Advanced

Investigating the effect of phosphorylation on DivIVA oligomerization in Streptomyces coelicolor

Kazakevych, Juri (2012) MOBM18 20121
Degree Projects in Molecular Biology
Abstract
Streptomyces belong to the phylum Actinobacteria, which are Gram-positive bacteria with GC-rich DNA. Most Streptomyces are strict aerobes and inhabit soil. They are known for production of a wide range of secondary metabolites, including antibiotics e.g. the macrolide erythromycin (S. erythraeus) or chloramphenicol (S. venezuelae). The typical smell of wet soil is owed to geosmin, also a secondary metabolite of Streptomyces.

Unlike most other bacteria, streptomycetes resemble fungi in the sense that they grow by tip extension and form a branching vegetative mycelium containing multiple chromosome copies in each hyphal cell. Shortage of nutrients induces formation of aerial mycelium. The aerial mycelium consists of long hyphae finally... (More)
Streptomyces belong to the phylum Actinobacteria, which are Gram-positive bacteria with GC-rich DNA. Most Streptomyces are strict aerobes and inhabit soil. They are known for production of a wide range of secondary metabolites, including antibiotics e.g. the macrolide erythromycin (S. erythraeus) or chloramphenicol (S. venezuelae). The typical smell of wet soil is owed to geosmin, also a secondary metabolite of Streptomyces.

Unlike most other bacteria, streptomycetes resemble fungi in the sense that they grow by tip extension and form a branching vegetative mycelium containing multiple chromosome copies in each hyphal cell. Shortage of nutrients induces formation of aerial mycelium. The aerial mycelium consists of long hyphae finally maturing and dividing into monochromosomal spores. S. coelicolor is the most used model organism for genetic and associated functional investigations of streptomycetes.

Regulation of tip extension in S. coelicolor
While many bacteria, e.g. Escherichia coli, grow by extending the lateral cell wall, S. coelicolor has a growth pattern restricted to hyphal tips. The essential protein DivIVA is mediating this localization as well as the emergence of new hyphal branches, shown by studies of fluorescent fusion proteins and time-lapse imaging: cell wall synthesis occurs only at DivIVA-foci. Homologs of DivIVA can also be found mediating apical growth in other actinobacteria e.g. Mycobacterium tuberculosis. Most Gram-positive bacteria possess DivIVA-homologs involved in cell growth, division or polarity. S. coelicolor DivIVA is a 41 kDa α-helical coiled-coil protein. Based on crystal structure and electron microscopy, Bacillus subtilis DivIVA is suggested to build tetrameric or higher order oligomeric structures.

S. coelicolor DivIVA was recently shown to be phosphorylated by the Ser/Thr-kinase AfsK with strong effect on DivIVA localization, apical growth and increased branching frequence. Phosphorylation of DivIVA is reversible in vivo with 5 identified phosphorylation in the C-terminal region. This α-helical region is suggested to overlap in di- and tetrameres and phosphorylation may therefore affect the regulation of oligomerization. Oligomerization level regulation is not restricted to Streptomyces, effects of phosphorylation on protein interaction and oligomerization are wide spread and can be observed e.g. on the yeast actin cytoskeleton.

Popular science summary:

Oligomerization of native and phosphorylated DivIVA

The aim of this project was to investigate how phosphorylation affects the properties of the DivIVA protein and specificially to determine whether oligomerization of DivIVA is affected by phosphorylation status. Oligomerization levels were analyzed by methods responding to the relative size of protein complexes, including light scattering assays, Blue Native/Clear Native Polyacrylamide Gel Electrophoresis (BN/CN-PAGE) and gel filtration followed by Dot Blot immunostaining. Phosphorylation status was analyzed by SDS-PAGE and Pro-Q-Diamond staining.

Two main routes led to generation of DivIVA-derivatives to be used for detection of effects related to phosphorylation: First, in vivo phosphorylation by co-production of native DivIVA with AfsK-kinase domain (AfsKK) in E. coli. Second, phosphomimic mutations of the 5 known phosphorylation sites in divIVA were generated by substitution of serines/threonines to aspartates. In both cases produced DivIVA was purified by the fused selfcleaving chitin-binding domain (CBD). The 5-fold phosphomimic DivIVA mutant completely resembles in vivo phosphorylated native DivIVA in all conducted experiments, so that it can be recommended as a convenient and mild alternative to modulation of (de)phosphorylation in further in vitro and in vivo experiments. The mutagenesis method enables also generation of selected phosphomimic mutations (e.g. 1-fold mutations, 31 in total) which can be used to link phenotypes to distinct phosphorylation sites.

Oligomerization analysis of phosphorylated, phosphomimic and native DivIVA revealed no major direct effect of phosphorylation on oligomerization level in the applied experimental system. This does not rule out a phosphorylation effect in general but emphasizes a possible involvment of further factors. This factors could e.g. be scaffold proteins binding to the DivIVA depending on oligomerization status.

