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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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Evidence of interaction between Bd0108 and Bd0109.A. Fluorescence quenching assay of W fluorescence of Bd0109 by addition of (naturally non-fluorescent) Bd0108 protein. Bd0109 at a concentration of 2.41 µM was titrated with Bd0108 at 1.95 mM in non-reducing buffer. Increasing concentrations of Bd0108 resulted in increased quenching of fluorescence indicating interaction between the two proteins.B. Protease protection assay with Bd0109 and Bd0108 using chymotrypsin. The monomer of Bd0108 is around 17 kDa and the monomer of Bd0109 is around 65 kDa. Extra bands begin to appear around 35 and 43 kDa (indicated by asterisks) in the Bd0109 lanes treated with chymotrypsin which represent degradation products of Bd0109. These products cannot be seen in the lanes containing a complex of the two proteins (labelled complex) indicating that Bd0108 is interacting with Bd0109 to protect it from chymotrypsin digestion. Times indicated at the top are in minutes from adding the chymotrypsin.
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pone-0079759-g006: Evidence of interaction between Bd0108 and Bd0109.A. Fluorescence quenching assay of W fluorescence of Bd0109 by addition of (naturally non-fluorescent) Bd0108 protein. Bd0109 at a concentration of 2.41 µM was titrated with Bd0108 at 1.95 mM in non-reducing buffer. Increasing concentrations of Bd0108 resulted in increased quenching of fluorescence indicating interaction between the two proteins.B. Protease protection assay with Bd0109 and Bd0108 using chymotrypsin. The monomer of Bd0108 is around 17 kDa and the monomer of Bd0109 is around 65 kDa. Extra bands begin to appear around 35 and 43 kDa (indicated by asterisks) in the Bd0109 lanes treated with chymotrypsin which represent degradation products of Bd0109. These products cannot be seen in the lanes containing a complex of the two proteins (labelled complex) indicating that Bd0108 is interacting with Bd0109 to protect it from chymotrypsin digestion. Times indicated at the top are in minutes from adding the chymotrypsin.

Mentions: As the bd0108 and bd0109 genes were co-transcribed, we postulated that they might work together in regulating the switch to HI growth by Bdellovibrio. Thus both genes were expressed and the proteins they encoded were purified. The natural tryptophan content of Bd0109 and the absence of tryptophan in Bd0108 allowed us to study the interactions of the purified gene products of the two adjacent hit locus genes (Figure 6). It was clear that addition of Bd0108 protein quenched the fluorescence of Bd0109 showing that the two proteins interact (Figure 6A). Also interaction of the proteins before the administration of the protease chymotrypsin protected Bd0109 against proteolytic digest (Figure 6B). Thus we propose that Bd0109 and Bd0108 interact in vivo. As both proteins have predicted N terminal secretion signals (see below) this interaction could take place in the Bdellovibrio periplasm or externally.


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)

Evidence of interaction between Bd0108 and Bd0109.A. Fluorescence quenching assay of W fluorescence of Bd0109 by addition of (naturally non-fluorescent) Bd0108 protein. Bd0109 at a concentration of 2.41 µM was titrated with Bd0108 at 1.95 mM in non-reducing buffer. Increasing concentrations of Bd0108 resulted in increased quenching of fluorescence indicating interaction between the two proteins.B. Protease protection assay with Bd0109 and Bd0108 using chymotrypsin. The monomer of Bd0108 is around 17 kDa and the monomer of Bd0109 is around 65 kDa. Extra bands begin to appear around 35 and 43 kDa (indicated by asterisks) in the Bd0109 lanes treated with chymotrypsin which represent degradation products of Bd0109. These products cannot be seen in the lanes containing a complex of the two proteins (labelled complex) indicating that Bd0108 is interacting with Bd0109 to protect it from chymotrypsin digestion. Times indicated at the top are in minutes from adding the chymotrypsin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818213&req=5

pone-0079759-g006: Evidence of interaction between Bd0108 and Bd0109.A. Fluorescence quenching assay of W fluorescence of Bd0109 by addition of (naturally non-fluorescent) Bd0108 protein. Bd0109 at a concentration of 2.41 µM was titrated with Bd0108 at 1.95 mM in non-reducing buffer. Increasing concentrations of Bd0108 resulted in increased quenching of fluorescence indicating interaction between the two proteins.B. Protease protection assay with Bd0109 and Bd0108 using chymotrypsin. The monomer of Bd0108 is around 17 kDa and the monomer of Bd0109 is around 65 kDa. Extra bands begin to appear around 35 and 43 kDa (indicated by asterisks) in the Bd0109 lanes treated with chymotrypsin which represent degradation products of Bd0109. These products cannot be seen in the lanes containing a complex of the two proteins (labelled complex) indicating that Bd0108 is interacting with Bd0109 to protect it from chymotrypsin digestion. Times indicated at the top are in minutes from adding the chymotrypsin.
Mentions: As the bd0108 and bd0109 genes were co-transcribed, we postulated that they might work together in regulating the switch to HI growth by Bdellovibrio. Thus both genes were expressed and the proteins they encoded were purified. The natural tryptophan content of Bd0109 and the absence of tryptophan in Bd0108 allowed us to study the interactions of the purified gene products of the two adjacent hit locus genes (Figure 6). It was clear that addition of Bd0108 protein quenched the fluorescence of Bd0109 showing that the two proteins interact (Figure 6A). Also interaction of the proteins before the administration of the protease chymotrypsin protected Bd0109 against proteolytic digest (Figure 6B). Thus we propose that Bd0109 and Bd0108 interact in vivo. As both proteins have predicted N terminal secretion signals (see below) this interaction could take place in the Bdellovibrio periplasm or externally.

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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