Monika Kern fl Microbial Diversity course MB!,, Woods Hole 1991 Isolation of Heliobacteriaceae The Heliobacteriaceae are anoxygenic photothrophic bacteria. In 1983 Howard Gest first isolated Heliobacterium chiorum from surface soil and until now only two species Heliobacterium and Heliobacillus are known. The Heliobacteriaceae are distinguished from all other anoxygenic phototrophs because off several aspects: - they contain bacterichlorophyll g with an absorption maximum at 790nm, which differs from various bacteriochlorophylls from green and purple bacteria (vinyl group on ring I of tertrapyrrole like in plant chl a; pyrrol ring II is reduced as in bcl a and b; as esterfying alcohol bchl g contains farnesol which also occurs in the bchl of green sulfur bacteria; hel g shares structural relationship of chl a and bchl), — Heliobacteria seem to be primary soil bacteria, — their green colour is due to the presence of bchl g and the green carotenoid neurosporene, — heliobateria lack differentiated structures like chlorosomes or intracytoplasmic membranes, the photopigment systems are located in the cytoplasmic membrane, — heliobacteria are the only anoxigenic phototrophic bacteria which can font endospores resitent to high temperature and dryness. This year during the Amherst Symposium on Photosynthesis the existence of the endospores could first be demonstrated. Photoheterotrophic growth only occurs under strict anaerobic conditions. They are able to fix molecular nitrogen. Until now heliobacteria have only been isolated from soil especially from paddy soil (Thailand, Tanzania) and they were found to withstand dryness much better than other phototrophic bacteria. They have not yet been isolated from aquatic habitats. Unlike purple nonsulfur bacteria only few carbonsources are photometabolized. Pyruvate is known to be the best substrate, besides pyruvate lactate, acetate (+ bicarbonate), butyrate and malate can be used. Methods For selective enrichment of Heliobacteria dry surface soils (Swope ground, garden soil, forest soil) as well as freshwater from a pond near the bikepath to Falmouth were used. Because Heliobacteria can form heat resistance endospores all samples were pasteurized (15 mm, 80°C) before inoculation of enrichment cultures. All experiments were performed under anaerobic conditions (in the anaerobic C Media hood). heat bacteria were Heliobacteriacea, MAM Pyruvate temperatures MAN—N: MAN: NaHCO 3 For Mixed vitamines, phosphates After — — after were electron - light garden garden sodium—lactate sodium—malate sodium—acetate — Enrichment Concentrated Swope sample 1mM Swope (lg/l) MIS: PMS—N: AMS—N: PMS: enrichment contains cystein treated used + sterilized autoclaving: acid intensity. ground autoclaving ground without Cultures pyruvate without as NH4C1 soil soil (lg/l) mineral without MIS enrichment sources: nitrogen (see mineral were NaHC0 3 cultures was pasteurized (no without tend samples. pasteurized a stock (lg/l) (no and 2g/l pyruvate, mixture cultures appendix). ammonia, lg/ lg/ were heat) separately autoclaved added (2g/l) Because salt ammonia, heat) NH4C1 to In: and all 1 1 source, salts solutions cultures for discourage incubated ammonia, medium media The cystein as as of media N2 the (lg/l), high media contains a N2 Prokaryotes carbon organic reducing and separately, as were isolation were as of N2 media NH4+ were according N1i4+ nitrogen P145, P148-N, P145, P145-N, light at added growth nitrogen the and cooled as 35°C also acetate acids MIS, MIS, or or filtersterilized. nitrogen intensities agent. following MIS—N, electron to P345-N, N2 N2 of 2nd and of carbon inoculated source to MAN MAN under the as Heliobacteracea source purple edition, (2.7g/l) Madigan at carbon MAN-N MAN—N basal plus plus source about source, nitrogen sources, components and and with medium and M.T. plus in high 8000 green NH4C1 press not and The lux forest forest slurry measurements was freshwater Results around No oxygen. Identification For

After sucrose probably 350 probably could growth some may had Rhodomicrobium garden some multiplies bacterium fresh of abundant References the — — —

(Desulfotomaculum).

Madigan,

Gest Brockmann,

growth

algae.

detecting

added. -

outcompeted

explain light

enrichments of

media be 1000

one

pasteurized soil

soil

790

of

soil

et

To

the

(final

due observed due purple

week After of purple which

microscop.

al.

nm). and avoid rim.

M.

by

pasteurized

enrichment

(no

it

its

(not

were

Heliobacteria

to to

N.;

heliobacterial

budding T.