With various phosphomimic DivIVA-mutants, inducible in vivo and in vitro (de)phosphorylation systems and several established oligomer analysis methods, a powerful toolkit is being assembled to approach the regulation of tip extension and branching in S. coelicolor.

Supervisor: Klas Flärdh
Master´s Degree Project 30 credits in Microbiology 2012
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Kazakevych, Juri
supervisor
organization
course
MOBM18 20121
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3633215
date added to LUP
2013-04-12 15:40:30
date last changed
2013-04-12 15:40:30
@misc{3633215,
  abstract     = {Streptomyces belong to the phylum Actinobacteria, which are Gram-positive bacteria with GC-rich DNA. Most Streptomyces are strict aerobes and inhabit soil. They are known for production of a wide range of secondary metabolites, including antibiotics e.g. the macrolide erythromycin (S. erythraeus) or chloramphenicol (S. venezuelae). The typical smell of wet soil is owed to geosmin, also a secondary metabolite of Streptomyces.

Unlike most other bacteria, streptomycetes resemble fungi in the sense that they grow by tip extension and form a branching vegetative mycelium containing multiple chromosome copies in each hyphal cell. Shortage of nutrients induces formation of aerial mycelium. The aerial mycelium consists of long hyphae finally maturing and dividing into monochromosomal spores. S. coelicolor is the most used model organism for genetic and associated functional investigations of streptomycetes.

Regulation of tip extension in S. coelicolor
While many bacteria, e.g. Escherichia coli, grow by extending the lateral cell wall, S. coelicolor has a growth pattern restricted to hyphal tips. The essential protein DivIVA is mediating this localization as well as the emergence of new hyphal branches, shown by studies of fluorescent fusion proteins and time-lapse imaging: cell wall synthesis occurs only at DivIVA-foci. Homologs of DivIVA can also be found mediating apical growth in other actinobacteria e.g. Mycobacterium tuberculosis. Most Gram-positive bacteria possess DivIVA-homologs involved in cell growth, division or polarity. S. coelicolor DivIVA is a 41 kDa α-helical coiled-coil protein. Based on crystal structure and electron microscopy, Bacillus subtilis DivIVA is suggested to build tetrameric or higher order oligomeric structures.

S. coelicolor DivIVA was recently shown to be phosphorylated by the Ser/Thr-kinase AfsK with strong effect on DivIVA localization, apical growth and increased branching frequence. Phosphorylation of DivIVA is reversible in vivo with 5 identified phosphorylation in the C-terminal region. This α-helical region is suggested to overlap in di- and tetrameres and phosphorylation may therefore affect the regulation of oligomerization. Oligomerization level regulation is not restricted to Streptomyces, effects of phosphorylation on protein interaction and oligomerization are wide spread and can be observed e.g. on the yeast actin cytoskeleton.

Popular science summary:

Oligomerization of native and phosphorylated DivIVA

The aim of this project was to investigate how phosphorylation affects the properties of the DivIVA protein and specificially to determine whether oligomerization of DivIVA is affected by phosphorylation status. Oligomerization levels were analyzed by methods responding to the relative size of protein complexes, including light scattering assays, Blue Native/Clear Native Polyacrylamide Gel Electrophoresis (BN/CN-PAGE) and gel filtration followed by Dot Blot immunostaining. Phosphorylation status was analyzed by SDS-PAGE and Pro-Q-Diamond staining.

Two main routes led to generation of DivIVA-derivatives to be used for detection of effects related to phosphorylation: First, in vivo phosphorylation by co-production of native DivIVA with AfsK-kinase domain (AfsKK) in E. coli. Second, phosphomimic mutations of the 5 known phosphorylation sites in divIVA were generated by substitution of serines/threonines to aspartates. In both cases produced DivIVA was purified by the fused selfcleaving chitin-binding domain (CBD). The 5-fold phosphomimic DivIVA mutant completely resembles in vivo phosphorylated native DivIVA in all conducted experiments, so that it can be recommended as a convenient and mild alternative to modulation of (de)phosphorylation in further in vitro and in vivo experiments. The mutagenesis method enables also generation of selected phosphomimic mutations (e.g. 1-fold mutations, 31 in total) which can be used to link phenotypes to distinct phosphorylation sites.

Oligomerization analysis of phosphorylated, phosphomimic and native DivIVA revealed no major direct effect of phosphorylation on oligomerization level in the applied experimental system. This does not rule out a phosphorylation effect in general but emphasizes a possible involvment of further factors. This factors could e.g. be scaffold proteins binding to the DivIVA depending on oligomerization status. 

With various phosphomimic DivIVA-mutants, inducible in vivo and in vitro (de)phosphorylation systems and several established oligomer analysis methods, a powerful toolkit is being assembled to approach the regulation of tip extension and branching in S. coelicolor.

Supervisor: Klas Flärdh
Master´s Degree Project 30 credits in Microbiology 2012
Department of Biology, Lund University},
  author       = {Kazakevych, Juri},
  language     = {eng},
  note         = {Student Paper},
  title        = {Investigating the effect of phosphorylation on DivIVA oligomerization in Streptomyces coelicolor},
  year         = {2012},
}