dissertation final version 521138

SCHOOL OF MEDICINE
YSGOL FEDDYGAETH
PM304 – Biomolecular Research Project 2011
Single Honours in Genetics
Name: Damianos Mavridis (521138)
Title: “Defining an in vivo model to functionally
characterise Streptomyces coelicolor DpsA
protein”.

Biomolecular Research Project
(D. Mavridis, 521138) Page 2
Defining an in vivo model to functionally characterise
Streptomyces coelicolor DpsA protein
Damianos G. Mavridis,
Supervisor: Dr. Ricardo Del Sol,
Institute of Life Science, School of Medicine, Swansea University.
Abstract
The current project was an in vivo investigation made in Streptomyces strains as an effort to
devise a model that could aid in the functional characterization of Dps proteins. The aims
were initially to develop a model that could verify the protective roles of DpsA proteins in
relation to Streptomyces susceptibility under osmotic stress factors using potassium chloride
and also test the functionality of truncated versions of the proteins. Previous studies have also
shown that DpsA proteins can cause changes in bacterial nucleoid condensation as a result of
their low-specificity DNA binding properties. These phenotypic changes were therefore
explored by live staining with Syto-9 under fluorescent microscope in order to investigate
functionality of the wild type DpsA protein versus a truncated version. We gathered some
evidence that a DpsA lacking its carboxyl tail can partially restore nucleoid compartment size
in Streptomyces coelicolor dpsA mutants. The experiments identified minor differences
between strains and therefore it has been underlined that there is still a necessity for further
investigation in regard to the functionality of these proteins in in vivo models.

1. Introduction
1.1 Dps proteins: General Overview
In their natural environment bacteria have to be able to survive under various environmental
changes and presumably to be able to cope against stress factors like nutrient depletion, heat
and osmotic stresses (Ohniwa et al., 2006). Dps proteins also known as DNA-binding protein
from starved cells (Ohniwa et al., 2006; Ceci et al., 2004), have been found to be of major
importance, functioning like a “shielding” mechanism that protects bacterial DNA from
external factors (Facey et al., 2009). This protection is mainly acquired by two mechanisms,
first by Dps-DNA interaction that hyper-condenses the genetic material, and secondly by
Fenton chemistry and storage of Fe(III) as a mineral into the protein cavity (Facey et al.,
2009). Dps proteins have a molecular weight of 19kD (Ohniwa et al., 2006) and belong to the
ferritin-like protein family. They consist out of 12 monomers combined in such way
structurally that form a dodecamer hollow sphere in which a Fenton-like reaction was found
to take place (Facey et al., 2009).
(Pulliainen et al., 2005)
By this reaction it is possible for the oxidation of Fe(II) into Fe(III) to be mediated, so it can
be stored as a mineral in the cavity of these proteins (Pulliainen et al., 2005). Iron is both
necessary but can also be highly toxic forming reactive oxygen radicals inside a cell (Facey et
al., 2009). From humans to plants and bacteria, iron should be constantly maintained under
strictly controlled concentrations. The oxidation of Fe(II) as a process prevents reactive
oxygen species (ROS) to be generated, therefore preventing their accumulation in high
concentrations in the bacterial cell, by storing iron [Fe(III)] as crystalline or amorphous
Figure 1:
A Dps-like 12mer dodecamer (Ilary et al., 2000)

