Role of Type IV Pili in Predation by Bdellovibrio Bacteriovorus

Bdellovibrio bacteriovorus, as an obligate predator of Gram-negative bacteria, requires contact with the surface of a prey cell in order to initiate the life cycle. After attachment, the predator penetrates the prey cell outer membrane and enters the periplasmic space. Attack phase cells of B. bacteriovorus have polar Type IV pili that are required for predation. In other bacteria, these pili have the ability to extend and retract via the PilT protein. B. bacteriovorus has two pilT genes, pilT1 and pilT2, that have been implicated in the invasion process. Markerless in-frame deletion mutants were constructed in a prey-independent mutant to assess the role of PilT1 and PilT2 in the life cycle. When predation was assessed using liquid cocultures, all mutants produced bdelloplasts of Escherichia coli. These results demonstrated that PilT1 and PilT2 are not required for invasion of prey cells. Predation of the mutants on biofilms of E. coli was also assessed. Wild type B. bacteriovorus 109JA and the pilT1 mutant decreased the mass of the biofilm to 35.4% and 27.9% respectively. The pilT1pilT2 mutant was able to prey on the biofilm, albeit less efficiently with 50.2% of the biofilm remaining. The pilT2 mutant was unable to disrupt the biofilm, leaving 92.5% of the original biofilm after predation. The lack of PilT2 function may impede the ability of B. bacteriovorus to move in the extracellular polymeric matrix and find a prey cell. The role of Type IV pili in the life cycle of B. bacteriovorus is thus for initial recognition of and attachment to a prey cell in liquid cocultures, and possibly for movement within the matrix of a biofilm

Editorial:Mechanisms of Prokaryotic Predation

No abstract available

Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

<p>Abstract</p> <p>Background</p> <p><it>Rhizobium leguminosarum </it>bv. <it>viciae </it>establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera <it>Vicia, Lathyrus, Pisum </it>and <it>Lens</it>. Motility and chemotaxis are important in the ecology of <it>R. leguminosarum </it>to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.</p> <p>Results</p> <p><it>R. leguminosarum </it>strains 3841 and VF39SM have seven flagellin genes (<it>flaA</it>, <it>flaB, flaC, flaD, flaE, flaH</it>, and <it>flaG</it>), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. <it>flaA, flaB, flaC</it>, and <it>flaD </it>are in tandem array and are located in the main flagellar gene cluster. <it>flaH </it>and <it>flaG </it>are located outside of the flagellar/motility region while <it>flaE </it>is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of <it>flaA </it>resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of <it>flaB </it>and <it>flaC </it>resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in <it>flaD, flaE, flaH</it>, and <it>flaG </it>were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in <it>flaD </it>and <it>flaG </it>were not significantly affected while <it>flaE </it>and <it>flaH </it>mutants exhibited shortened filaments and reduced swimming motility.</p> <p>Conclusion</p> <p>The results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for <it>R. leguminosarum </it>strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.</p

Individual nutrition therapy and exercise regime: A controlled trial of injured, vulnerable elderly (INTERACTIVE trial)

© 2008 Thomas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Proximal femoral fractures are amongst the most devastating consequences of osteoporosis and injurious accidental falls with 25–35% of patients dying in the first year post-fracture. Effective rehabilitation strategies are evolving however, despite established associations between nutrition, mobility, strength and strength-related functional outcomes; there has been only one small study with older adults immediately following fragility fracture where a combination of both exercise and nutrition have been provided. The aim of the INTERACTIVE trial is to establish whether a six month, individualised exercise and nutrition program commencing within fourteen days of surgery for proximal femur fracture, results in clinically and statistically significant improvements in physical function, body composition and quality of life at an acceptable level of cost and resource use and without increasing the burden of caregivers. Methods and Design This randomised controlled trial will be performed across two sites, a 500 bed acute hospital in Adelaide, South Australia and a 250 bed acute hospital in Sydney, New South Wales. Four hundred and sixty community-dwelling older adults aged > 70 will be recruited after suffering a proximal femoral fracture and followed into the community over a 12-month period. Participants allocated to the intervention group will receive a six month individualised care plan combining resistance training and nutrition therapy commencing within 14 days post-surgery. Outcomes will be assessed by an individual masked to treatment allocation at six and 12 months. To determine differences between the groups at the primary end-point (six months), ANCOVA or logistic regression will be used with models adjusted according to potential confounders. Discussion The INTERACTIVE trial is among the first to combine nutrition and exercise therapy as an early intervention to address the serious consequence of rapid deconditioning and weight loss and subsequent ability to regain pre-morbid function in older patients post proximal femoral fracture. The results of this trial will guide the development of more effective rehabilitation programs, which may ultimately lead to reduced health care costs, and improvements in mobility, independence and quality of life for proximal femoral fracture sufferers. Trial registration Australian Clinical Trials Registry: ACTRN12607000017426

PCR oligonucleotide primers used for amplification of <i>pilT1</i> and <i>pilT2</i>.

<p><b>*</b>restriction sites are underlined.</p><p>PCR oligonucleotide primers used for amplification of <i>pilT1</i> and <i>pilT2</i>.</p

Presence of type IV pili on the surface of cells as assessed by electron microscopy and immunofluorescence microscopy.

<p>Statistical significance determined using the Chi-Square test, *p<0.005.</p><p>Presence of type IV pili on the surface of cells as assessed by electron microscopy and immunofluorescence microscopy.</p

Scanning electron microscopy of predation on pre-formed biofilms of <i>E. coli</i>.

<p>(<b>A</b>) Biofilms of <i>E. coli</i> CO1 (arrows) were formed for 48 h on polyvinyl chloride plastic coverslips. Predator cultures (arrowheads) of (B) <i>B. bacteriovorus</i> 109JA, (C) Δ<i>pilT1</i>, (D) Δ<i>pilT2</i> (E) Δ<i>pilT1pilT2</i> were added for a further 24 h.</p

Biofilm predation assay.

<p>Biofilms of <i>E. coli</i> CO1 were pre-formed for 48 h in 96-well microtiter plates. Predator cultures containing either <i>B. bacteriovorus</i> 109JA or a <i>pilT</i> mutant were added and the plates incubated for a further 24 h. Residual biofilm cells were stained with crystal violet and the optical density at 600 nm (OD600) determined (*p<0.005). To exclude secreted factors contributing to the decrease in remaining biofilm, a 0.45 µm filtrate of a <i>B. bacteriovorus</i> 109JA culture used as a control. The percent biofilm remaining relative to the 109JA filtrate is shown on the secondary axis. Data presented were an average of 12-wells per replicate repeated in triplicate. Statistical significance was measured using a 1-way ANOVA with a Bonferonni corrected post-hoc Students T-test, *p<0.005.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p