Molecular Microbiology (2013) 90(5), 919–922 ■
doi:10.1111/mmi.12433 First published online 30 October 2013
MicroCommentary A new route for polar navigation Lori L. Burrows* Department of Biochemistry and Biomedical Sciences, McMaster University, Rm 4H18, 1280 Main St. W., Hamilton, Ontario, Canada, L8S4K1.
Summary Placement of motility structures at the poles of rodshaped bacteria is a common engineering problem with a variety of potential solutions. While investigating the mechanisms for positioning of the single polar flagellum of Pseudomonas aeruginosa, Cowles and colleagues discovered a new membrane-bound threecomponent system related to TonB–ExbB–ExbD that they named ‘Poc’ for polar organelle co-ordinator, which controls polar localization of both the flagellum and type IV pili. The Poc complex itself is not found at the poles, and is required for increased expression of pilus genes upon surface association, suggesting a new paradigm of localization control. Motility is a widespread bacterial trait, allowing them to seek out favourable environments and avoid unpleasant ones. Flagella are long helical filaments whose rotation propels bacteria through fluids via swimming motility (Berg, 2003). Type IV pili (T4P) are thinner filaments whose repeated extension (assembly) and retraction (disassembly) is used – similar to a grappling hook – for twitching motility on surfaces (Burrows, 2012). In rod-shaped bacteria, both these organelles are located at the poles, so that motility occurs preferentially in the direction of the long axis of the cell. This is the most efficient arrangement for reducing shear forces on the cell body, as it presents the smallest surface area going forward. In addition, localization of adhesive organelles such as T4P to the poles lets bacteria adhere more efficiently to surfaces by initially presenting a smaller surface area at a distance, minimizing electrostatic repulsion (Young, 2006). Although the flagellum and T4P are both located at the poles in Pseudomonas aeruginosa, their specific localization patterns differ. The single flagellum is located only at Accepted 15 October, 2013. *For correspondence. E-mail burrowl@ mcmaster.ca; Tel. (+1) 905 525 9140 x22029; Fax (+1) 905 522 9033.
© 2013 John Wiley & Sons Ltd
the old pole, while T4P assembly systems are typically bipolar, though pilus filaments can be assembled at either or both poles. A previous study by Cowles and Gitai (2010) showed that patterns of flagellum and T4P regulation were different, also suggesting that the two complexes become polar via independent routes. This initial hypothesis was supported in their current study (Cowles et al., 2013) by the results of time-lapse imaging of dividing cells. Fluorescent fusions to flagellar proteins FlhF and CheA – shown previously to be polarly localized, and in the case of FlhF, to control localization of the flagellum – formed bright, immotile foci at old poles, with new puncta appearing over time only at sites of incipient cell division. A fluorescent fusion to the T4P retraction ATPase PilT was also polar, though its localization was less predictable and sometimes motile, consistent with a previous study showing that its localization depends on the dynamic cytoskeletal protein, MreB (Cowles and Gitai, 2010). To identify determinants of polar flagellum localization in P. aeruginosa, Cowles et al. (2013) looked for transposon insertion mutants with reduced swimming motility, a phenotype associated with mislocalized flagella. Among more than 240 such mutants – most of which had no flagella – they found those with mutations in genes encoding known regulators FlhF and FleN (which controls the number of flagella produced), and three new mutants with insertions in PA0406, PA2982 and PA2983. The first two genes were identified previously in a screen for P. aeruginosa mutants that were avirulent in a Drosophila infection model (D’Argenio et al., 2001). Although both are required for twitching motility, other T4P mutants were fully virulent in Drosophila, hinting even then that PA0406 and PA2983 had additional roles (Whitchurch, 2006). PA0406 encodes a TonB homologue named TonB3 (Huang et al., 2004), while the others encode homologues of ExbB (PA2983) and ExbD (PA2982). In Escherichia coli, TonB–ExbB–ExbD form a complex that is important for energizing siderophore uptake (Braun, 1995). Based on Cowles and colleagues’ subsequent detailed characterization that revealed effects on both flagellum and T4P localization, PA2983 and PA2982 were named PocA and PocB (polar organelle co-ordination). Like their E. coli homologues, PocAB could be co-precipitated, suggestive of complex formation. Their interaction did not depend on