ferrihydrate-like cores (Pulliainen et al., 2005). It has been suggested that the iron as Fe(II)
can enter the cavity of such proteins through pores that exist due to a three-fold symmetry of
12meric Dps proteins (Zeth et al., 2004). These ferritin-like proteins have also the ability to
bind the DNA molecule non-specifically. Studies have suggested that a property as such can
confer DNA protection also by altering the bacterial nucleoid condensation (Facey et al.,
2009; Ohniwa et al., 2006). In certain bacterial species Dps proteins can be induced under
stresses (i.e. hydrogen peroxide in Escherichia coli and Mycobacterium smegmatis)
(Chowdhury et al., 2007). It has been demonstrated that Dps proteins were not induced in
Streptomyces strains while stimulation with hydrogen peroxide (H2O2) as a factor (Facey et
al., 2009). In general, there has been much effort towards the investigation of Dps proteins in
many organisms. Understanding how Dps provide protection to various pathogens may be
proven to be of significant importance for better targeted antibiotic production (Schwartz et
al., 2010). Similarly like S. coelicolor Dps proteins Bacillus anthracis found to have two
versions (homologues), Dps1 and Dps2 that provide the bacterium protection and specifically
against peroxide stress (Schwartz et al., 2010). Iron binding proteins have proven very
important for mediating the metabolism of iron and also for preventing accumulation of
reactive oxygen radicals (ROS) (Wiedenheft et al., 2005). Dps structure has been found
closely affiliated with this of bacterioferritins, consisting by a four helix bundle comprised by
two interconnected helix-turn-helix motifs (Gauss et al., 2006). In contrast with ferritin and
bacterioferritin proteins the Dps diversify mainly because of their distinctive oligomere
assembly, to form dodecamers whereas bacterioferritins and ferritins are found as 24-mers
(Gauss et al., 2006). From a gene expression aspect, Dps induction under stress conditions
has been found to be controlled by several transcription factors (Facey et al., 2011).
Regarding studies in Bacillus subtilis, an example of this expression control is mediated by
the SigmaB
transcription factor, which controls in total 200 genes (Facey et al., 2011). Further
analyses revealed that DpsA protein orthologue in Streptomyces coelicolor is up-regulated
under osmotic stress conditions and transcription is mediated from a SigmaB
-like promoter
(Facey et al., 2011). Furthermore there have been identified 9 SigmaB
-like paralogues in
Streptomyces coelicolor distinguishing the expression control mechanism from that of
Bacilus subtilis regulon which is controlled by a single SigmaB
(Facey et al., 2011). The norm
of current research has been driven towards analysing further how each ferritin-like protein
and Dps proteins could assemble into complexes, understanding in every step, by in vitro or
in vivo methods, the importance of illuminating the pathway of Fenton-like reactions
(Wiedenheft et al., 2005), especially when realizing that these proteins are commonly shared
among such a variety of species and organisms.
1.2 S. coelicolor DpsA protein structure:
There are 3 Dps orthologues in S. coelicolor A3 (2) (strain M145), DpsA, DpsB and DpsC.
This study has been focussed on DpsA only, because of its inducibility under osmotic stress
conditions (Facey et al., 2009). The low specificity DNA binding properties of Dps proteins
thought to be affected by the amino- end (N-tail) terminal domain of Dps proteins because
the surface of the protein dodecamer is negatively charged (Facey et al., 2009). DNA exhibits
also a negative charge so the only possible way that could explain the Dps-DNA binding
would be the fact that tails positively charged would somehow mediate this process (Facey et
al., 2009). In a previous study, it had been mentioned that DpsA protein in Streptomyces
contains 2 tails, an 15 residue long amino tail (N-tail) and a 25 residue long carboxyl tail (C-
tail) both having a single residue that exhibits a positive charge (Facey et al., 2009). Both
tails are also thought to affect the structure of DpsA dodecamer and how the protein

assembles in space (Facey et al., 2009). Ongoing in vitro studies made by Streptomyces
coelicolor research group showed no indications though that DpsA could bind DNA, so the
truncated version used in the current research was only used for testing the functionality of
DpsA in terms of protein’s in vivo role in relation under stress conditions.
1.3 Objectives:
The experiments of this study were made in an attempt to devise a model that could
functionally characterise DpsA proteins in Streptomyces. First it was necessary to assess the
inducibility of DpsA in four different Streptomyces strains. S. coelicolor A3(2) (strain M145),
was used as a control because it naturally possesses all three Dps orthologues (DpsA, DpsB
and DpsC). The second strain chosen for the experiment was a mutant version of S.
coelicolor lacking the dpsA gene (dpsA-
) (Facey et al., 2009). This strain was chosen to asses
both susceptibility and phenotypic changes such as nucleoid condensation. The third strain
used was Streptomyces venezuelae, which naturally lacks a DpsA- like orthologous protein.
This last strain was chosen for the artificial incorporation of dpsA gene using a plasmid as a
vector. The specific character that this strain possesses was that naturally it does not have the
gene encoding DpsA and therefore would be a good choice as a model organism for testing
the hypothesis. The susceptibility of all strains was measured using colony forming units
(C.F.U.) on bacteria plated on MS medium (mannitol and soya flour medium) containing
potassium chloride (KCl) at various concentrations. Two recombinant versions were
produced using two plasmids. One was containing the gene expressing the full length DpsA
protein (pDpsA7) (Facey et al., 2009) and the second (pDpsADCT) containing the gene
lacking the area that encodes for the carboxyl–tail domain of the protein (C-tail deletion
produced by deletion in the 3’ region of the gene). This incorporation of the plasmids in the
previously discussed strains was thought to be leading to observation of any potential
differences in the susceptibility under osmotic stress conditions. Contrasting the wild type
and partial protein expression of the bacterial strain under investigation, it was speculated that
it could give an insight of how the carboxyl tail of the protein could alter potentially the
structure of the dodecamer, hence it’s function, and therefore what could be the different
outcomes of such expression. Also the main hypothesis was that Streptomyces venezuelae
expressing the dpsA gene would be less susceptible than the wild type. In addition
Streptomyces coelicolor dpsA-
and M145 strains would help the differential observations as
controls.

