The twin-arginine translocation (Tat)system is essential for Rhizobium-legume

symbiosisS. Meloni, et al. 2003. Molecular Microbiology 48(5): 1195-1207

Presented by Denise AslettMicrobiology Journal Club

March 19, 2007

Rhizobium leguminosarum

• _-proteobacteria

• Symbiont of legumes

• 3 biovars that differ in hostspecificity– viciae (peas and broad

beans)

– trifolii (clovers)

– phaseoli (kidney beans)

• Host specificity genescarried on large plasmidsthat also carry genes fornitrogen fixation

• tat genes on chromosomeSource: Genome Biology 2006, 7:R34

Twin-arginine Transporter (Tat)

Lee, P., Tullman-Ercek, D. and Georgiou, G. 2006. The Bacterial Twin-Arginine Translocation Pathway. Annual Review of Microbiology. 60:373-395.

Ser/Thr-Arg-Arg-x-Phe-Leu-Lys, where x = polar aa

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 athread: invasion of legume plants by rhizobia. Current Opinion in Microbiology. 3:613-617.

Hypothesis and Experiments

• The Tat system is essential for Rhizobium-legume symbiosis.– Characterized tat gene cluster and analyzedtat expression

– Examined hydrogenase translocation andactivity

– Examined N2 fixation phenotype in a tatBCmutant

– Analyzed outer membrane integrity in a tatmutant

Figure 1tat gene cluster in R. leguminosarum

Genomic tat configuration from NCBITat protein domains

Table 1Hydrogenase activity in tat mutants is

significantly diminished

SPF25 = mutant expressing hydrogenase in vegetative cells

SP61 = Tat deficient mutant of SPF25

Figure 2Membrane targeting of Hydrogenasesubunits is disrupted in tat mutants

SPF25 SP61

mutantexpressing

hydrogenasein vegetative

cells =

Tatdeficientmutant ofSPF25 =

Hydrogenase structure fromDesulfovibrio gigas courtesy of

www.chem.ox.ac.uk/icl/faagroup/dgigasx.gif

Figure 3APlants inoculated with a tatBC mutant were

deficient in N2 fixation

Used to assessnitrogenase activity in

environmentalsamples

Acetylene Reduction Assay

UPM791=Wild type

SM61 =tatBC mutant

Small, round,white noduleslacking acetylenereduction activity

Pink, normalnodules expressinghigh levels ofacetylene reductionactivity (data notshown)

Left: chlorotic peas fromwww.soilhealth.com

Figure 3BPlants inoculated with a tatBC mutant had lowernumbers of infected cells and no infection threads

UPM791 SM61 Wild type tatBC mutants

Figure 3BPlants inoculated with a tatBC mutant had lower

numbers of infected cells

UPM791 SM61 Wild type tatBC mutants

Figure 4 - Rieske protein

• Forms theiron sulphurcomponent ofcytochromebc1 complex

• Has a Tatdependentsignal peptide

N- terminal sequences from different Rhizobiaceae

Table 2Effects 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 3BSS113 mutant with cytochrome bc1

deficiency can still infect plant cells

Wild type tatBC mutant bc1 mutant UPM791 SM61 SS113

Figures 5A & 5BEffect of tat mutation on R. leguminosarum

outer membrane

Wild type tatBC mutant

Rel

ativ

e O

D60

0

Increasing SDS _

wt tatBCB

LPS fractions

Conclusions• Tat genes are constitutively expressed in R.

leguminosarum vegetative cells.

• The mutation of tatBC genes abolished membranetargeting of the large and small hydrogenase subunitsand the effect is at the post-translational level.

• Tat mutations interrupt the symbiotic process beforebacteroid release in the cortex and the observedphenotype cannot be fully explained by a cytochromebc1 deficiency.

• A functional Tat pathway is essential for biologicalnitrogen fixation by the Rhizobium-legume symbiosis.