Study of paralogous SSB proteins from Streptomyces
coelicolor
Ž.
1
Filić ,
T.
1
Paradžik , N.
Ivić2,M.
Luić2,
D.
1
Vujaklija
1Laboratory
for Molecular Genetics, Division of Molecular Biology, Ruđer Bošković Institute
2Laboratory for chemical and biological crystallography, Division of Physical Chemistry, Ruđer Bošković Institute
Streptomyces species (Fig.1) are filamentous Actinobacteria mostly known for their production of numerous
secondary metabolites that have a wide array of applications, such as antibiotics, immunosupresors or
anticancer drugs. They grow predominantly in soil where they form a vegetative hyphal network. Nutrient
depletion activates the extrusion of areal hyphae and the formation of spores that enable resistance to low
nutrient and water availability.
Figure 1. A typical phenotype of Streptomyces coelicolor
colonies on SM agar plates.
(http://www.flickr.com/photos/ajc1/3288213824/)
Single stranded DNA binding proteins (SSBs)
are essential for cell survival from humans to
bacteria and viruses. SSBs participate in DNA
replication, recombination and repair
processes protecting single stranded DNA
intermediates (ssDNA) disrupting undesired
secondary structures (Fig.2) and interacting
with numerous other essential proteins
modulating their activities.
Figure 2. The role of SSB protein in a replication fork
http://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP
SSB proteins from most prokaryotic species
exist as tetramers which bind to ssDNA
through structurally conserved folding
motifs (OB folds) at the N-terminus, while
the C-terminal domain is responsible for
protein interaction. We have solved the 3D
structure of two SSBs from S. coelicolor.
Their structures mostly
resemble
mycobacterial SSB with unique variations,
clamp like structure reported previously
and S-S bridges as depicted in Fig.3.
Figure 3. Superposition of SSB-B (green) and SSB-A (grey) from S. coelicolor.
Disulphide bridges of SSB-B are shown as sticks and coloured in yellow, while the
clamp mechanism between strands 9 characteristic for SSB-A is coloured in pink.
Streptomyces coelicolor contains two paralogus genes encoding single stranded DNA binding proteins: SSB-A and SSB-B.
ssbA - 4 303 613 - 4 304 212 bp
In order to asses the biological
role of SSB-A a recombineering
method developed by Gust et
al. was performed using
transposon-mutagenised
cosmids (Fig.4).
rpS6
rpS18
ssbA
rpL9
ssbB - 2 924 515 - 2 924 985 bp
ssbB
Clones with a ‘’scar’’ mutation of
the ssbA gene gave no viable
mutants, while the mutation of
ssbB locus produced white areal
mycelium (whi – like phenotype)
(Fig.5) and the elucidation of this
phenomena is underway.
Figure 4. Red arrows indicate the insertion position of
transposon tn5062 causing disruption of ssbA and ssbB
genes
S. coelicolor M 145
(wild – type)
Inactivated SSB-B
(whi – like phenotype)
Figure 5. The phenotype of S. coelicolor M145 (wt )
and a mutant with a scar mutation of ssbB gene
One of major aims of this study was to identify the interacting partners of the SSB proteins from S. coelicolor. This is being performed using
TAP (tandem affinity purification) technology.
Preparation of TAP constructs consists of cloning two tags (protein A and
Calmodulin binding peptide - CBP, divided by tobacco etch virus - TEV protease
cleavage site) at the 5’ terminus of ssb gene. These constructs are further
subcloned onto expression vectors (Fig.6) and transformed into S. coelicolor.
Overall protein extraction is then isolated from the exponential growth phase
and purified as presented in the Fig.7.
Figure 7.
Tandem-affinitypurification (TAP)
scheme.
After generating the TAP
tagged protein(s), cell extracts
are subjected to two step
purification. The first column
consists of IgG beads which
bind proteinA and bound
proteins. TEV protease cleaves
the immobilized multiprotein
complexes
that
undergo
another round of binding on
calmodulin beads. The native
complex is then eluted by
chelating calcium using EGTA.
Figure 6. TAP - tag constructs
at the 5’ terminus of ssB gene
The interacting partners from SSB-A were identified by the MS analysis. At
present, identified interactants of SSB-A protein are clasified based on their
cellular function (Table 1). Some of the identified proteins have been reported
previously, such as ATP-dependent DNA helicase, RecG, DNA topoisomerase I,
recombinase A, RecQ helicase, RNA polymerase beta and molecular
chaperone DNaK.
Our aim is to confirm interactions of SSB-A protein with other proteins that
were not published previously applying the two-hybrid system and
immunoprecipitation techniques. In parallel, identification of SSB-B proteins is
underway. It is of our special intrerest to identify proteins that possibly
interact with SSB-B since our novel and unpublished result clearly pointed out
that this protein plays an important role in bacterial cytokinesis.
Translation and posttranslational
modification
9
DNA metabolism (transcription,
replication, repair)
4
Cell metabolism and transport
8
Hypotetical proteins
10
Table 1. Proteins identified by
MS analysis. Proteins are
classified based on their
cellular function.
References:
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Acknowledgements:
Thanks to all the members of Laboratory for Molecular Genetics at the
Ruđer Bošković institute for their kind support, assistance and collaboration.