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Activity of Bdellovibrio hit locus proteins, Bd0108 and Bd0109, links Type IVa pilus extrusion/retraction status to prey-independent growth signalling.

Capeness MJ, Lambert C, Lovering AL, Till R, Uchida K, Chaudhuri R, Alderwick LJ, Lee DJ, Swarbreck D, Liddell S, Aizawa S, Sockett RE - PLoS ONE (2013)

Bottom Line: We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type.Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108.These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to prey-independent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation backgrounds.

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Related in: MedlinePlus

Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.A. Operonal structure of the bd0108hit locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.B. In wild-type cells bd0108 and bd0109 are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.C. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.D. In HI strains containing the 42 bp deletion variant of bd0108, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.
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pone-0079759-g011: Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.A. Operonal structure of the bd0108hit locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.B. In wild-type cells bd0108 and bd0109 are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.C. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.D. In HI strains containing the 42 bp deletion variant of bd0108, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.

Mentions: Thus we propose a model (Figure 11) in which extrusion/retraction of a Type IVa pilus fibre sends signalling information into to the Bdellovibrio cell to cause it to grow and replicate. We postulate that this signal is sent when the Bdellovibrio invade prey (a Type IVa-pilus-dependent process); but also when growing as HI cultures in a (possibly pilus-associated) contact-dependent process that occurs at high cell densities and not in dilute cell suspensions. We propose that Bd0109 protein, modulated by Bd0108, regulates/monitors pilus extrusion/retraction post translationally. We propose that Bd0109 protein has an essential role by one of two means; 1) it is a recognition mechanism which transmits a growth signal by monitoring the extrusion/retraction of the pilus and without Bd0109, Bdellovibrio are non-viable as HI or HD cells because they don’t receive the growth signal. This signal could be an absence of normal extrusion/retraction that the attack phase Bdellovibrio would carry-out while seeking prey. This absence would be found in ∆bd0108 mutants which have overly-retracted pili, the 42 bp deletion mutants of bd0108 which have overly-extruded pili, and in wild-type attack phase cells having entered prey where pili would then be in a fixed state, possibly attached to the prey cell walls. The signal from Bd0109 could be transmitted into the Bdellovibrio cell by interactions with an as yet unidentified membrane protein. In this model, the Bd0109 is sequestered by Bd0108, and released in a stochastic manner to either interact at the cell wall with the extruding/retracting pilus, or in the presence of a static pilus, interact with an unknown membrane protein to signal cell growth.


Activity of Bdellovibrio hit locus proteins, Bd0108 and Bd0109, links Type IVa pilus extrusion/retraction status to prey-independent growth signalling.

Capeness MJ, Lambert C, Lovering AL, Till R, Uchida K, Chaudhuri R, Alderwick LJ, Lee DJ, Swarbreck D, Liddell S, Aizawa S, Sockett RE - PLoS ONE (2013)

Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.A. Operonal structure of the bd0108hit locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.B. In wild-type cells bd0108 and bd0109 are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.C. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.D. In HI strains containing the 42 bp deletion variant of bd0108, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818213&req=5

pone-0079759-g011: Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.A. Operonal structure of the bd0108hit locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.B. In wild-type cells bd0108 and bd0109 are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.C. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.D. In HI strains containing the 42 bp deletion variant of bd0108, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.
Mentions: Thus we propose a model (Figure 11) in which extrusion/retraction of a Type IVa pilus fibre sends signalling information into to the Bdellovibrio cell to cause it to grow and replicate. We postulate that this signal is sent when the Bdellovibrio invade prey (a Type IVa-pilus-dependent process); but also when growing as HI cultures in a (possibly pilus-associated) contact-dependent process that occurs at high cell densities and not in dilute cell suspensions. We propose that Bd0109 protein, modulated by Bd0108, regulates/monitors pilus extrusion/retraction post translationally. We propose that Bd0109 protein has an essential role by one of two means; 1) it is a recognition mechanism which transmits a growth signal by monitoring the extrusion/retraction of the pilus and without Bd0109, Bdellovibrio are non-viable as HI or HD cells because they don’t receive the growth signal. This signal could be an absence of normal extrusion/retraction that the attack phase Bdellovibrio would carry-out while seeking prey. This absence would be found in ∆bd0108 mutants which have overly-retracted pili, the 42 bp deletion mutants of bd0108 which have overly-extruded pili, and in wild-type attack phase cells having entered prey where pili would then be in a fixed state, possibly attached to the prey cell walls. The signal from Bd0109 could be transmitted into the Bdellovibrio cell by interactions with an as yet unidentified membrane protein. In this model, the Bd0109 is sequestered by Bd0108, and released in a stochastic manner to either interact at the cell wall with the extruding/retracting pilus, or in the presence of a static pilus, interact with an unknown membrane protein to signal cell growth.

Bottom Line: We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type.Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108.These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to prey-independent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation backgrounds.

Show MeSH
Related in: MedlinePlus