15977Bacteroides fragilis ATCC 25285T

Porphyromoas asaccharolytica ATCC 25260T

Sporocytophaga myxococcoides DSM 11118T

10 nucleotide substitutions per 100 bases Maximum likelihood 1298 bp.

Bacteroidetes

-Proteobacteria

10 m

10 m

AN-BI4 (Bacteroidetes)

AN-BI3A (-Proteobacteria)

•Isolated from the same enrichment from Bannock interface

•Anaerobic

•Moderate halophiles

•Equivalent tRFs in brine and interface samples

•Fermentative growth

•Organic acids as carbon and energy source

•Uses a variety of S-compounds as terminal electron acceptors

An Important Team in the Interface

Paper 4 Taxonomy / ecology of Bacteroidetes and -Proteobacteria

Solar Salterns on the shore of the Great Salt Lake

Halite crystals forming around lab-grown colonies of halococci (Sabine Castanier, University of Nantes)

Halite crystal forming around microbial cells in the Berre salt works (Sabine Castanier, University of Nantes)

Haloarchaea enhance halite precipitation

Mean water activity of brines at 25 C (+/- 2 s.e, n= 3)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Sea water Discovery l'Atalante Urania west Bannock

Sample

a w

Water activities of the brines

Gillion, J., McGenity, T.J and Hallsworth, J.E.

Activity of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides in the presence of different

concentrations of MgCl2 (♦), NaCl (■) (± se, n= 2)

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Total concentration of salts (M)

Pe

rce

nta

ge

of

hig

he

st

init

ial r

ate

of

rea

cti

on

(%

)

Gillion, J., McGenity, T.J and Hallsworth, J.E.

Indications of metabolic activity and growth:•Ectoenzyme activity in Discovery Basin (Marseille)•Methanogenesis in Discovery Basin (Marseille)•Sulphate reduction in Discovery Basin (Marseille)

Indications of the presence of cells/DNA (not necessarily signs of life):•DNA in Discovery Basin (Several)•Cells in Discovery Basin (Milano)

Indications of microbial survival in Discovery brine:•Isolates from Discovery Basin (Several)

Has anyone isolated RNA from Discovery Basin?

Life in Discovery hypersaline brine

Can organisms grow in 5 Molar MgCl2?

Strain Origin Rationale Phylogenetic group Next cultured relative

AN-AI3 AI I, inc DI; common; fac an Archaea - Haloarchaea Halorhabdus utahensis

DS 16 DS D Firmicutes - Bacillales Bacillus alcalophilus

DS 9 DS D Firmicutes - Bacillales Bacillus simplex

DI 1 DI I, inc DI Firmicutes - Bacillales Bacillus sp. 19500

DS 5 DS I, inc DI; common Firmicutes - Bacillales Bacillus sp. MB-5

DS 14 DS D Firmicutes - Bacillales Bacillus sp. MB-9

DS 1 DS D Firmicutes - Bacillales Bacillus sp. MN-003

DS 10 DS D Firmicutes - Bacillales Glacial ice bacterium G200-N5

AS 2 AS fac an Firmicutes - Bacillales Halobacillus salinus

US 13 US fac an Firmicutes - Bacillales Virgibacillus marismortui

AN-BS5A BS I, inc DI; common; an enrich Firmicutes - Bacillales Virgibacillus pantothenticus

AN-AS6E AS fac an; halotol Firmicutes - Clostridiales Thermohalobacter berrensis

AN-BI2D BI I; an enrich Alpha - Alpha proteobacterium GMDJE10F1

LC 25 DI I, inc DI; common Gamma - Alteromonadaceae Alteromonas macleodii

LC 7 DB I Gamma - Alteromonadaceae Pseudoalteromonas sp. A28

DS 8 DS common Gamma - Halomonadaceae Halomonas aquamarina

LC 26 BI I Gamma - Halomonadaceae Halomonas sp. MBIC2031

AN-BI1D BI I; an enrich Gamma - Idiomarina Slope strain DIII1c

AN-BI1B BI I; common; an enrich Gamma - Marino-Alcani Marinobacter hydrocarbonoclasticus

