Nitrogen Fixation Island and Rhizosphere Competence Traits in the Genome of
Root-associated Pseudomonas stutzeri A1501
Søren Damkiær (s041877), Nicholas Jochumsen (s041852), Rune T. Nordvang (s041872) and Rune Jensen (s041859)
Course 27101 - Comparative Microbial Genomics: A Bioinformatics Approach
Introduction
Nitrogen fixation island (49kb)
Glu
pe ta
ro thio
xid n
as e
Ou
e
pr ter
ot m
ein e
m
br
an
e
Inspection of the locus where the nif genes were located
indicated that the nif genes have been acquired as part of a
genomic island.
tiv
e
co pro
bS
te
in
P. stutzeri
A1501
The nif region in P. stutzeri A1501 is located
at a putative insertional hotspot
Pu
ta
A closer look at the region
of interest confirmed the
presence of genes being
shared between P. stutzeri
and nitrogen-fixing
bacteria. Interestingly, it
was found that the genes
actually encoded products
of a notorious nitrogenase
complex essential for
nitrogen fixation.
co
bP
P. stutzeri A1501 carries a nitrogen fixation island
In order to identify genes possibly involved in nitrogenfixation, the genome of P. stutzeri A1501 was compared to the
genomes of known nitrogen-fixing organisms and other
Pseudomonas spp. The BLAST atlas from this comparison is
shown in figure 1
co
bU
Nitrogen is the largest single constituent of the Earth's atmosphere
(78 %) and an important element for living organisms. Despite the
availability of nitrogen from the atmosphere, plants often suffer from
nitrogen-limitation. Plants are only able to take up nitrogen through
their roots, primarily in the form of nitrates and ammonium. The supply
of nitrogenous compounds in the soil is being continually replenished
by microorganisms which fix atmospheric nitrogen [1]. Plants secrete
large amounts of various compounds through the roots (root exudats)
to support growth of nitrogen-fixing microorganisms in the
rhizosphere. In agriculture, a beneficial symbiotic relationship between
plants and nitrogen-fixing microorganisms is of major interest since it
diminishes the need of fertilizer and as a result the production cost and
the environmental impact.
Results and Discussion
P. stutzeri A1501
P. aeruginosa PAO1
From the BLAST atlas
it was possible to
identify a region
containing genes
unique to nitrogenfixing bacteria
P. stutzeri A1501
4,567,418 bp
nifH
P. fluorescens Pf-5
nifK
P. stutzeri
A1501
P. fluorescens Pf0-1
P. syringae
- pv. tomato DC3000
Figure 1 Illustration of the symbiotic relationship between nitrogen-fixing bacteria and plants
The capacity to fix nitrogen is widely distributed in phyla of Bacteria
and Archaea but has long been considered absent from the
Pseudomonas genus [2]. However, resent studies have indicated that
specific strains of Pseudomonas stutzeri indeed are able to fix nitrogen
[3]. In this study we investigated the newly sequenced genome of
Pseudomonas stutzeri A1501 by comparative genomics. The aim of the
analysis is primarily to identify genes involved in nitrogen-fixation and
secondly to identify genes associated with life in the rhizosphere.
Materials and Methods
nifD
- pv. phaseolicola 1448A
NifH
NifD
Figure 2 BLAST atlas comparing P, stutzeri to other bacteria. P. stutzeri A1501 (outer
BLAST lane), P. aeruginosa PAO1, P. entomophila L48, P. fluorescens Pf-5, P. fluorescens
Pf0-1, P. mendocina ymp, P. putida KT2440, P. syringae pv. phaseolicola 1448A, P.
syringae pv. tomato str. DC3000, Azoarcus sp. BH72, A. caulinodans ORS 571, K.
pneumoniae 342, R. etli CFN 42, R. leguminosarum bv. trifolii WSM2304 (inner BLAST
lane). Lanes 14 to 17 show genes, rRNAs and tRNAs on leading strand and lagging
strand (lane 14), percent AT (lane 15), global directed repeats (lane 16) and global
inverted repeats (lane 17).