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TonB3, and the investigators were unable to demonstrate TonB3 interaction with either Poc protein. However, their common phenotypes suggested a functional association. Clean deletions confirmed that all three genes were necessary for normal swimming, and for polar placement of the flagellum. The GTPase FlhF was previously considered the topmost component in the polar localization hierarchy for the flagellum of P. aeruginosa, controlling not just localization, but also function (Kazmierczak and Hendrixson, 2013; Schniederberend et al., 2013). However, FlhF has now been dethroned by the Poc complex, as a FlhF–GFP fusion traded its wild-type bipolar localization for non-polar sites – correlated with the positions of non-polar flagella – when any of TonB3, PocA or PocB was missing. The previous identification of TonB3 as a component affecting twitching motility prompted Cowles and coworkers to take a closer look at T4P localization in the new mutants, leading to some surprising results. T4P localization was markedly affected, with approximately 70% of cells having non-polar pili, but only in the PocA mutant – the other two had no pili. This finding was consistent with previous observations that only tonB3 and pocB mutants were avirulent in Drosophila (D’Argenio et al., 2001). Subsequent studies of pilin gene regulation showed that the tonB3 and pocB mutants failed to upregulate pilA expression on solid surfaces, while lack of pocA had no effect. Huang et al. (2004) reported previously that tonB3 mutants had substantially reduced (though not completely absent) motility and less surface pili than the wild type, while the current study reported no motility and almost complete lack of piliation when tonB3 and pocB mutants were imaged by electron microscopy. These minor discrepancies may relate to the different P. aeruginosa strains used (PAK in the 2004 study, versus PAO1 in the current one). Although the pocA mutant had non-polar T4P, its twitching motility was impaired. The investigators suggested that retraction of mis-localized pili along multiple and opposing vectors resulted in essentially zero net motility, although pilus functionality was not formally tested. The discrepancy in pilus expression between the three mutants could not be readily explained by looking at markers of T4P complex localization, as both GFP–PilT and PilQ–mCherry – normally polar – were diffuse in all cases. Flagella and T4P are large multiprotein complexes that span all compartments of the cell envelope, including the covalently closed peptidoglycan layer (Scheurwater and Burrows, 2011). The finding that the loss of the Poc proteins caused aberrant placement of these complexes begs the question of how they could be assembled at places where they’re not normally found. Flagellum synthesis typically occurs at the old pole after cell division and involves a muramidase (FlgJ) that creates an opening for the complex in the PG layer (Nambu et al., 1999;
Kazmierczak and Hendrixson, 2013). It is possible that the muramidase can allow the flagellum to pass through the cell envelope in non-polar locations. In contrast, T4P systems in general lack PG-degrading enzymes. However, if T4P assembly were normally co-ordinated with cell division, nascent complexes could be targeted to the site of septum synthesis for incorporation during synthesis of new poles, a ‘pre-installation’ strategy, instead of the ‘retrofitting’ strategy used by flagella. The association of flagellar and T4P components with septa was noted by Cowles and co-workers when they generated filamentous cells using the cell division inhibitor, cefsulodin. The recruitment of T4P systems to developing septa might be driven by the similarity of specific components to cell division proteins. For example, PilM – a component of the transenvelope T4P assembly complex – has striking structural similarity to FtsA, an early recruit to the contractile Z-ring that directs inward synthesis of the cell envelope (van den Ent and Lowe, 2000; Karuppiah and Derrick, 2011; Tammam et al., 2013). PilM interactions with the Z-ring could position T4P systems at the appropriate location for incorporation into the new poles of each daughter cell (Fig. 1A). With this scenario in mind, it is possible to imagine that disruption of such a co-ordinated process might lead to lack of pili as seen in the tonB3 and pocB mutants. However, it is less clear how T4P assembly complexes might be mis-incorporated into the cell envelopes of pocA mutants, particularly since all three mutants showed complete delocalization of PilT and PilQ, instead of colocalization of these markers with non-polar pili in the pocA background. Careful investigation of the state of the T4P assembly system in pocA mutants will be useful, since the process by which it is normally directed to the poles and assembled through all layers of the cell envelope remains largely mysterious. Many studies of polar localization mechanisms have suffered from a chicken and egg problem; if protein X localizes to the pole via its interactions with polar component Y, then the question becomes how Y became localized to the pole, and so on. Intriguingly, Cowles and co-workers found that the Poc complex itself is not polar (the localization of TonB3 could not be determined due to low levels of expression), leaving the mechanism by which it directs localization of downstream proteins an open question. In the case of flagella, the TonB3–PocAB system appears to control the placement of FlhF; when it ends up at a non-polar site, that’s where the flagellum is made (Fig. 1B). The effects of the TonB3–PocAB system on T4P expression are more complicated, and are linked to transcriptional regulation of pilus genes in response to surface association, as well as to placement of T4P. The lack of transcriptional upregulation of pilus genes in response to surface contact was interesting, and may point to a con© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 90, 919–922
