Download New regulators of cell division in Streptomyces coelicolor

Antje Marie Wollkopf
New regulators of cell division in Streptomyces coelicolor
Streptomycetes are soil bacteria that play an important role in the industrial production of
antibiotics as well as other secondary metabolites. They grow in long, branching filaments
called hyphae, which form a mycelium.
Streptomyces coelicolor shows two different modes of hyphal growth: a vegetative substrate
mycelium and a grey spore-forming aerial mycelium. During sporulation of the aerial
mycelium, dramatic reorganization occurs. Regularly spaced sporulation septa divide the
hyphae into unigenomic prespore units. Eventually these mature and are released as free
spores. Sporulation septa differ a lot from the vegetative septa formed in the substrate
mycelium. Furthermore, they are responsible for complete separation and detachment of the
resulting daughter cells. This thesis deals with the developmental regulation of cell division in
S. coelicolor, in particular sporulation septation in the aerial mycelium.
The protein FtsZ is the first identified compound of the cytoskeletton in prokaryots and plays a
key role in cell division in virtually all bacteria. In S. coelicolor, FtsZ is required for septum
formation in the substrate and aerial hyphae but is not essential for growth and viability. Before
sporulation septation in the aerial hyphae, FtsZ assembles in the cytoplasm of the sporogenic
hyphae and forms regularly spaced cytokinetic rings, the Z rings. These recruit other proteins
to the division site that contribute to the formation of the septum. Once stable rings have
formed, septum formation is triggered. Unigenomic prespore units develop, mature, and are
released as free spores. So far, it has not been shown which mechanisms control timing and
location of the FtsZ assembly and Z ring formation during the cell cycle in S. coelicolor.
Homologues of the known regulators of FtsZ assembly from other bacteria have so far not been
found in the genome of S. coelicolor. A long-term goal to which this thesis contributes is to
identify and characterize new regulators of cell division in S. coelicolor.
Non-sporulating S. coelicolor strains have been isolated that have specific mutations in the ftsZ
allele resulting in the disruption of sporulation and the failure of grey spore pigment
production. The in vivo effects of mutations on FtsZ are not fully understood, but it is assumed
that the balance between assembling and disassembling of FtsZ is shifted in some ways. This
could i.e. result in disturbed general properties of the FtsZ protein itself or in altered
interactions between FtsZ and other cell division proteins. In this thesis genomic libraries of
the S. coelicolor wild type genome that ideally represent the entire genomic DNA sequence
were constructed to be able to introduce and overexpress wild type genes in S. coelicolor ftsZ
mutants. These genomic libraries were used to look for strains in which wild type genes that
directly control FtsZ assembly may restore the function of the mutated ftsZ allele and thereby
suppress the sporulation-deficiency. Such strains were successfully isolated. One of these
strains was analysed microscopically and showed restored sporulation to a certain degree. It
was observed that some irregularities accumulated in the sporogenic aerial hyphae of this
strain, including odd-shaped spores, partial septation, and separation. Further analysis will
clarify the cause of the observed restored sporulation phenotypes. This will help to identify and
characterize wild type genes that may encode novel regulators of cell division proteins that
control Z ring assembly and cell division in S. coelicolor.
Supervisor: Klas Flärdh
Lund University
Faculty of Science
Department of Cell and Organism Biology
Degree Project in Microbiology, 20 Swedish Credit Points
Antje Marie Wollkopf
A search for new regulators of cell division in Streptomyces coelicolor:
Screening for multicopy suppressors of ftsZ(Spo) mutants
Abstract
Streptomycetes are mycelial-forming, gram-positive soil bacteria that play an important role in
the industrial production of antibiotics as well as other secondary metabolites. This thesis
focuses on the developmental regulation of cell division in Streptomyces coelicolor, in
particular the regulation of the key cell division protein FtsZ in sporulation septation of aerial
hyphae. FtsZ is involved in at least two cell division processes during development, sporulation
septation in the aerial hyphae and formation of hyphal crosswalls in the vegetative hyphae.
Specific missense mutations in ftsZ, resulted in disruption of sporulation and failure of grey
spore pigment production, but have no detrimental effects on growth. One particular mutant
strain was chosen for this study. The ftsZ17(Spo) mutant K102 carries in the ftsZ codon 249 a
mutation leading to a change of the amino acid alanine to threonine. The overall goal of this
project is to identify and characterize wild type genes that directly control FtsZ assembly and
that when expressed at elevated levels would allow the ftsZ17(Spo) allele to behave normally,
thereby suppressing the phenotype. For this purpose five genomic libraries from S. coelicolor
A3(2) were constructed in shuttle vector pKC1218. These libraries were transferred by
interspecies conjugation from Escherichia coli into S. coelicolor A3(2) ftsZ17(Spo) mutant
K102. Transconjugants containing library plasmids were screened for suppression of the
sporulation defect. Within this thesis, twenty strains with restored colony phenotypes were
successfully isolated. One of these strains was analysed microscopically and showed restored
sporulation of the aerial hyphae. However, it was observed that some irregularities
accumulated in the sporogenic aerial hyphae of this strain including odd-shaped spores and
partial separation. Further analysis will show whether the suppressing activity of the introduced
library constructs is the cause of these phenotype changes. Eventually genes will be identified
that may encode yet unknown proteins that control Z ring assembly and cell division in S.
coelicolor.
Supervisor: Klas Flärdh
Lund University
Faculty of Science
Department of Cell and Organism Biology
Degree Project in Microbiology, 20 Swedish Credit Points