Limits...
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.

Show MeSH

Related in: MedlinePlus

RT-PCR expression of the sRNA downstream of bd0108 and expression of a control gene dnaK.Expression of a small, non-coding RNA downstream of bd0108 was shown to be different in HI strains carrying different mutations in bd0108 and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of bd0108. Strain HID22 (bd0108∆42bp), had slightly lower expression, while those strains with a wild-type bd0108; HD100 and HID2, show much lower expression of the sRNA. Expression of dnaK was uniform across the samples indicating a matched amount of total RNA was used for the experiment.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3818213&req=5

pone-0079759-g010: RT-PCR expression of the sRNA downstream of bd0108 and expression of a control gene dnaK.Expression of a small, non-coding RNA downstream of bd0108 was shown to be different in HI strains carrying different mutations in bd0108 and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of bd0108. Strain HID22 (bd0108∆42bp), had slightly lower expression, while those strains with a wild-type bd0108; HD100 and HID2, show much lower expression of the sRNA. Expression of dnaK was uniform across the samples indicating a matched amount of total RNA was used for the experiment.

Mentions: RNA-seq analysis identified a small non-coding RNA located between the transcriptional units of bd0103 and bd0108 (bases 96436-96822 of HD100). Manual inspection of the intergenic region suggested the presence of a sRNA-encoding gene between positions 96455 and 96716 (261 bp). RT-PCR analysis using primers designed to anneal to this RNA shows that in all HI strains, this was significantly up-regulated relative to the HD100 attack phase and to strain HID2 which each have a wild-type bd0108 gene (Figure 10). In the HID22 (bd0108∆42bp) strain the level of upregulation was slightly, but repeatably, lower than in the ∆bd0108 strains and in the HID13 strain which has a start codon point mutation in the bd0108 gene. This fits with the hypothesis that the small non-coding RNA is regulated in association with bd0108 expression.


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)

RT-PCR expression of the sRNA downstream of bd0108 and expression of a control gene dnaK.Expression of a small, non-coding RNA downstream of bd0108 was shown to be different in HI strains carrying different mutations in bd0108 and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of bd0108. Strain HID22 (bd0108∆42bp), had slightly lower expression, while those strains with a wild-type bd0108; HD100 and HID2, show much lower expression of the sRNA. Expression of dnaK was uniform across the samples indicating a matched amount of total RNA was used for the experiment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079759-g010: RT-PCR expression of the sRNA downstream of bd0108 and expression of a control gene dnaK.Expression of a small, non-coding RNA downstream of bd0108 was shown to be different in HI strains carrying different mutations in bd0108 and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of bd0108. Strain HID22 (bd0108∆42bp), had slightly lower expression, while those strains with a wild-type bd0108; HD100 and HID2, show much lower expression of the sRNA. Expression of dnaK was uniform across the samples indicating a matched amount of total RNA was used for the experiment.
Mentions: RNA-seq analysis identified a small non-coding RNA located between the transcriptional units of bd0103 and bd0108 (bases 96436-96822 of HD100). Manual inspection of the intergenic region suggested the presence of a sRNA-encoding gene between positions 96455 and 96716 (261 bp). RT-PCR analysis using primers designed to anneal to this RNA shows that in all HI strains, this was significantly up-regulated relative to the HD100 attack phase and to strain HID2 which each have a wild-type bd0108 gene (Figure 10). In the HID22 (bd0108∆42bp) strain the level of upregulation was slightly, but repeatably, lower than in the ∆bd0108 strains and in the HID13 strain which has a start codon point mutation in the bd0108 gene. This fits with the hypothesis that the small non-coding RNA is regulated in association with bd0108 expression.

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