A new route for polar navigation 921
Fig. 1. Co-ordination of flagellum and type IV pilus complex assembly with cell division.A. The GTPase FlhF (square symbol) positions the flagellum at the old pole (dark blue). When cells begin the division cycle, it is recruited to the ‘new old’ pole of the flagellum-less daughter cell (light blue), where a flagellum will subsequently be assembled. The exact determinants for polar localization of type IV pili (T4P) in P. aeruginosa are not known, but the complex (triangle symbol) might be recruited to the septum of dividing cells, resulting in the insertion of T4P systems at the new poles (pink) of each daughter cell after division. T4P can be assembled at either or both poles.B. The TonB3, PocA and PocB proteins are predicted to be localized to the inner membrane (IM), and to have similar topologies to their homologues TonB, ExbD and ExbB respectively. When any of the three is missing, FlhF is mislocalized and the flagellum is assembled at non-polar sites (left). When TonB3 or PocB is missing, no T4P are produced, but lack of PocA results in production of non-polar pili (right).
sequence, rather than cause, of not making pili. P. aeruginosa has complex and interconnected regulatory networks controlling its rapid adaptation to changes in lifestyle, including switching from detached to attached states (Coggan and Wolfgang, 2012). Loss of T4P has been linked to decreased synthesis of cAMP, an important secondary messenger that controls the expression of surface-associated virulence factors, including T4P themselves (Fulcher et al., 2010). Those data imply a feedforward mechanism, where T4P are needed to trigger a signal leading to the synthesis of – among other things – more pili. The TonB3–PocAB system may represent a specialized layer of this surface-sensing repertoire, as Cowles and colleagues found that it was mainly genes associated with pilus biogenesis – rather than the larger set that are altered by surface association – whose expression became non-responsive in tonB3 and pocB backgrounds. One might imagine that TonB3–PocAB are part of the earliest response to surface contact, which pili can mediate at a distance while the cell body is still in a © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 90, 919–922
liquid environment. P. aeruginosa may use its T4P as ‘whiskers’ to report when a surface is initially detected – perhaps via forces generated on the pilus assembly system upon tethering of a pilus filament – triggering downstream regulatory events that could lead eventually to more robust surface interactions. This idea is consistent with TonB3 and PocB’s predicted periplasmic location, where they could be influenced by interactions with the peptidoglycan skeleton or the pilus assembly system, while PocA is mostly cytoplasmic (Fig. 1B). The sequence and architectural similarities of TonB3– PocAB to the canonical TonB–ExbBD system, which relies on the proton motive force for its function, hint at a possible energy transduction role. Cowles et al. noted a previous report stating that perturbations of membrane potential in Bacillus subtilis affect the localization of proteins such as MinD, FtsA and MreB that control cell elongation and division (Strahl and Hamoen, 2010). Thus, loss of TonB3, PocA and/or PocB could have membrane potential-related effects on localization of these and related proteins such as PilM (above). Interestingly, FlhF, which is mislocalized when any of the three proteins of interest is missing, has been linked to the control of septum placement in Campylobacter jejuni. In that species, flhF mutants form minicells, which typically result from formation of Z-rings at sites other than mid-cell (Balaban and Hendrixson, 2011). In the current study, loss of any of tonB3, pocA or pocB resulted in cells that were 15% shorter than wild type, implying a possible disconnect in the co-ordination of cell elongation with cell division. Interestingly, a double knockout of pocAB could not be made, suggesting potential lethality that might relate to disruptions in membrane potential or cell division; these ideas remain to be tested. Cowles and co-workers have produced a thoughtprovoking study which emphasizes that bacteria, formerly thought to be relatively homogenous in spatial organization, instead have sophisticated and interconnected mechanisms for ensuring that things get to where they need to be, when they need to be there (Kirkpatrick and Viollier, 2011). The fact that close to 40% of the tonB3, pocA and pocB mutants still had correctly localized flagella, coupled with the finding that the three gene products have dissimilar effects on T4P biogenesis, show that there are multiple and redundant mechanisms for polar localization of motility structures that remain to be discovered.
