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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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Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.For Host Dependent cells (A) with a genomic copy of the wild-type bd0108 gene, carrying the pSUP404.2 plasmid encoding either the wild-type bd0108 gene or the 42 bp deletion variant of bd0108 had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more Bdellovibrio initially added and shows that the extra copies of bd0108 or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (B) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type bd0108 gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type bd0108, but not the 42 bp deletion variant, intrans restores the HI cells to a predatory lifestyle.
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pone-0079759-g002: Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.For Host Dependent cells (A) with a genomic copy of the wild-type bd0108 gene, carrying the pSUP404.2 plasmid encoding either the wild-type bd0108 gene or the 42 bp deletion variant of bd0108 had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more Bdellovibrio initially added and shows that the extra copies of bd0108 or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (B) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type bd0108 gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type bd0108, but not the 42 bp deletion variant, intrans restores the HI cells to a predatory lifestyle.

Mentions: HI grown Bdellovibrio are morphologically diverse and this makes matching their cell numbers difficult as discussed in the previous section. To get around this we used a predation assay on standardised numbers of luminescent E. coli prey over the course of 48 hours. From this it was possible to assay the efficiency of predation as it correlated with the initial numbers of starting Bdellovibrio (Figure 2), without the need for matching initial amounts by centrifuge concentration such as Percoll gradients [21] which could have a profound effect on the physiological state of the cells. This method has previously been used to measure more subtle deficiencies in predation of populations of mutant Bdellovibrio [22-24] and also avoids any potential bias in matching cells of different size by total protein.


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)

Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.For Host Dependent cells (A) with a genomic copy of the wild-type bd0108 gene, carrying the pSUP404.2 plasmid encoding either the wild-type bd0108 gene or the 42 bp deletion variant of bd0108 had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more Bdellovibrio initially added and shows that the extra copies of bd0108 or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (B) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type bd0108 gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type bd0108, but not the 42 bp deletion variant, intrans restores the HI cells to a predatory lifestyle.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079759-g002: Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.For Host Dependent cells (A) with a genomic copy of the wild-type bd0108 gene, carrying the pSUP404.2 plasmid encoding either the wild-type bd0108 gene or the 42 bp deletion variant of bd0108 had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more Bdellovibrio initially added and shows that the extra copies of bd0108 or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (B) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type bd0108 gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type bd0108, but not the 42 bp deletion variant, intrans restores the HI cells to a predatory lifestyle.
Mentions: HI grown Bdellovibrio are morphologically diverse and this makes matching their cell numbers difficult as discussed in the previous section. To get around this we used a predation assay on standardised numbers of luminescent E. coli prey over the course of 48 hours. From this it was possible to assay the efficiency of predation as it correlated with the initial numbers of starting Bdellovibrio (Figure 2), without the need for matching initial amounts by centrifuge concentration such as Percoll gradients [21] which could have a profound effect on the physiological state of the cells. This method has previously been used to measure more subtle deficiencies in predation of populations of mutant Bdellovibrio [22-24] and also avoids any potential bias in matching cells of different size by total protein.

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