2. Results
2.1 Construction of a DpsA lacking its Carboxyl-tail:
Using plasmid pDpsA7 (Facey et al., 2009) expressing the full length (wild type) DpsA
protein it was possible to develop a plasmid that could express a truncated version of the
protein. A carboxyl-tail truncated version (pDpsADCT) was made using restriction digestion
by Xho1 enzyme. The pDpsA7 plasmids were extracted from an overnight culture of E. coli
in LB medium using a plasmid DNA purification kit (Zyppy™ Plasmid Miniprep Kit).
Following the purification process a total of 10µl of plasmid DNA was digested using
restriction enzyme Xho1 (1µl), 5µl restriction buffer (10x), 34µl of H2O and 1µl BSA
(Promega company; Kieser et al., 2000). The initial pDpsA7 plasmid (Facey et al., 2009) had
two Xho1 sites at the 3’ region of the gene making it possible to cut the specified area that
corresponded to a truncation of the C-tail of the protein, preserving the correct reading frame.
Ligation of the plasmids was made using T4 DNA ligase as per protocol (Kieser et al.,
2000).The recombinant plasmid generated was named pDpsADCT and was transferred into
Streptomyces strains as mentioned in Materials and Methods section via intergeneric
conjugation techniques (Kieser et al., 2000).
Figure 2: On top there is a representation of a wild type DpsA monomer having its two tails (an
amino tail and a carboxyl tail). At the bottom of the box a representation of a truncated DpsA
monomer expressed lacking a carboxyl tail. The genetic maps indicate the restriction enzymatic

Figure 3:
Western blot analysis of S. venezuelae recombinant (having the
pDpsA7 plasmid) that shows induction under osmotic stress
conditions.
 Top panel: No production of DpsA protein when grown
on MS medium without potassium chloride (S.
coelicolor M145 [S.c+] used as a control)
 Second panel: shows production of DpsA protein when
S. venezuelae left to propagate on MS containing KCl
reaction used by Xho1 enzyme, to produce a new plasmid lacking the DNA region which expresses
the C-tail DpsA protein version.
Six histidine tags cloned in the initial plasmid pDpsA7, were maintained in the final product
pDpsADCT, ensuring that the expression of the protein could be monitored by Western
Blotting analysis in the recombinants incorporated (Figure 3, only shows S. venezuelae
containing pDpsA7). Clearly therefore, figure’s 3 Western Blotting results verify that there
was induction of DpsA protein in Streptomyces venezuelae recombinant under osmotic stress
conditions and also ensure that the incorporation of the plasmid pDpsA7 has been made
successfully.
2.2 Osmotic stress susceptibility studies:
All strains were cultivated in MS (mannitol-soya flour medium) containing various potassium
chloride concentrations (0mM, 200mM, 300mM) for 3days at 300
C. After incubation time the
colonies formed were counted (C.F.U). The measurements included four replicates of S.
coelicolor M145, S .coelicolor dpsA-
, dpsA-
/pDpsADCT, S. venezuelae w/t, S.
venezuelae/pDpsADCT and S. venezuelae/pDpsA7 at each concentration of KCl (0mM,
200mM and 300mM).The C.F.U collected in an array of excel format tables and the average
at each concentration of KCl has been collected. The error bars illustrated in figure 4 have
been produced by standard deviation of the colony forming units at each concentration.
Following that, the histogram that had been produced was further refined using a logarithmic
scale format (Figure 4). Data investigations were made and the resulting graph (Figure 5) was
used to depict clearly the most of the significant results of this study’s observations. The
averages of each strain have been used to normalize each of the 4 initial replicates, by a
simple division of the replicates (C.F.U.) by the average concentration at 200mM. Following
this treatment of the results, another collection of 4 normalised replicates has been made and
their values were again averaged to produce a single measurement at each concentration of
each strain. These results showing the change of the colony forming units normalised at
200mM concentration of KCL can be seen in the graph of figure 5. It is clearly presented that
there is a minor difference in the susceptibility between S. venezuelae w/t, S.
venezuelae/pDpsA7 and S. venezuelae/pDpsADCT. Either one of the recombinants of S.
venezuelae produced have shown to be somewhat less prone to potassium chloride induced
osmotic stress than the wild type.