References Balaban, M., and Hendrixson, D.R. (2011) Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni. PLoS Pathog 7: e1002420. Berg, H.C. (2003) The rotary motor of bacterial flagella. Annu Rev Biochem 72: 19–54. Braun, V. (1995) Energy-coupled transport and signal trans-
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duction through the gram-negative outer membrane via TonB–ExbB–ExbD-dependent receptor proteins. FEMS Microbiol Rev 16: 295–307. Burrows, L.L. (2012) Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu Rev Microbiol 66: 493– 520. Coggan, K.A., and Wolfgang, M.C. (2012) Global regulatory pathways and cross-talk control Pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol 14: 47–70. Cowles, K.N., and Gitai, Z. (2010) Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa. Mol Microbiol 76: 1411–1426. Cowles, K.N., Moser, T.S., Siryaporn, A., Nyakudarika, N., Dixon, W., Turner, J.J., and Gitai, Z. (2013) The putative Poc complex controls two distinct Pseudomonas aeruginosa polar motility mechanisms. Mol Microbiol 90: 923– 938. D’Argenio, D.A., Gallagher, L.A., Berg, C.A., and Manoil, C. (2001) Drosophila as a model host for Pseudomonas aeruginosa infection. J Bacteriol 183: 1466–1471. van den Ent, F., and Lowe, J. (2000) Crystal structure of the cell division protein FtsA from Thermotoga maritima. EMBO J 19: 5300–5307. Fulcher, N.B., Holliday, P.M., Klem, E., Cann, M.J., and Wolfgang, M.C. (2010) The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity. Mol Microbiol 76: 889–904. Huang, B., Ru, K., Yuan, Z., Whitchurch, C.B., and Mattick, J.S. (2004) tonB3 is required for normal twitching motility and extracellular assembly of type IV pili. J Bacteriol 186: 4387–4389. Karuppiah, V., and Derrick, J.P. (2011) Structure of the
PilM–PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus. J Biol Chem 286: 24434–24442. Kazmierczak, B.I., and Hendrixson, D.R. (2013) Spatial and numerical regulation of flagellar biosynthesis in polarly flagellated bacteria. Mol Microbiol 88: 655–663. Kirkpatrick, C.L., and Viollier, P.H. (2011) Poles apart: prokaryotic polar organelles and their spatial regulation. Cold Spring Harb Perspect Biol 3: pii: a006809. doi:10.1101/cshperspect.a006809 Nambu, T., Minamino, T., Macnab, R.M., and Kutsukake, K. (1999) Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium. J Bacteriol 181: 1555–1561. Scheurwater, E.M., and Burrows, L.L. (2011) Maintaining network security: how macromolecular structures cross the peptidoglycan layer. FEMS Microbiol Lett 318: 1–9. Schniederberend, M., Abdurachim, K., Murray, T.S., and Kazmierczak, B.I. (2013) The GTPase activity of FlhF is dispensable for flagellar localization, but not motility, in Pseudomonas aeruginosa. J Bacteriol 195: 1051–1060. Strahl, H., and Hamoen, L.W. (2010) Membrane potential is important for bacterial cell division. Proc Natl Acad Sci USA 107: 12281–12286. Tammam, S., Sampaleanu, L.M., Koo, J., Manoharan, K., Daubaras, M., Burrows, L.L., and Howell, P.L. (2013) PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA. J Bacteriol 195: 2126–2135. Whitchurch, C.B. (2006) Biogenesis and function of type IV pili in Pseudomonas species. In Pseudomonas. Levesque, J.L.R.a.R.J. (ed.). New York, NY: Kluwer Academic/ Plenum Publishers, pp. 139–188. Young, K.D. (2006) The selective value of bacterial shape. Microbiol Mol Biol Rev 70: 660–703.
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 90, 919–922
doi:10.1111/mmi.12433 First published online 30 October 2013
MicroCommentary A new route for polar navigation Lori L. Burrows* Department of Biochemistry and Biomedical Sciences, McMaster University, Rm 4H18, 1280 Main St. W., Hamilton, Ontario, Canada, L8S4K1.