(1985)

of fresh flchl

two 60%)

vannielii. seemed

heat)

Bchl

the growth

two the non

survival

is

exposure

pasteurized, non

inoculated

Lipinski

In:

within heliobacterial

(measurements

enrichments able

g

and growth

and

algae. sulfur

water

g

sulfur

The

Ferns It

is

that

and

cultures in of

as

a to

is

very

the

Prokaryotes,

in

vivo to

half endospore Rhodomicrobium Microbiol A. cells

a

of the bacteria in

It

form the

with

bacteria

dry

growth 02 reducing

anaerobic

(1983)

PMS,

NH4C1 PMS—N, PMS,

sensitive media: any Clostridia. turned

which shows week

purple

only

high samples of

can soils. exospore

growth

pateurized

of

ANS,

ANS or

spectra,

or

spectra

Arch. purple and forming

Ecol. easily in

AXS—N,

an the MIS-N, were gas

accompained out

N2

agent non

2nd

N2 MAN

chamber

this

MAN

towards

absorption for

several

production

enrichment

that vannielii

like

H2S

Microbiol. sulfur edition

31, inoculated

plus

non

identified

MAN—N

plus

were

microscopy).

spectral MAX—N)

0.05% samples sulfate enrichments

production

the

cysts

317—322

mixed

sulfur

damage

transfers

run

by bacteria

most ascorbate

(in

maximum

showed

was

reducers cultures growth

occured,

and

with

with

(range

press) under

by

was

this

In

in

to cultures

than

eliminates

overgrow

heliobacteria:

After

cysteine,

MEDIUM

LNM4C1

CaCl2.2HO

enrichments

Medium

Distilled

Ethylenediaminetetraacetate—Na—salt MgS04.7H20— MODIFICATIONS

1C2HP04

MEDIUM

Trace Sodium

Yeast Vitamin

Adjust

12-IS

PHS—N

MIS employing.N2

cooling

heliobacteria

This

1

interfernce

=

—

- I

or

element

extract

—

—

2 pyruvate

flIS

and

N2:C0 ig PYRUVATh

PMS pH

enrichment

12

0.9g water

thiosulfate

PMS

briefly containing -

—

to

minus

2

minus

MiXED

200mg 75mg

—

OF

minus have

6.B

— solution

20

(95:5) — as

—

MEDIUM

in O.lg

MINERAL

1 from

pyruvate

with pg

pyruvate ACID add sole 2.2g

been

liter medium

pyruvate—based NIi4C1.

ammonia,, and

to

fermentative

NaOI{

nitrogen MINERAL

PMS —

successfully

SALTS

final transfer

1

avoids

plus

plus

-

ml

Beadspace

or

but

-

14C1

—12—

concentration

SALTS (pus)

2OmN

20mM

source

the

-

to the

enrichments.

and

—

anaerobes

sodium

employed

sodium - anaerobic use (HAMS)

10mg

of

-

use

sterilize

usually

enrichment

of

of

lactate -

acetate

pyruvate

of

N2

which

for

chamber develop

1mM generally

-

by

-

enrichment

-

occasionally should

and autoclaving

either

and

until

more

0.1%

- Pa-ic1

employs

be

methionine,

-

-

slowly

NaHCO3]

cul€ure

used.

- 20

a

-

mm.

variety

of

J}-L

-

- •

of

•

• •

•

cooled,

final •

transfer

hflgba

organic

jtthylenediaminetetraacetate—Na—salt—lOmg

Lj(112P04 ¶jH4Cl NaCl

‘HgSO4.71120

MODIFICATIONS iYeast fNaRCO3—lg Distilled

/Trace taCl2.2110

MEDIUM

Vitamin

Sodium

Sodium

Adjust Sodium

Distilled

concentration

add

—

HANS—N

teria

to acids

—

elements

extract

0.4g

3

—

—

malate acetate This

lactate 0.8 p11 NaIICO3

anaerobic E17—ZO

—

— O.45g

water O.3g

PYRUVATE—TEAST —

—

but

to

sa ig

=

water

200mg

medium 75mg

OF HANS

be

6.8

— potential

—

is from

—

I

—

—

of HANS

0.2g N2:C0

2g

ml

I

ig lg

unsuitable — and Ig

minus chamber

luft1

liter

is

a 1

sterilize liter filter

suitable

either

(95:5)

NN4C1. photoheterotrophic

EXTRACt

until

for

sterilized

methionine,

for

by

—13— used. Neadspace

enrichment

(pm)

autoclaving

growth -

?iEDIUN

stock -

cysteine,

of -

of

substrates. -

enrichment

pure

solution

20

mm.

