Encellede Analyse af Bacillus subtilis Biofilm Brug fluorescensmikroskopi og Flowcytometri
Summary
Mikrobielle biofilm udgøres generelt af forskellige subpopulationer af specialiserede celler. Enkelt-celle-analyse af disse subpopulationer kræver anvendelse af fluorescerende reportere. Her beskriver vi en protokol til at visualisere og overvåge flere subpopulationswithin<em> B. subtilis</em> Biofilm med fluorescensmikroskopi og flow-cytometri.
Abstract
Biofilmdannelse er en generel egenskab for næsten alle bakterier 1-6. Når bakterier danner biofilm celler er indkapslet i ekstracellulære matrix, der hovedsagelig udgøres af proteiner og exopolysaccharider, blandt andre faktorer 7-10. Den mikrobielt samfund indkapslet i biofilmen ofte viser differentieringen af særskilte underpopulation af specialiserede celler 11-17. Disse delpopulationer eksistere side om side og ofte viser rumlige og tidslige organisation i biofilmen 18-21.
Biofilm dannelse i model organisme Bacillus subtilis kræver differentiering af forskellige subpopulationer af specialiserede celler. Blandt dem er den subpopulation af matrix producenter ansvarlige for at producere og udskille den ekstracellulære matrix af biofilmen afgørende for biofilmdannelse 11,19. Derfor, differentiering af matrix producenter er kendetegnende for biofilmdannelse i B. subtilis.
Vi har brugt fluorescerende reportere til at visualisere og kvantificere subpopulationen af matrix producenter i biofilm af B. subtilis 15,19,22-24. Konkret har vi observeret, at subpopulationen af matrix producenter differentierer i respons på tilstedeværelsen af selv-produceret ekstracellulært signal surfactin 25. Interessant nok er surfactin fremstilles ved en subpopulation af specialiserede celler forskellige fra den subpopulation af matrix producenter 15.
Vi har beskrevet i denne rapport tekniske tilgang er nødvendige for at visualisere og kvantificere subpopulationen af matrix producenter og surfactin producenter inden for biofilm fra B. subtilis. For at gøre dette, er fluorescerende indberettere af gener, der kræves for matrix produktion og surfactin produktion indsættes i kromosomet af B. subtilis. Reportere udtrykkes kun i en subpopulation af specialiserede celler. Derefter kan subpopulationer væreovervåges ved hjælp af fluorescensmikroskopi og flowcytometri (se figur 1).
Den kendsgerning, at forskellige subpopulationer af specialiserede celler eksisterer i flercellede samfund af bakterier giver os et andet perspektiv om reguleringen af genekspression i prokaryoter. Denne protokol omhandler dette fænomen eksperimentelt, og det kan let tilpasses enhver anden bearbejdning model, at belyse de molekylære mekanismer, der ligger til grund for fænotypiske heterogenitet inden for en mikrobielle samfund.
Protocol
Discussion
Det faktum, at bakterielle samfund viser subpopulationer af celler, der udtrykker specifikt sæt af gener beviser kompleksiteten af mikrobielle samfund 33,34. Denne protokol skal hjælpe til at fastslå, om ekspressionen af ethvert gen af interesse er begrænset til en bestemt underpopulation af specialiserede celler i det mikrobielle samfund. Visualisering af disse delpopulationer kræver udvikling af nye teknikker, fordi de traditionelle metoder til at overvåge genekspression eller microarray a…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde er finansieret af Young Investigator Research Program, fra Center for Infectious Disease Research (ZINF) fra universitetet i Würzburg. Juan C Garcia-Betancur er ph.d.-stipendiat fra Graduate School of Life Sciences (GSL'er) af universitetet i Würzburg.
Materials
| Technique | Name of the reagent | Company | Catalog number |
| MSgg composition | potassium phosphate 5mM | Roth | 6878 |
| MOPS 100mM | Sigma-Aldrich | M1254 | |
| Magnesium chloride 2mM | Roth | 2189.1 | |
| Calcium chloride 700μM | Roth | A119.1 | |
| Ferric chloride 50μM | Sigma-Aldrich | 157740 | |
| Zinc chloride 1μM | Applichem | A2076 | |
| Thiamine 2μM | Sigma-Aldrich | 74625 | |
| Glycerol 0.5% | Roth | 7533 | |
| Glutamate 0.5% | Sigma-Aldrich | 49621 | |
| Tryptophan 50μg/ml | Sigma-Aldrich | T0254 | |
| Phenylalanine 50μg/ml | Sigma-Aldrich | P2126 | |
| Cell fixation | Paraformaldehyde | Roth | 0335 |
| Name of the equipment | Company | Catalog number | |
| Sonication | Cell Sonicator | Bandelin | D-1000 |
| Fluorescence Microscopy | Fluorescence Microscope | Leica | DMI6000B |
| Name of the software | Company | Catalog Number | |
| Fluorescence Microscopy | AsaF | Leica | |
| Flow cytometry | FCASDiva | BD | |
| Flow cytometry | FlowJo | Treestar |
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