Summary Placement of motility structures at the poles of rodshaped bacteria is a common engineering problem with a variety of potential solutions. While investigating the mechanisms for positioning of the single polar flagellum of Pseudomonas aeruginosa, Cowles and colleagues discovered a new membrane-bound threecomponent system related to TonB–ExbB–ExbD that they named ‘Poc’ for polar organelle co-ordinator, which controls polar localization of both the flagellum and type IV pili. The Poc complex itself is not found at the poles, and is required for increased expression of pilus genes upon surface association, suggesting a new paradigm of localization control. Motility is a widespread bacterial trait, allowing them to seek out favourable environments and avoid unpleasant ones. Flagella are long helical filaments whose rotation propels bacteria through fluids via swimming motility (Berg, 2003). Type IV pili (T4P) are thinner filaments whose repeated extension (assembly) and retraction (disassembly) is used – similar to a grappling hook – for twitching motility on surfaces (Burrows, 2012). In rod-shaped bacteria, both these organelles are located at the poles, so that motility occurs preferentially in the direction of the long axis of the cell. This is the most efficient arrangement for reducing shear forces on the cell body, as it presents the smallest surface area going forward. In addition, localization of adhesive organelles such as T4P to the poles lets bacteria adhere more efficiently to surfaces by initially presenting a smaller surface area at a distance, minimizing electrostatic repulsion (Young, 2006). Although the flagellum and T4P are both located at the poles in Pseudomonas aeruginosa, their specific localization patterns differ. The single flagellum is located only at Accepted 15 October, 2013. *For correspondence. E-mail burrowl@ mcmaster.ca; Tel. (+1) 905 525 9140 x22029; Fax (+1) 905 522 9033.
© 2013 John Wiley & Sons Ltd
the old pole, while T4P assembly systems are typically bipolar, though pilus filaments can be assembled at either or both poles. A previous study by Cowles and Gitai (2010) showed that patterns of flagellum and T4P regulation were different, also suggesting that the two complexes become polar via independent routes. This initial hypothesis was supported in their current study (Cowles et al., 2013) by the results of time-lapse imaging of dividing cells. Fluorescent fusions to flagellar proteins FlhF and CheA – shown previously to be polarly localized, and in the case of FlhF, to control localization of the flagellum – formed bright, immotile foci at old poles, with new puncta appearing over time only at sites of incipient cell division. A fluorescent fusion to the T4P retraction ATPase PilT was also polar, though its localization was less predictable and sometimes motile, consistent with a previous study showing that its localization depends on the dynamic cytoskeletal protein, MreB (Cowles and Gitai, 2010). To identify determinants of polar flagellum localization in P. aeruginosa, Cowles et al. (2013) looked for transposon insertion mutants with reduced swimming motility, a phenotype associated with mislocalized flagella. Among more than 240 such mutants – most of which had no flagella – they found those with mutations in genes encoding known regulators FlhF and FleN (which controls the number of flagella produced), and three new mutants with insertions in PA0406, PA2982 and PA2983. The first two genes were identified previously in a screen for P. aeruginosa mutants that were avirulent in a Drosophila infection model (D’Argenio et al., 2001). Although both are required for twitching motility, other T4P mutants were fully virulent in Drosophila, hinting even then that PA0406 and PA2983 had additional roles (Whitchurch, 2006). PA0406 encodes a TonB homologue named TonB3 (Huang et al., 2004), while the others encode homologues of ExbB (PA2983) and ExbD (PA2982). In Escherichia coli, TonB–ExbB–ExbD form a complex that is important for energizing siderophore uptake (Braun, 1995). Based on Cowles and colleagues’ subsequent detailed characterization that revealed effects on both flagellum and T4P localization, PA2983 and PA2982 were named PocA and PocB (polar organelle co-ordination). Like their E. coli homologues, PocAB could be co-precipitated, suggestive of complex formation. Their interaction did not depend on
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TonB3, and the investigators were unable to demonstrate TonB3 interaction with either Poc protein. However, their common phenotypes suggested a functional association. Clean deletions confirmed that all three genes were necessary for normal swimming, and for polar placement of the flagellum. The GTPase FlhF was previously considered the topmost component in the polar localization hierarchy for the flagellum of P. aeruginosa, controlling not just localization, but also function (Kazmierczak and Hendrixson, 2013; Schniederberend et al., 2013). However, FlhF has now been dethroned by the Poc complex, as a FlhF–GFP fusion traded its wild-type bipolar localization for non-polar sites – correlated with the positions of non-polar flagella – when any of TonB3, PocA or PocB was missing. The previous identification of TonB3 as a component affecting twitching motility prompted Cowles and coworkers to take a closer look at T4P localization in the new mutants, leading to some surprising results. T4P localization was markedly affected, with approximately 70% of cells having non-polar pili, but only in the PocA mutant – the other two had no pili. This finding was consistent with previous observations that only tonB3 and pocB mutants were avirulent in Drosophila (D’Argenio et al., 2001). Subsequent studies of pilin gene regulation