cultures

or

and thiosulfate

When

should

add

of

partially

to

and Adjust

dissolved

stoppered scrth—capped aseptically

TRACE

‘- then r VNicl2.6uo ‘-7eCl2.4110 V

4fnCl2.4110

1,/CuCl2.2H0

LNa2WO4.2110 HgSO4.7H20

CaC12.2H0 Na2S03.5H0—

Yeast Sodium

Distilled Ethylenediaminetetraacetate—Na—salt—5.2g

ZnC12

}13B0

CoC12.6H0—

VoSO4.2H20

Add

Growth

Growth

to

sealed

ELEMENTS

p1-

compounds

extract

— —

before

pyruvate

culture

7,

Vessels 70mg

of 6mg

with

tubes with water

— —20mg

autoclave

—

—

pure —

—

-

200mg

—

adding 1.5g

100mg

— 190mg 17mg — 25rng

sterile 30mg

100mg

tubes

stoppers 2mg in

—

or 8g

—

cultures

188mg

2.2g

1

the

bottles.

0CLL—’ -

liter,

remaining -.

and (Bellco - -

prereduced

above - - - --

or -

store

:

of -

screw - -

/t5a

order; - -.

heliobacteria

anaerobic Sterile - -

components. -

in

V03

—14—

media

taps

anaerobic

make - -. empty

before

culture within

2C1Th&

sure

can vessels

Store

chamber -.

removal.

that -

the

be

tubes, -

at

accomplished

anaerobic

the

can

until

4°C.

18

EDTA

be

Screw

x -

filled

used.

142mm)

is

chamber

cap

fully

in -

tubes

rubber

or

and

in

or nioni252 0.200 .---1 --.——-I -- No. Wavelength (nm.) Abs. 965.00 0.026 2 857 .00 0.119 3 802.00 0.085 4 591.00 0.059 5 512.00 0.100 6 479.50 0.114 6

A K b 0.100 3 S

4.

- 1.

0.000— . I • I • I • I 350.0 675.0 1000 .0 Wavelength (nm.) Created: 10: 32 07/27/91 Data: Original I IOp’n Scan Speed: Fast Slit Width: 1.0 enrichment25

PhadomicyosQean, tann,g 0/ C 0 0 C) Microbial Monika MBL, )naerobic TMA trimethylantine respiration TMAO: TMAO TMA—assays compounds TMAO bacteria have example TI4AO nitrogen. phosphorylation. the concentration Anaerobic several marine parahaemolvticus, to electron anaerobic TMA bacteria thvphimurium. methane equivalents TMA acceptor involves or electron TMAO—reductases as Some production bound Organisms can Shewanella in The gain carbon—, C—source. the wet and and be Woods and to reduction is so methanogenes Trimethylamine Trimethylamine TMAO—reductase Kern, periplasm. detected fish mainly marine used weight. ATP face called TMAO TMAO from TMA especially was an acceptor acceptor like Diversity growth are growth In respiration like Hole in species of during to electron nitrogen— are Juergen high are their as TMA. oxidation found is very general Rhodobacter TMAO in alkaline N—oxide contributes TMAO fish facilitate E.coli 1991 a It oxidized also by in waste of TMAO fish of TMAO osmolyte during some and The e.g. abundant has natural anaerobic general in in an and bioluminescent those reducers excrete several N-oxide Breitung transport which able are tissue with general neither can e.g.: and ability or increase products as concentrations. been Methanosarcina Pseudomonas TMA. R.sphaeroides anaerobic R.sphaeroides by a which used capsulatus, to fermentation also energy—source. to by habitats TMAO are is terminal TMAO in bacteria. observed E.coli; some TMAO growth can is oxidize the some THAO of is involved marine to be membrane system of grow of frequently as to use methylotrophs only reductases organisms odor used evaluate the bacteria protein marine growth. terminal species, nor marine eliminate by some electron TMAO that in TMAO. contain environments. used H. and pH species of For processes. TMA as electron posses in fish bound purple sphaeroides; of Zooplancton TMAO organisms spoiling as enables a TMAO metabolism. more TMAO bioluminescent example fish to as is with Salmonella media sink electron from tissues a molybdenum. acceptor: excess used reduce a or can a terminal facilitates respiration. and transport than freshness. membrane reduction terminal nonsulfur of E.coli, localized produce bacteria due TMAO fish as is THA reducing and often TMAO 1% and used Vibrio to a These TMAO for and N— of the The activity of molybdenum dependent enzymes can specifically inhibited by tungsten. Tungsten competitively inhibits molybdenum transport and incorporation of molybdenum in protein. Anaerobic respiration systems in bioluminescent bacteria so far are only poorly investigated. Some luminescent Alteromonas species can use nitrate as a terminal electron acceptor during anaerobic growth. TMAO is a very important compound in marine environments. The idea of our project was to look if luminescent bacteria as an adaptation to their natural environment are able of anaerobic TMAO respiration. Material and Methods

Samples: marine water samples from Garbage beach and MBL beach, fish gut.

