FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids

FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids

The crystal structure of FusC reveals structural homology to MPP.

<p>(A) The crystal structure of FusC (top panel) with the four M16 protease domains colored yellow, red, green, and blue from N- to C-terminus: the halves of FusC form a partially open clamshell structure strikingly similar to the structure of MPP (bottom panel). The MPP (PDB 1HR6) heterodimer has a β-subunit (pink and peach) and α-subunit (pale green and blue). (B) A top-down view of the N-terminal half of FusC showing how domain 1 (labeled “D1”; presented in a cartoon and stick representation) interacts with domain 2 (labeled “D2”; shown with electrostatic surface rendering). (C) The catalytic site of the inactive E⁸³A mutant of FusC contains a catalytic zinc ion typical of M16 family proteases. (D) The catalytic site of EDTA-inactivated FusC lacks this metal ion, consistent with its chelation by EDTA. MPP, mitochondrial processing peptide; PDB, Protein Data Bank.</p

A Brownian ratchet model for ferredoxin import into <i>Pectobacterium</i>.

<p>The TBDT FusA is a β-barrel protein in the outer membrane (labeled “OM”), with the M16 peptidase FusC on the <i>trans</i> side of the outer membrane, in the periplasm. Initial interactions of plant ferredoxin with the <i>cis</i> face of FusA have been modeled, and complementary surfaces would provide for a docking reaction [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.ref009" target="_blank">9</a>]. As discussed in the text, some degree of unfolding of ferredoxin is required in order to allow its entry into the translocation channel of the FusA β-barrel. Engagement of an unfolded ferredoxin segment to FusC would establish the conditions for a Brownian ratchet to drive vectorial movement and thereby power protein import through the channel of the FusA β-barrel domain. Given the size of ferredoxin, a complete unfolding of the protein would not be required. Liberation of iron would require proteolysis of ferredoxin by either FusC or another protease in the periplasm. TBDT, TonB-dependent transporter.</p

HMM search results on TBDTs in <i>Pectobacterium</i>.

<p>The HMM score reflects the statistical significance of the hit, the asterisk denotes a lack of statistical significance in the match for the FusA homologue, but structural analysis [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.ref009" target="_blank">9</a>] confirms the FusA sequence as the 23rd example of a TBDT encoded in the genome of <i>P</i>. <i>carotovorum</i> subsp. <i>cartorvorum</i> Waldee RMIT1.</p

SAXS shows that FusC is flexible in solution and undergoes a conformational change on ferredoxin binding.

<p>(A) SEC-SAXS analysis of FusC and FusC(E⁸³A) with and without the addition of ferredoxin. FusC is shown in the absence (black) and presence (blue) of ferredoxin; FusC(E⁸³A) is shown in its absence (red) and presence (green). The Kratky plot of FusC data suggests a multidomain structure with some interdomain flexibility. Perturbance of FusC(E⁸³A) in the presence of ferredoxin is observed. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s010" target="_blank">S2 Data</a>. (B) Scattering angle versus intensity plot for FusC. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s010" target="_blank">S2 Data</a>. (C) P_r distribution showing that FusC has a maximum dimension of 129 Å, adopting an elongated conformation in solution. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s010" target="_blank">S2 Data</a>. (D) A plot of the Guinier region of FusC SAXS showing the particle has a radius of gyration of 37.4 Å. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s010" target="_blank">S2 Data</a>. (E) An ITC isotherm of a titration of ferredoxin. Saturatable heats of binding were observed through the titration of ferredoxin into a sample of FusC (blue) but not for a titration of the equivalent buffer into a sample of FusC (green) nor for a titration of ferredoxin into a sample of buffer lacking FusC (red). The fitted binding curve yielded reaction kinetics (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.t002" target="_blank">Table 2</a>) and a disassociation constant (Kd) of 1.7 μM. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s010" target="_blank">S2 Data</a>. (F) Ab initio model of FusC in solution, generated using DAMMIF, shows that FusC can adopt an elongated confirmation in solution. (G) A surface model of the crystal structure of FusC, which has a maximum dimension of 94 Å. All SAXS data (used to generate plots and models in panels A, B, C, D, and F) have been deposited at the SASBDB, accession: SASDDQ6, SASDDR6, SASDDS6, SASDDT6. ITC, isothermal titration calorimetry; SASBDB, Small Angle Scattering Biological Databank; SAXS, small-angle X-ray scattering; SEC-SAXS, size-exclusion chromatography coupled with small-angle X-ray scattering.</p

SAXS scattering shows that FusC(E⁸³A) undergoes major conformational change upon ferredoxin binding but remains elongated in solution.