AN-BI7C BI I; an enrich Gamma - Marino-Alcani Gamma proteobacterium GMD16F03

AN-BI1C BI I; an enrich Gamma - Vibrionales Vibrio natriegens

Selection of Isolates to Test for Growth at Different MgCl2 Concentrations

Conc NaCl in non-buffered media (M)

Conc MgCl2 in non-buffered media

(M)

Strain 0 0.43 0.86 1.72 2.59 3.45 4.31 5.17 0 0.26 0.52 0.79 1.05 1.47

AN-BI1D - + + + + + - - + + - - - -

LC26 + + + + + + + - + + + + - -

DS8 + + + + + - - - + + + - - -

DI1 + + + + + + - - + + + + - -

DS9 + + + - - - - - + + - - - -

AN-BI1C - + + + - - - - - + - - - -

DS5 - + + + - - - - + + + - - -

AN-BI2D + + + + + - - - - - - - - -

LC25 + + + + - - - - + + - - - -

LC7 - + + + + - - - - + - - - -

AS2 - - - - - - - - + + - - - -

DS1 + + + + + - - - + + + + - -

AN-BS5A + + + + + + - - + + + + + -

US13 - - - + + + - - - - - - - -

AN-BI1B - + + + + + - - + + - - - -

AN-BI7C - + + + + + - - + + - - - -

DS16 - + + + - - - - - - - - - -

DS10 + + + - - - - - + + - - - -

10D + + + + + + - - + + + + - -

21D + + + + + + - - + + + + - -

Virgibacillus pantothenticus•All strains prefer NaCl•No growth beyond 1.05 M MgCl2

Growth of Isolates on Different Concentrations of NaCl and MgCl2

•But, these isolates had been pre-grown on NaCl-rich media

•Also, we had only a bulk sample of Discovery interface

•And so, obtained a dissection of Discovery interface and inoculated into MgCl2-rich media (November 2003 Cruise R/V Urania)

------32501.3989.1------29501.3939.2------24001.3849.3------20251.3772.1------19001.3752.2------17001.3702.3----?-14501.3652.4---?+?11501.3602.5---+++9001.3552.6---+++6001.3482.7--?+++~5001.3452.8---+++~4501.3442.9---+++~4501.3442.10

21011680126084042048

Mg2+ concentration in medium (mM)Approx. [MgCl2]

(mM)RI

Interfacefraction

Dis

cove

ry I

nte

rfac

e

------52001.38210.1------50501.37810.3------49251.3765.1------45751.3735.2------42251.3705.3----??38751.3675.4----++24501.3555.5---?++13001.3455.6---?++8251.3425.7--??++7001.3405.10

21011680126084042048

Mg2+ concentration in medium (mM)Approx. [NaCl] (mM)

RIInterface fraction

Ata

lan

te I

nte

rfac

e

Samples Inoculated into CPS medium (unbuffered)

RI = refractive index

- = no growth + = growth? = possible growth

1000 mM MgCl2 = 95.2 gl-1

•Growth only from the top of Discovery interface

•Discovery microbes grow in media with higher MgCl2 concentrations

Preliminary data indicate that there is a point beyond which microbes cannot grow (~1.5 M MgCl2), but:

•Only a restricted number of media tested

•This is MgCl2 not Discovery basin

•Microbes continue to surprise us

However, beyond this point life is extremely stressful

•Examine isolates

•tRFLP across interface

•Quantitative real-time PCR to look at distribution of key isolates

•Other?

Future Work

Paper 5 Why there is microbial life in Discovery brine, but 1 to 1.5 Molar MgCl2 seems to inhibit life

PhD Studentship at Essex

Documents

Terry McGenity, Andrea Sass, Gayle Harris, Jenny Gillion, Boyd McKew, Flavia Alves, John Hallsworth, Ken Timmis Milano May 2004 Latest from Essex Strain