We tried to detect TMAO reduction in media with pH indicator dyes because the product TMA is highly alkaline. We looked for anaerobic growth in liquid media in the presence and absence of TMAO and compared growth (measurement of the optical density at 540 mu) and pH changes under different culture conditions.

Mo dependence and specific inhibition of TMAO reduction by WO was analyzed. Media

1. SWC (for enrichments)

80% glycerol 3 ml/l yeast 3 gIl tryptone 5 g/l agar 15 g/l filtered seawater 700 ml distilled water 350 ml, pH 7.5 2. PLBS distilled water 950 ml 1M Tris’HCl, pH 7.6 50 ml tryptone 10 gIl Yeast 5 gIl 80 % glycerol 3.75 ml NaCl 20 g/l - add 15 g agar/l if desired - after autoclaving add 1 ml of filter sterilized FAC (ferric ammonium citrate 28 mg/l)

PLBS-HT: PLBS with 5 mM TrisHCl, 30mM TMAO 0 PLBS-LT: PLBS with 5 mM TrisHCl, 1 g/1 triptone, 0.5 g/l YE PH indicators: Cresol red 0.025% final concentration. Thymol blue 0.075% final concentration.

Influence of No and Wo on TMAO reduction

Mo (final concentration 100 jtM) or Wo (final concentration 10 mM) or No + Wo were added to liquid cultures (PLBS—HT). Growth conditions

Aerobic growth: SWC or PLBS plates at room temperature. Anaerobic growth: PLBS—HT and PLBS-LT plates in the anaerobic chamber, or liquid cultures (PLBS and PLBS-HT) in screw cap tubes gassed with Ar. C without Table Vibrio Photobac. FG11 FG1O FG9 FG2 FG1 20 fischerii FG8 FG7 FG6 leiognathi FG5 FG4 Isolate FG3 14 4 kESULTS 12 11 10 3 2 19 18 17 9 8 1 16 15 7 5 6 1. TMAO. Anaerobic 5 5 5 5 5 5 5 5 5 pH 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 - 5 (-) TMAO = growth no growth. 0.273 0.242 0.97 0.41 0.47 0.395 0.39 0.4 0.42 0.38 0.49 0.4 0.545 0.32 0.38 0.36 0.45 0.95 0.72 0.5 0.5 0.24 0.24 0.42 0.5 0.64 1.26 0.52 0.48 0.58 0.71 0.73 of luminescent 8 7 7 7 7 7 8 7 7 8 7.5 7 7 7 pH 7 7 7 7 7 7 >7 7 7 7 7 7.5 + 7 8 8 7.5 6 8 ThAO bacteria — 0.54 — — — — — 0.62 — — — — — — — — — — 0.78 0.8 — — — — — — — — — — 0.82 0.73 with and 0 0 0

nI-3 ob. II CD H 0 U) ft 0• 0 0 H P1 I-i txj p1 I—i I-,. ft •0 0 CD it CD CD frb 0 ‘3’ 1 ft 0 I 1%

0 + + + + + ÷ + ÷ + DI 00 E 00 S 00 S 0 0 0 0 0 0 a + + + 0 0 p3 0 ‘- a ?rP i w o 0 HO’ wfrj

0’ It H H H H -‘ (3 0 H M

‘d H -1 0 -J -J MDI . . . . . I-,) In a a 0 0 C. (31 0 P FjØ (nil Discussion There is strong evidence that some luminescent bacteria are able to use TMAO as a terminal electron acceptor for anaerobic respiration. Anaerobic growth of some isolates as well as of Vibrio fischerii and Photobacterium leiqnothii taic itlflg could be enhanced by the addition of TMAO to media. The increase of the pH indicates TMA formation. Unfortunately we could not quantify TMA (eq by HPLC). Especially the specific inhibition of TMAO reduction by Wo and the restoration of the ability to reduce TMAO by Mo confirm the idea of TMAO respiration by luminescent bacteria.

References: — Kelly, D.J. et al. (1988) Arch. Microbiol. 150, 138—144 — Arata et al. (1988) J. Biochem. 103, 1011—1015 — Satoh, T.; Kurihara, F. N. (1987) J. Biochem. 102, 191—197