1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
0 100 200 300 400
COLONYFORMINGUNITS(C.F.U.)
KCl CONCENTRATIONS (mM)
Streptomyces strains C.F.U. against various
concentrations of KCl
M145
Dpsaˉ
dpsaˉ/pDpsADCT
S.venezuelae
S.venezuelae/pDpsADCT
S.venezuelae/pDpsA7A
Figure 4: The data collected of S. coelicolor M145, DpsA-
, S. venezuelae, contrasted to recombinant
strains under susceptibility testing. [all bacteria were grown on MS (manitol soya flour medium) under 3
distinct concentrations of potassium chloride (KCl)] and an incubation time of 3 days. The error bars
shown represent standard deviation.
In this study we observed a delay of bacterial growth of every Streptomyces strain used at
potassium chloride (KCl) concentrations above 300mM. Especially S. venezuelae showed to
be the most susceptible, with a non uniform and delayed incubation time ranged up to 5 days.
Therefore it has been decided to test susceptibilities up to 3 days fixed incubation time and up
to 300mM KCl. As showed in figure 4, it has been observed a minor difference between
Streptomyces venezuelae having the plasmid expressing the DpsA protein (pDpsA7) and S.
venezuelae wild type. Especially at 300mM concentrations the wild type C.F.U count was
almost 1,000 colonies, whereas the recombinant version having the dpsA gene was found
higher in C.F.U (≈14,000 forming units) with almost no decline in numbers of colonies in
contrast to the other KCl concentrations. Streptomyces coelicolor strain M145 (wild type) had
a significant higher C.F.U count in all concentrations but showed a characteristic decline in
higher concentrations of KCl. This experiment didn’t show significant and conclusive results
indicating that Streptomyces venezuelae can be used as a model organism to assess DpsA
proteins functionality. Its natural character lacking the dpsA gene had been hypothesized to
make it suitable for testing in contrast to the artificially mutated version S. coelicolor dpsA-
which shows no differences at all. The data collected were closely affiliated stating that there
is still a necessity to find a more robust method that could promise larger differences in terms
of susceptibility changes. It has been showed by this study, that the incorporation of the
recombinant plasmids (pDpsA7 and pDpsADCT) producing the DpsA protein (either whole
length or with the deleted carbon tail) had no effect in the susceptibility of S. coelicolor dpsA-
mutant.

Figure 5: This graph shows the difference of the susceptibility of the spores observed. The values had been
normalised, dividing the average of colonies counted by the C.F.U observed at 200mM concentration of each
strain (more detailed explanation in Results).

2.3 Nucleoid morphology analyses:
S. coelicolor mutant (dpsA-
), S. coelicolor M145 and dpsA-
/pDpsADCT, having the
recombinant plasmid (pDpsADCT- standing for deleted C-tail domain) were further analysed
using fluorescent microscopy. It was possible to visualise the nucleoids by staining with
Syto-9 live stain, and identify any phenotypic changes between the wild type of S. coelicolor
and dpsA mutants. Figure 6 shows, as expected that S. coelicolor M145 nucleoids display no
irregularities in bacterial nucleoid condensation (Facey et al., 2009). It exhibits a uniform
pattern of nucleoids with almost identical diameter. In contrast Streptomyces coelicolor dpsA-
nucleoids were found irregular in terms of shape and diameter of spore compartments, as
described also in a previous study (Facey et al., 2009). It was significant in fact that it has
been observed a partial restoration of nucleoid irregularities regarding dpsA-
mutant carrying
the plasmid pDpsADCT. A challenging part of this procedure was the difficulty in estimating
the age of the aerial hyphae analysed. Even though the bacteria were similarly incubated at
300
C on MS under specific length of time, it was roughly estimated which spores to include
in the collection of the data. Overall on a controlled incubation Petri plate there was no
similar maturation pattern of spores, and there have been observed both immature and mature
spores. Under those circumstances the collection of spores that would have been suitable
(similar maturation stage) for the following spore measurements, was made empirically by
appearance of spore compartment size and formation and consequently data may have been
prone to human error. It should be therefore mentioned that at this particular stage of the
experiment there was no protocol or method that could ensure a better data collection system
than this empirical method. Figure 7 shows the resulting histogram obtained after measuring
≈700 individual nucleoids of each strain. The measurements of the length of each spore were
collected, expressed into frequencies (%) and afterwards plotted against nucleoid size range
in micrometers. The dpsA-
/pDpsA7 recombinant was not included in the phenotypic
investigation because it has been previously identified that pDpsA7 complements
Streptomyces coelicolor dpsA-
mutant (Facey et al., 2009).
Figure 6: M145 strain stained using Syto-9 live stain, visualization of uniform shapes of spore
compartments. DpsA-
strain exhibiting irregularities in shape and diameter of spore compartments.
DpsA-
/pDpsADCT recombinant, having the plasmid that encodes for the truncated DpsA protein
(deleted carbon tail domain) - partial restoration of nucleoid irregularities.
dpsA -
M145
dpsA - /
pDpsA7DCT