showed that the tonB3 and pocB mutants failed to upregulate pilA expression on solid surfaces, while lack of pocA had no effect. Huang et al. (2004) reported previously that tonB3 mutants had substantially reduced (though not completely absent) motility and less surface pili than the wild type, while the current study reported no motility and almost complete lack of piliation when tonB3 and pocB mutants were imaged by electron microscopy. These minor discrepancies may relate to the different P. aeruginosa strains used (PAK in the 2004 study, versus PAO1 in the current one). Although the pocA mutant had non-polar T4P, its twitching motility was impaired. The investigators suggested that retraction of mis-localized pili along multiple and opposing vectors resulted in essentially zero net motility, although pilus functionality was not formally tested. The discrepancy in pilus expression between the three mutants could not be readily explained by looking at markers of T4P complex localization, as both GFP–PilT and PilQ–mCherry – normally polar – were diffuse in all cases. Flagella and T4P are large multiprotein complexes that span all compartments of the cell envelope, including the covalently closed peptidoglycan layer (Scheurwater and Burrows, 2011). The finding that the loss of the Poc proteins caused aberrant placement of these complexes begs the question of how they could be assembled at places where they’re not normally found. Flagellum synthesis typically occurs at the old pole after cell division and involves a muramidase (FlgJ) that creates an opening for the complex in the PG layer (Nambu et al., 1999;
Kazmierczak and Hendrixson, 2013). It is possible that the muramidase can allow the flagellum to pass through the cell envelope in non-polar locations. In contrast, T4P systems in general lack PG-degrading enzymes. However, if T4P assembly were normally co-ordinated with cell division, nascent complexes could be targeted to the site of septum synthesis for incorporation during synthesis of new poles, a ‘pre-installation’ strategy, instead of the ‘retrofitting’ strategy used by flagella. The association of flagellar and T4P components with septa was noted by Cowles and co-workers when they generated filamentous cells using the cell division inhibitor, cefsulodin. The recruitment of T4P systems to developing septa might be driven by the similarity of specific components to cell division proteins. For example, PilM – a component of the transenvelope T4P assembly complex – has striking structural similarity to FtsA, an early recruit to the contractile Z-ring that directs inward synthesis of the cell envelope (van den Ent and Lowe, 2000; Karuppiah and Derrick, 2011; Tammam et al., 2013). PilM interactions with the Z-ring could position T4P systems at the appropriate location for incorporation into the new poles of each daughter cell (Fig. 1A). With this scenario in mind, it is possible to imagine that disruption of such a co-ordinated process might lead to lack of pili as seen in the tonB3 and pocB mutants. However, it is less clear how T4P assembly complexes might be mis-incorporated into the cell envelopes of pocA mutants, particularly since all three mutants showed complete delocalization of PilT and PilQ, instead of colocalization of these markers with non-polar pili in the pocA background. Careful investigation of the state of the T4P assembly system in pocA mutants will be useful, since the process by which it is normally directed to the poles and assembled through all layers of the cell envelope remains largely mysterious. Many studies of polar localization mechanisms have suffered from a chicken and egg problem; if protein X localizes to the pole via its interactions with polar component Y, then the question becomes how Y became localized to the pole, and so on. Intriguingly, Cowles and co-workers found that the Poc complex itself is not polar (the localization of TonB3 could not be determined due to low levels of expression), leaving the mechanism by which it directs localization of downstream proteins an open question. In the case of flagella, the TonB3–PocAB system appears to control the placement of FlhF; when it ends up at a non-polar site, that’s where the flagellum is made (Fig. 1B). The effects of the TonB3–PocAB system on T4P expression are more complicated, and are linked to transcriptional regulation of pilus genes in response to surface association, as well as to placement of T4P. The lack of transcriptional upregulation of pilus genes in response to surface contact was interesting, and may point to a con© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 90, 919–922
A new route for polar navigation 921
Fig. 1. Co-ordination of flagellum and type IV pilus complex assembly with cell division.A. The GTPase FlhF (square symbol) positions the flagellum at the old pole (dark blue). When cells begin the division cycle, it is recruited to the ‘new old’ pole of the flagellum-less daughter cell (light blue), where a flagellum will subsequently be assembled. The exact determinants for polar localization of type IV pili (T4P) in P. aeruginosa are not known, but the complex (triangle symbol) might be recruited to the septum of dividing cells, resulting in the insertion of T4P systems at the new poles (pink) of each daughter cell after division. T4P can be assembled at either or both poles.B. The TonB3, PocA and PocB proteins are predicted to be localized to the inner membrane (IM), and to have similar topologies to their homologues TonB, ExbD and ExbB respectively. When any of the three is missing, FlhF is mislocalized and the flagellum is assembled at non-polar sites (left). When TonB3 or PocB is missing, no T4P are produced, but lack of PocA results in production of non-polar pili (right).
