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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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Plaque assay of predation by Host Independent mutants compared to HD100.Dilution of predatory cultures of Bdellovibrio strains on double layer YPSC overlay plates containing prey E. coli S17-1 in the top layer. Dilution was carried out in Ca/HEPES with 10 µl of the resulting dilution series spotted at positions indicated in the diagram. Plates show there is no significant effect on HD100 having extra copies of the bd0108 gene or the 42 bp deletion variant of bd0108 on plaquing ability. Complementation tests for the HID13 strain (ATG->ATA mutation in bd0108) with the plasmid pSUP404.2 containing the wild-type bd0108 gene showed an increase in plaquing, however this was not observed for the Δbd0108 deletion strain. Plates are representative of at least three independent repeats.
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pone-0079759-g003: Plaque assay of predation by Host Independent mutants compared to HD100.Dilution of predatory cultures of Bdellovibrio strains on double layer YPSC overlay plates containing prey E. coli S17-1 in the top layer. Dilution was carried out in Ca/HEPES with 10 µl of the resulting dilution series spotted at positions indicated in the diagram. Plates show there is no significant effect on HD100 having extra copies of the bd0108 gene or the 42 bp deletion variant of bd0108 on plaquing ability. Complementation tests for the HID13 strain (ATG->ATA mutation in bd0108) with the plasmid pSUP404.2 containing the wild-type bd0108 gene showed an increase in plaquing, however this was not observed for the Δbd0108 deletion strain. Plates are representative of at least three independent repeats.

Mentions: To compare our data to that of Roschanski and Strauch [12] for a bd0108 transposon mutant which they had complemented with a wild-type bd0108 gene, plaquing capability of each strain was also measured. To do this, we used serial dilutions to 10-4 of complemented strains grown in predatory cultures and spotted 10 µl upon dual layer overlay plates containing E. coli in the top layer of agar. For the spontaneous HI isolates tested, HID13 (and HID22 - data not shown) complementation with pSUP404.2-108 enhanced the plaquing capability compared to both that of pSUP404.2 alone and pSUP404.2-Δ42 (Figure 3). It had previously been reported that complementation of the reduced plaquing in HI mutants could be achieved by crossover [26] as well as in trans by the addition of wild-type bd0108 [12]. We observed a similar complementation effect, except we found that plaquing ability was a very variable phenotype. Here we show a strain with a bd0108 point mutation was clearly complemented (as in previous reports), but the Δbd0108 markerless deletion strains had strong HI growth, masking the area of clearing in both control and complemented strains and thus making the assay hard to read. It was also noted that in some cases, the lower dilution spots of Bdellovibrio gave clearer plaquing, again indicating that the presence of more HI cells in the higher dilutions was impeding predatory 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)

Plaque assay of predation by Host Independent mutants compared to HD100.Dilution of predatory cultures of Bdellovibrio strains on double layer YPSC overlay plates containing prey E. coli S17-1 in the top layer. Dilution was carried out in Ca/HEPES with 10 µl of the resulting dilution series spotted at positions indicated in the diagram. Plates show there is no significant effect on HD100 having extra copies of the bd0108 gene or the 42 bp deletion variant of bd0108 on plaquing ability. Complementation tests for the HID13 strain (ATG->ATA mutation in bd0108) with the plasmid pSUP404.2 containing the wild-type bd0108 gene showed an increase in plaquing, however this was not observed for the Δbd0108 deletion strain. Plates are representative of at least three independent repeats.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079759-g003: Plaque assay of predation by Host Independent mutants compared to HD100.Dilution of predatory cultures of Bdellovibrio strains on double layer YPSC overlay plates containing prey E. coli S17-1 in the top layer. Dilution was carried out in Ca/HEPES with 10 µl of the resulting dilution series spotted at positions indicated in the diagram. Plates show there is no significant effect on HD100 having extra copies of the bd0108 gene or the 42 bp deletion variant of bd0108 on plaquing ability. Complementation tests for the HID13 strain (ATG->ATA mutation in bd0108) with the plasmid pSUP404.2 containing the wild-type bd0108 gene showed an increase in plaquing, however this was not observed for the Δbd0108 deletion strain. Plates are representative of at least three independent repeats.
Mentions: To compare our data to that of Roschanski and Strauch [12] for a bd0108 transposon mutant which they had complemented with a wild-type bd0108 gene, plaquing capability of each strain was also measured. To do this, we used serial dilutions to 10-4 of complemented strains grown in predatory cultures and spotted 10 µl upon dual layer overlay plates containing E. coli in the top layer of agar. For the spontaneous HI isolates tested, HID13 (and HID22 - data not shown) complementation with pSUP404.2-108 enhanced the plaquing capability compared to both that of pSUP404.2 alone and pSUP404.2-Δ42 (Figure 3). It had previously been reported that complementation of the reduced plaquing in HI mutants could be achieved by crossover [26] as well as in trans by the addition of wild-type bd0108 [12]. We observed a similar complementation effect, except we found that plaquing ability was a very variable phenotype. Here we show a strain with a bd0108 point mutation was clearly complemented (as in previous reports), but the Δbd0108 markerless deletion strains had strong HI growth, masking the area of clearing in both control and complemented strains and thus making the assay hard to read. It was also noted that in some cases, the lower dilution spots of Bdellovibrio gave clearer plaquing, again indicating that the presence of more HI cells in the higher dilutions was impeding predatory 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