References
Acknowledgements
We would like to thank David W. Ussery and Peter F. Hallin for guidance and help during the making of this poster.
We would especially like to thank Peter F. Hallin for his skillful supervision and patience in the making of the
BLAST atlases used on this poster.
NifH
N2
Figure 4 Physical organization of the highly conserved region containing the cob
gene cluster and putative glutathione peroxidase in sequenced Pseudomonas
strains. Genes of the same color indicate corresponding orthologous genes with
high homology (80%) at the nucleic acid level and arrow shows transcriptional
directions of genes. Inserted genes are marked in white.
NifK
NH3
Figure 3 Simple illustration of the core nitrogenase complex.
The complex consists of three different subunits encoded by
the genes nifH, nifD and nifK found on the nif island.
The P. stutzeri A1501 genome carries many rhizosphere competence traits
Complete genome sequences of the strains investigated in this study
were downloaded from the NCBI Genome database [4]. BLAST atlases
was generated by the CBS DTU online tool, gwBrowser 0.91 server [5].
To identify unique genes encoding transporters and chemotaxis
proteins in P. stutzeri A1501 and P. aeruginosa PAO1, a comparative
search was performed using a “reciprocal best BLAST” approach [6].
[1] Cheng, G. (2008). Journal of Integrative Plant Biology 50 (7): 786–798.
[2] Desnoues,N., Lin,M., Guo,X., Ma,L., Carreno-Lopez,R., and Elmerich,C. (2003). Microbiology 149: 2251-2262.
[3] Vermeiren,H., Willems,A., Schoofs,G., de,M.R., Keijers,V., Hai,W., and Vanderleyden,J. (1999). Syst Appl Microbiol 22: 215-224.
[4] http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi
[5] http://www.cbs.dtu.dk/services/gwBrowser/index.php
[6] www.pseudomonas.com.
[7] Lugtenberg, B.J.J. and Bloemberg, G.V. (2004). Pseudomonas, vol 1, edited by Ramos, J, Kluwer Academic.
P. syringae
The four major genetic islands in P. stutzeri A1501 were investigated for rhizosphere competence traits.
The islands contained several transporter encoding genes, including transporters associated with
amino acids, carbohydrates and C4-dicarboxylates, which are the major components of root exudates
[7].
Genomic islands
To investigate if P. stutzeri A1501 carries additional genes related to a lifestyle in the rhizosphere a
comparative search between P. stutzeri A1501 and P. aeruginosa PAO1 was performed.
Transporter encoding genes
80
220
170
Figure 5 Comparison of transporter encoding genes in P. stutzeri A1501
( ) and P. aeruginosa PAO1 ( ). The genome of P. stutzeri A1501 contains
80 unique transporter encoding genes, which are predicted to be
involved in Na+, SO42-, Pi, NO3-, NH4+, amino acid, C4-dicarboxylate and
maltose transport – all common root exudates [7].
Chemotaxis related genes
14
20
16
Figure 6 Comparison of chemotaxis related genes in P. stutzeri
A1501 ( ) and P. aeruginosa PAO1 ( ). The genome of P. stutzeri
A1501 contains 14 unique genes, which encode proteins involved in
chemotaxis. Some of these may be involved in the sensing of root
exudates, e.g. maltose.
Concluding remarks
In this study we investigated the genome of P. stuzeri
A1501. The comparative analysis revealed that P.
stutzeri A1501 carries a nitrogen fixation island at a
putative insertional hotspot. Besides this P. stutzeri
A1501 carries at least four major genomic islands.
Investigation of these islands revealed the presence
of numerous genes encoding transporters of amino
acids, carbohydrates and C4-dicarboxylates, the main
components of root exudates. In addition it was
found by a comparative search between P. stutzeri
A1501 and P. aeruginosa PAO1 that P. stutzeri A1501
carries more than 80 unique transporter encoding
genes, most of which surprisingly were located
outside the genetic islands. Many of these
transporters were predicted to be involved in uptake
of root exudates. The presence of the nif island
together with the genes associated to rhizosphere
competence strongly suggests that P. stutzeri A1501
can adapt to a life in the rhizosphere.