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Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

Lallemand M, Login FH, Guschinskaya N, Pineau C, Effantin G, Robert X, Shevchik VE - PLoS ONE (2013)

Bottom Line: We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane.Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS.These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

View Article: PubMed Central - PubMed

Affiliation: INSA-Lyon, Villeurbanne, France ; CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, Lyon, France.

ABSTRACT
The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

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Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM.(A), secretion activity of OutL/M variants. (B and C), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies (A), or with GST-OutL antibodies (B), or with OutM antibodies (C), as in Figure 4. The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.
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pone-0079562-g005: Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM.(A), secretion activity of OutL/M variants. (B and C), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies (A), or with GST-OutL antibodies (B), or with OutM antibodies (C), as in Figure 4. The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.

Mentions: OutLP380C was non-functional (Figure S2B, lane 8), suggesting that the proline substitution caused significant structural alterations. Consequently, this variant was excluded from any further analysis. To examine the presumed OutL/M interface, OutLI378C and OutLG381C were co-expressed in the D. dadantii ΔoutL strain with one of the OutM variants, L122C, A126C, L139C, V144C, P145C, L122C/L139C or A126C/P145C. Most of the combinations restored pectinase secretion at, or near, the wild-type level except OutLG381C/OutML122C/L139C and OutLG381C/OutMA126C/P145C which were more severely affected (Figure S2B, lanes 11 and 12). Surprisingly, expression of the two latter combinations in the wild-type D. dadantii A4229 strain also dramatically diminished pectinase secretion (Figure 5A, lanes 10 and 13). These data indicate a dominant negative interference of these variants with the secretion system. When separated in a non-reducing gel, the OutLG381C/OutMP145C pair generated an additional species of about 60 kDa that cross-reacted with both OutL and OutM antibodies and this is consistent with an OutL-M complex (Figure 5B and C, lane 8). Such a complex was much less abundant with the OutLG381C/OutMV144C pair (Figure 5C, lane 9) indicating that contrary to OutMP145C, OutMV144C is not close enough to OutLG381C. Remarkably, when co-expressed with OutLG381C, OutMP145C generated both hetero- and homodimers, indicating that β3-cβC (OutM/L) and β3-β3 (OutM/M) interactions occur simultaneously or alternate in the course of secretion (Figure 6A).


Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

Lallemand M, Login FH, Guschinskaya N, Pineau C, Effantin G, Robert X, Shevchik VE - PLoS ONE (2013)

Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM.(A), secretion activity of OutL/M variants. (B and C), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies (A), or with GST-OutL antibodies (B), or with OutM antibodies (C), as in Figure 4. The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815138&req=5

pone-0079562-g005: Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM.(A), secretion activity of OutL/M variants. (B and C), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies (A), or with GST-OutL antibodies (B), or with OutM antibodies (C), as in Figure 4. The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.
Mentions: OutLP380C was non-functional (Figure S2B, lane 8), suggesting that the proline substitution caused significant structural alterations. Consequently, this variant was excluded from any further analysis. To examine the presumed OutL/M interface, OutLI378C and OutLG381C were co-expressed in the D. dadantii ΔoutL strain with one of the OutM variants, L122C, A126C, L139C, V144C, P145C, L122C/L139C or A126C/P145C. Most of the combinations restored pectinase secretion at, or near, the wild-type level except OutLG381C/OutML122C/L139C and OutLG381C/OutMA126C/P145C which were more severely affected (Figure S2B, lanes 11 and 12). Surprisingly, expression of the two latter combinations in the wild-type D. dadantii A4229 strain also dramatically diminished pectinase secretion (Figure 5A, lanes 10 and 13). These data indicate a dominant negative interference of these variants with the secretion system. When separated in a non-reducing gel, the OutLG381C/OutMP145C pair generated an additional species of about 60 kDa that cross-reacted with both OutL and OutM antibodies and this is consistent with an OutL-M complex (Figure 5B and C, lane 8). Such a complex was much less abundant with the OutLG381C/OutMV144C pair (Figure 5C, lane 9) indicating that contrary to OutMP145C, OutMV144C is not close enough to OutLG381C. Remarkably, when co-expressed with OutLG381C, OutMP145C generated both hetero- and homodimers, indicating that β3-cβC (OutM/L) and β3-β3 (OutM/M) interactions occur simultaneously or alternate in the course of secretion (Figure 6A).

Bottom Line: We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane.Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS.These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

View Article: PubMed Central - PubMed

Affiliation: INSA-Lyon, Villeurbanne, France ; CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, Lyon, France.

ABSTRACT
The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Show MeSH
Related in: MedlinePlus