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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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PFU to CFU ratio for pooled spontaneous HI strains and the markerless bd0108 deletion HI mutants of Bdellovibrio.In the spontaneous generated HI strains; HID2 and HID26 (wild-type for bd0108) HID6, HID13, HID18, (point mutations in bd0108) and HID22 (bd0108∆42bp), and the markerless deletion mutants of bd0108, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type bd0108 is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of bd0108. The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).
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pone-0079759-g004: PFU to CFU ratio for pooled spontaneous HI strains and the markerless bd0108 deletion HI mutants of Bdellovibrio.In the spontaneous generated HI strains; HID2 and HID26 (wild-type for bd0108) HID6, HID13, HID18, (point mutations in bd0108) and HID22 (bd0108∆42bp), and the markerless deletion mutants of bd0108, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type bd0108 is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of bd0108. The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).

Mentions: There was significant reduction in the number of colony forming units (CFU) on PY agar plates compared to plaque forming units (PFU) on overlayered prey-lawn plates. This was observed for all spontaneous and directed deletion bd0108 mutants carrying pSUP404.2-108 compared to both pSUP404.2 alone, and pSUP404.2-Δ42 (Figure 4). The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced in strains containing pSUP404.2-Δ42; 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and pSUP404.2-Δ42 was also significant (P<0.01). The results of this CFU:PFU assay indicates that expression of a wild-type copy of the bd0108 gene inhibits the ability of Bdellovibrio to grow axenically as an HI culture to some extent in diverse bd0108 mutant backgrounds, an observation in agreement with Roschanski and co-workers who observed that complementation with bd0108 inhibited growth on autoclaved prey [12].


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)

PFU to CFU ratio for pooled spontaneous HI strains and the markerless bd0108 deletion HI mutants of Bdellovibrio.In the spontaneous generated HI strains; HID2 and HID26 (wild-type for bd0108) HID6, HID13, HID18, (point mutations in bd0108) and HID22 (bd0108∆42bp), and the markerless deletion mutants of bd0108, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type bd0108 is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of bd0108. The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818213&req=5

pone-0079759-g004: PFU to CFU ratio for pooled spontaneous HI strains and the markerless bd0108 deletion HI mutants of Bdellovibrio.In the spontaneous generated HI strains; HID2 and HID26 (wild-type for bd0108) HID6, HID13, HID18, (point mutations in bd0108) and HID22 (bd0108∆42bp), and the markerless deletion mutants of bd0108, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type bd0108 is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of bd0108. The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).
Mentions: There was significant reduction in the number of colony forming units (CFU) on PY agar plates compared to plaque forming units (PFU) on overlayered prey-lawn plates. This was observed for all spontaneous and directed deletion bd0108 mutants carrying pSUP404.2-108 compared to both pSUP404.2 alone, and pSUP404.2-Δ42 (Figure 4). The ratio of CFU:PFU was reduced from 2.16 x 10-1 in strains containing the pSUP404.2 alone to 3.21 x 10-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced in strains containing pSUP404.2-Δ42; 2.56 x 10-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and pSUP404.2-Δ42 was also significant (P<0.01). The results of this CFU:PFU assay indicates that expression of a wild-type copy of the bd0108 gene inhibits the ability of Bdellovibrio to grow axenically as an HI culture to some extent in diverse bd0108 mutant backgrounds, an observation in agreement with Roschanski and co-workers who observed that complementation with bd0108 inhibited growth on autoclaved prey [12].

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