sequence, rather than cause, of not making pili. P. aeruginosa has complex and interconnected regulatory networks controlling its rapid adaptation to changes in lifestyle, including switching from detached to attached states (Coggan and Wolfgang, 2012). Loss of T4P has been linked to decreased synthesis of cAMP, an important secondary messenger that controls the expression of surface-associated virulence factors, including T4P themselves (Fulcher et al., 2010). Those data imply a feedforward mechanism, where T4P are needed to trigger a signal leading to the synthesis of – among other things – more pili. The TonB3–PocAB system may represent a specialized layer of this surface-sensing repertoire, as Cowles and colleagues found that it was mainly genes associated with pilus biogenesis – rather than the larger set that are altered by surface association – whose expression became non-responsive in tonB3 and pocB backgrounds. One might imagine that TonB3–PocAB are part of the earliest response to surface contact, which pili can mediate at a distance while the cell body is still in a © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 90, 919–922
liquid environment. P. aeruginosa may use its T4P as ‘whiskers’ to report when a surface is initially detected – perhaps via forces generated on the pilus assembly system upon tethering of a pilus filament – triggering downstream regulatory events that could lead eventually to more robust surface interactions. This idea is consistent with TonB3 and PocB’s predicted periplasmic location, where they could be influenced by interactions with the peptidoglycan skeleton or the pilus assembly system, while PocA is mostly cytoplasmic (Fig. 1B). The sequence and architectural similarities of TonB3– PocAB to the canonical TonB–ExbBD system, which relies on the proton motive force for its function, hint at a possible energy transduction role. Cowles et al. noted a previous report stating that perturbations of membrane potential in Bacillus subtilis affect the localization of proteins such as MinD, FtsA and MreB that control cell elongation and division (Strahl and Hamoen, 2010). Thus, loss of TonB3, PocA and/or PocB could have membrane potential-related effects on localization of these and related proteins such as PilM (above). Interestingly, FlhF, which is mislocalized when any of the three proteins of interest is missing, has been linked to the control of septum placement in Campylobacter jejuni. In that species, flhF mutants form minicells, which typically result from formation of Z-rings at sites other than mid-cell (Balaban and Hendrixson, 2011). In the current study, loss of any of tonB3, pocA or pocB resulted in cells that were 15% shorter than wild type, implying a possible disconnect in the co-ordination of cell elongation with cell division. Interestingly, a double knockout of pocAB could not be made, suggesting potential lethality that might relate to disruptions in membrane potential or cell division; these ideas remain to be tested. Cowles and co-workers have produced a thoughtprovoking study which emphasizes that bacteria, formerly thought to be relatively homogenous in spatial organization, instead have sophisticated and interconnected mechanisms for ensuring that things get to where they need to be, when they need to be there (Kirkpatrick and Viollier, 2011). The fact that close to 40% of the tonB3, pocA and pocB mutants still had correctly localized flagella, coupled with the finding that the three gene products have dissimilar effects on T4P biogenesis, show that there are multiple and redundant mechanisms for polar localization of motility structures that remain to be discovered.
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