<p>Static-SAXS titration of ferredoxin into FusC (30 μM) mapping conformational changes that occur in FusC upon ferredoxin binding. (A) Scattering angle versus intensity plot for FusC(E⁸³A) with increasing concentration of ferredoxin. (B) Kratky plot of FusC(E⁸³A) scattering showing a shift from a multidomain structure to a single domain upon ferredoxin binding. (C) P_r distribution of FusC(E⁸³A) in the absence of ferredoxin (0 μM), partially saturated (50 μM), and saturated (100 and 150 μM), showing a conformational tightening of FusC upon ferredoxin binding. (D) A plot of the Guinier region of FusC(E⁸³A) at increasing ferredoxin concentration. (E) A plot of volume of FusC(E⁸³A) with increasing ferredoxin concentration. The observed increase and plateau of Porod volume is indicative of ferredoxin binding and saturation of FusC(E⁸³A). (F) A plot of the maximum dimension of FusC(E⁸³A) in solution, showing that the protease remains elongated in solution when in complex with ferredoxin. All SAXS data used to generate plots in this figure have been deposited at SASBDB, accession: SASDDU6. SASDDV6, SASDDW6, SASDDX6, SASDDY6, SASDDZ6, SASDD27, SASDD37, SASDD47, SASDD57, SASDD67, SASDD77. SASBDB, Small Angle Scattering Biological Databank; SAXS, small-angle X-ray scattering.</p

Kinship and iron acquisition in the known species and subspecies of <i>Pectobacterium</i>.

<p>(A) Molecular phylogeny of <i>Pectobacterium</i> species, showing the relationship of the Australian isolate RMIT1 as being <i>P</i>. <i>carotovorum</i> subsp. <i>cartorvorum</i> Waldee. In the analysis shown here, the Australian isolate is closely related to published genomes of <i>P</i>. <i>carotovorum</i> subsp. <i>cartorvorum</i> from China (red branch). Recent whole genome–based phylogenetic analysis suggests that the four recognized subspecies of <i>P</i>. <i>carotovorum</i> (<i>actinidiae</i>, <i>odoriferum</i>, <i>carotovorum</i>, and <i>brasiliense</i>) should be elevated to distinct species [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.ref063" target="_blank">63</a>]. (B) CLANS similarity network analysis depicts homology in protein datasets via all-against-all pairwise BLAST to cluster representations (dots) of individual protein sequences. Gray scale lines are shown between samples, with the most similar sequences shown as black, with an E-value cutoff of 1e−15. The analysis shows that M16 proteases from diverse plants species (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s008" target="_blank">S3 Table</a>) cluster into four groups: the MPP group (green), the plastid SPP group (blue), the PreP group (yellow), and a previously unnoticed M16 group we refer to as plant FusC (red). The M16 proteins from Enterobacteriaceae (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006026#pbio.2006026.s009" target="_blank">S1 Data</a>) cluster into five main groups: the PqqF-like sequences, the PtrA-like sequences, the sequences similar to YhjJ from <i>E</i>. <i>coli</i>, the sequences similar to YhjJ2 from <i>Yersinia</i> spp., and a cluster containing FusC proteins mainly from <i>Pectobacterium</i> and <i>Klebsiella</i> spp. (a minor cluster containing a very conserved set of sequences represented by <i>E</i>. <i>coli</i> PqqL sits between the FusC, YhjJ2, and YhjJ1 clusters). The CLANS analysis places the bacterial FusC sequence as being most related to the plant FusC sequences. CLANS, cluster analysis of sequences; MPP, mitochondrial processing peptide; PreP, presequence protease; SPP, stromal processing peptidase.</p

ITC-derived binding parameters for FusC(E⁸³A) and ferredoxin.

<p>ITC-derived binding parameters for FusC(E⁸³A) and ferredoxin.</p