Nitrogen Fixation by Symbiotic and Free-Living
Spirochetes
T.G. Lilburn, K.S. Kim, N.E. Ostrom, K.R. Byzek, J.R. Leadbetter, J.A.
Breznak (2001) Science 292:2495-2498
http://www.worldatlas.com/
Nitrogen Fixation by Symbiotic and
Free-Living Spirochetes

Exploratory/Hypothesis
ZAS-1, ZAS-2 and ZAS-9 (phylogenetically related to the
Treponemes)

Nitrogen fixation?

nifH and ability to fix N2
Spirochetes the likely origin of certain nifHs in termite guts
and perhaps in other environments?
Kingdom:Animalia
Phylum:Arthropoda
Class:Insecta
Order:Isoptera
Families:
Mastotermitidae
Kalotermitidae
Termopsidae
Hodotermitidae- Zootermopsis angusticollis
(pacific dampwood termite)
Rhinotermitidae
Serritermitidae
Termitidae
Termites and Nitrogen
•
•
•
•
Decomposers-lignocellulosic plant material
Gut microbiota is dense and diverse
Biogeochemical (nutrient) cycling-Carbon, Nitrogen. Release of
energy
Bigger role in N cycling than previously thought
www.epa.gov/maia/images/nitro.jpg
Termites and Nitrogen
 Nitrogen poor diet
 Symbiotic interactions with gut microbes to augment
nitrogen economy
-Recycling of excretory products
-Nitrogen fixation
 60% of nitrogen from N2 fixation
 Few strains:
-Citrobacter freundii, Pantoea agglomerans, and
Desulfovibrio spp. ? N2 fixation
 Survey for nifH in termite guts implied that diversity of
N2 fixing microbes was much greater than by culture
isolation and most of the a.a. sequences of NifH were
different from known microbial taxa.
Nitrogenase and nifH
N2 + 8e- + 8H+ + 16ATP ⇒ 2NH3 + H2 + 16ADP + 16Pi
 Organisms that are capable of fixing atmospheric dinitrogen (N2) possess
the enzyme nitrogenase, which reduces N2 to ammonia (NH3), a product
that is subsequently assimilated into biomass. Such organisms have
access to a ubiquitous source of nitrogen. Nitrogenase is found in
prokaryotic organisms, including many species of cyanobacteria.
Nitrogenases catalyzes the reduction of not only dinitrogen, but also a
variety of other substrates
 Nif genes found in free-living and
Symbiotic nitrogen fixers
 nifH encodes nitrogenase
Iron protein subunit
www.jic.bbsrc.ac.uk/staff/simon-george/
 Nitrogenase synthesis can be repressed in the
presence of NH4+ in medium
http://www.rcsb.org/pdb/explore.do?structureId=1NIP
PDB Nitrogenase Fe Azotobacter vinelandii
Acetylene reduction assay (AR)
 Measure nitrogenase activity.
 Nitrogenase can use other substrates with triple bonds
-Gaseous Nitrogen N≡N
(converted to NH3)
- Acetylene HC ≡CH
(converted to CH2=CH2)
 The use of nitrogenase-catalyzed reduction of acetylene (C2H2)
to ethylene (C2H4) as a sensitive and simple assay to measure
biological nitrogen fixation.
 ARA owes its popularity to its low cost, sensitivity and fast
response time.
Image
courtesy:
John
Breznak,
Michigan
State
University
Diversity of termite gut spirochetes
Role of Spirochetes in N2 Fixation?
•Free living or host associate
helical bacteria
•Class Spirochaetes
Order Spirochetales
Familes:
-Spirochaetaceae
w/genera:Treponema, Borrelia,
Brevinema, Cristispira,
spirochaeta
-Brachyspiraceae
-Leptospiraceae
•50% of gut microbes are
spirochetes
Role of Spirochetes in N2 Fixation?
•
Previously Isolated strains ZAS-1, ZAS-2, and ZAS-9 (in genera
Treponema)
• Acetogenesis:
4 H2 + 2 CO2 --> CH3COOH + 2 H2O
• First, examined gDNA for presence of nifH
-nifH encodes a nitrogenase enzyme
-catalyzes the reduction of N2 to NH3 (ammonia) which can then be
assimilated to biomass
• Second, examined their ability to fix N2
-Using the acetylene reduction assay (developed in the late 1960s by
Stewart et al. (1967; 1968) and hardy et al. (1968).
-Grow spirochetes under different gases 15N2, Ar, N2/CO2, Ar/CO2
Presence of nifH?
•Two nifH homologs in each
termite gut treponeme
•nifH homologs found in other
treponemes
•A.A. seq. of NifH had motifs
most like iron nitrogenase
•NifHs were phylogenetically
diverse and not congruent with
spirochete phylogeny based on
16S rRNA sequences
•No nifH found in halophile
spirochete, swine pathogen,
syphilis, or lyme disease
spirochete
Fig. 2 Phylogenetic tree of NifH sequences
from Spirochetes, other prok/termite gut, enviro.
15N/15N
used
2
89.0129/99.30
15N/Exp15N
6.3789/38
•SA of nitrogenase in S. aurantia was sufficient to provide virtually all the N needed by cells during
exponential growth on N2. Could not grow S. zuelzerae or ZAS-9 w/o YA.
•15N content of ZAS-9 was less than that for S. aurantia b/c diluted by 14N from YA. Has less 15N,
implies that N2 fixation enabled cells to assimilate nitrogenous compounds in YA
•Same for S. zuelzerae, SA is low, would only supply 20% of N needed for each doubling in biomass.
Suggest that nitrogenase activities are probably not max. attainable by cells.
S. aurantia
ZAS-9
+YA
•All show N2 dependent growth, and
NH4+-repressible AR activity
•ZAS-9 & S. zuelzerae could not be
grown w/o YA (source of fixed N)
•Can still see N limited growth in
these organisms by using media
w/2% YA w/no added NH4Cl. AR
activity commenced with the onset
of N2 dept. growth
•ZAS-1, ZAS-2 revealed no
enhancement of growth in N2 and
little nitrogenase activity.
S. zuelzerae
+YA
Authors Conclusions
 Show spirochetes can be N2-fixers
 S. aurantia, ZAS-9, S. zuelzerae highly correlated nifH
presence and nitrogen fixation
 Spirochetes have a role in termite nitrogen nutrition
-50% of prokaryotes in gut are spirochetes,
-spirochete NifHs were identical to NifH clones from a
variety of termites
 Potential contribution of spirochetes to N2 fixation might be very
high in termite guts. KO spirochetes = decreased termite
survival
 Free-living spirochetes may contribute substantially to N2
fixation in a variety of habitats.
Questions?