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Detection of c-di-GMP at single cell and population levels in Bacillus subtilis

Ezzati, Yasamin (2022) MOBN02 20221
Degree Projects in Molecular Biology
Popular Abstract
Detection of c-di-GMP level in Bacillus subtilis with Biosensors

Cyclic dinucleotides are signaling molecules that control several critical processes in bacteria. The most studied example is cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP) which is a universal bacterial second messenger. C-di-GMP is known to affect several cellular processes such as biofilm formation and dispersion, regulation of the cell cycle, differentiation, motility, and virulence. C-di-GMP is formed by joining two molecules of guanosine triphosphate (GTP) and is degraded by phosphodiesterases (PDEs). Bacillus subtilis has three DGCs: DgcP, DgcK, and DgcW and one PDE (PdeH). To detect c-di-GMP levels in vivo various fluorescence-based biosensors have been... (More)
Detection of c-di-GMP level in Bacillus subtilis with Biosensors

Cyclic dinucleotides are signaling molecules that control several critical processes in bacteria. The most studied example is cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP) which is a universal bacterial second messenger. C-di-GMP is known to affect several cellular processes such as biofilm formation and dispersion, regulation of the cell cycle, differentiation, motility, and virulence. C-di-GMP is formed by joining two molecules of guanosine triphosphate (GTP) and is degraded by phosphodiesterases (PDEs). Bacillus subtilis has three DGCs: DgcP, DgcK, and DgcW and one PDE (PdeH). To detect c-di-GMP levels in vivo various fluorescence-based biosensors have been developed.

In this work, two different biosensors were evaluated for use in the Gram-positive model bacterium Bacillus subtilis. The first biosensor called CensYBL is based on split fluorescent protein technology. When CBldD binds tetrameric c-di-GMP, it dimerizes, which leads to the complementation of the two YPet fragments and results in the restoration of the yellow fluorescent protein (YFP), and the fluorescence can be detected by using a fluorescence microscope. The second c-di-GMP biosensor is based on a c-di-GMP responsive riboswitch named lchAA and has already been shown to function in B. subtilis. The lchAA riboswitch is located downstream of a constitutive promoter (Pconst) and in front of a gene encoding YFP. YFP expression is inhibited by the riboswitch terminator when intracellular c-di-GMP levels are high. Low c-di-GMP levels will cause YFP to be expressed and fluorescence to be observed. The overall aim of the project was to evaluate and validate two c-di-GMP biosensors (CensYBL and riboswitch) for use in B. subtilis. Our observation and data show that only the riboswitch biosensor provided reliable estimates of the c-di-GMP levels in vivo; the CensYBL biosensor gave reliable result only at low levels of c-di-GMP.

Master’s Degree Project in Molecular Biology/ Microbiology 45 credits 2022
Department of Biology, Lund University

Supervisour: Claes von Wachenfeldt / department of Biology/ Lund University (Less)
Please use this url to cite or link to this publication:
author
Ezzati, Yasamin
supervisor
organization
course
MOBN02 20221
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9113581
date added to LUP
2023-04-18 12:56:13
date last changed
2023-04-18 12:56:13
@misc{9113581,
  author       = {{Ezzati, Yasamin}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Detection of c-di-GMP at single cell and population levels in Bacillus subtilis}},
  year         = {{2022}},
}