The twin-arginine translocation (Tat)
system is essential for Rhizobium-legume
symbiosis
S. Meloni, et al. 2003. Molecular Microbiology 48(5): 1195-1207
Presented by Denise Aslett
Microbiology Journal Club
March 19, 2007
Rhizobium leguminosarum
• _-proteobacteria
• Symbiont of legumes
• 3 biovars that differ in host
specificity
– viciae (peas and broad
beans)
– trifolii (clovers)
– phaseoli (kidney beans)
• Host specificity genes
carried on large plasmids
that also carry genes for
nitrogen fixation
• tat genes on chromosome
Source: Genome Biology 2006, 7:R34
Twin-arginine Transporter (Tat)
Ser/Thr-Arg-Arg-x-Phe-Leu-Lys, where x = polar aa
Lee, P., Tullman-Ercek, D. and Georgiou, G. 2006. The Bacterial Twin-Arginine Translocation Pathway. Annual Review of Microbiology. 60:373-395.
Rhizobium-Legume Symbiosis Overview
Sources: 1) Legume-Rhizobium Symbiosis from http://quorumsensing.ifas.ufl.edu/HCS200/LegRhiz.html, and 2) Gage, D. J. and Margolin, W. 2000. Hanging by a
thread: invasion of legume plants by rhizobia. Current Opinion in Microbiology. 3:613-617.
Hypothesis and Experiments
• The Tat system is essential for Rhizobiumlegume symbiosis.
– Characterized tat gene cluster and analyzed
tat expression
– Examined hydrogenase translocation and
activity
– Examined N2 fixation phenotype in a tatBC
mutant
– Analyzed outer membrane integrity in a tat
mutant
Figure 1
tat gene cluster in R. leguminosarum
Genomic tat configuration from NCBI
Tat protein domains
Table 1
Hydrogenase activity in tat mutants is
significantly diminished
SPF25 = mutant expressing hydrogenase in vegetative cells
SP61 = Tat deficient mutant of SPF25
Figure 2
Membrane targeting of Hydrogenase
subunits is disrupted in tat mutants
mutant
expressing
hydrogenase
in vegetative
cells =
SPF25
Hydrogenase structure from
Desulfovibrio gigas courtesy of
www.chem.ox.ac.uk/icl/faagroup/dgigasx.gif
Tat
deficient
mutant of
SPF25 =
SP61
Figure 3A
Plants inoculated with a tatBC mutant were
deficient in N2 fixation
Pink, normal
nodules expressing
high levels of
acetylene reduction
activity (data not
shown)
UPM791=
Wild type
SM61 =
tatBC mutant
Small, round,
white nodules
lacking acetylene
reduction activity
Acetylene Reduction Assay
Left: chlorotic peas from
www.soilhealth.com
Used to assess
nitrogenase activity in
environmental
samples
Figure 3B
Plants inoculated with a tatBC mutant had lower
numbers of infected cells and no infection threads
UPM791
Wild type
SM61
tatBC mutants
Figure 3B
Plants inoculated with a tatBC mutant had lower
numbers of infected cells
UPM791
Wild type
SM61
tatBC mutants
Figure 4 - Rieske protein
• Forms the
iron sulphur
component of
cytochrome
bc1 complex
• Has a Tat
dependent
signal peptide
N- terminal sequences from different Rhizobiaceae
Table 2
Effects of tat mutation on cytochrome bc1
functionality
“Nadi” test
Nadi reagents:
• _-naphthol + dimethyl-pphenylenediamine+ O2 indophenol blue + H2O
• Oxidatively converted to the blue dye by cytochrome c
Figure 3B
SS113 mutant with cytochrome bc1
deficiency can still infect plant cells
Wild type
UPM791
tatBC mutant
SM61
bc1 mutant
SS113
Figures 5A & 5B
Effect of tat mutation on R. leguminosarum
outer membrane
B
tatBC
Relative OD600
Wild type tatBC mutant
wt
Increasing SDS _
LPS fractions
Conclusions
• Tat genes are constitutively expressed in R.
leguminosarum vegetative cells.
• The mutation of tatBC genes abolished membrane
targeting of the large and small hydrogenase subunits
and the effect is at the post-translational level.
• Tat mutations interrupt the symbiotic process before
bacteroid release in the cortex and the observed
phenotype cannot be fully explained by a cytochrome
bc1 deficiency.
• A functional Tat pathway is essential for biological
nitrogen fixation by the Rhizobium-legume symbiosis.