Figure 7:
Nucleoid condensation analysis after measuring the diameters of approximately 700 nucleoid
compartments per strain
The resulting histogram shows that a truncated version of DpsA partially restores nucleoid
irregularities in S. coelicolor dpsA-
mutant
 DpsA appearing in grey illustrates S. coelicolor dpsA-
 dpsA/DCT is used as an abbreviation in the histogram standing for S. coelicolor
recombinant having the pDpsADCT truncated version of DpsA protein.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6
Nucleoid size range (micrometers)
Frequency(%)
M145
dpsA
dpsA/DCT
3. Discussion
Summarising the findings of this study the susceptibility experiments in Streptomyces
coelicolor M145, S. coelicolor dpsA-
and S venezuelae recombinants did not show any
indications that show significance. The minor changes observed in the susceptibility of S.
venezuelae recombinants in contrast to the wild type, show slight beneficial behaviour in
terms of how the recombinant organisms coped better to an extent, against high
concentrations of potassium chloride. Both pDpsA7 and pDpsADCT versions had almost
identical effects when incorporated in S. venezuelae, indicating that the carboxyl tail had no
different effect in the functionality of the protein while protecting against osmotic stress. The
difference in change of susceptibility from 200mM – 300mM KCl among Streptomyces
venezuelae and Streptomyces recombinants is shown in figure 5, representing the normalised
values as a ratio of C.U.F. divided by the 200mM average count in each strain. The truncated
version of DpsA protein (C-tail deletion) showed no change in the susceptibility of S.
coelicolor dpsA-
and dpsA-
had no measurable change in susceptibility in contrast to S.
coelicolor M145 strain.
The phenotypic changes presented on dpsA-
could be summarised as a partial restoration of
nucleoid morphology, showing that the truncated version of DpsA had an effect in the higher
order DNA organisation of S. coelicolor mutant. As human error has always been a critical
variable on the outcome of any experimental procedure, challenges such as the different
maturation stages in sporulation septa should be minimised. Specifically if the differences
observed had been as much as 0.5 of a micrometer that could in fact lead to a totally different
result for an analysis and especially in a process like the data collection of the nucleoid

diameters. Unfortunately there wasn’t any sophisticated method for this data collection than
simply trying to distinguish by appearance the spore maturation stage. Other observations
with less significance were that of the characteristic appearance of the recombinant bacterial
colonies when let to propagate on MS media, having a characteristic pale colour and a mild
difference in colony texture, in the first stages of colony establishment and also a mild delay
in the production of antibiotics (Facey et al., 2009). These morphological differences had
been also observed in a previous study made by Dr. Facey, Dr. R. Del Sol and colleagues.
Primarily the aims were to test whether a model like that presented above could test the
functionality of DpsA protein and whether it could clarify some of the questions made
previously on the role of the DpsA proteins in vivo and secondly try to distinguish any
differences that could indicate an alteration in protein function mediated by the truncation of
the carboxyl domain. None of the above arguments had been answered underlining that
probably DpsA functionality cannot be studied by a model like this. It has been shown that
further studies are needed on these ferritin-like proteins to be made. Alternative routes that
could give a further insight to this subject would include that of the production of an array of
different types of truncated versions of DpsA protein, having both amino-tail and carboxyl-
tail deletions and/or another amino tail deletion. In other studies including Dps proteins from
other organisms (Ceci et al., 2003; 2005), it was mentioned that there must be an importance
on the amino tail of the protein because of its high percentage in positively charged residues
that could mediate protein to DNA binding, but further in vitro studies with Streptomyces
coelicolor DpsA showed no indications at all that the protein possess the ability of binding
DNA (Del Sol, personal communication). It would be wise to incorporate an N-tail and dual-
tail deletion versions of DpsA protein in Streptomyces venezuelae and look for phenotypic
differences. If the N-tail would indeed be a crucial factor in DpsA monomer assembly to a
dodecamer it would also have tremendous differential results affecting the phenotype of the
host expressing it. Furthermore it would be wise to investigate the potassium chloride
mediated delay in growth of the recombinant bacteria. It would also demand investigations to
establish a proper liquid medium for such studies, because as highlighted in this study
Streptomyces strains had the tendency to grow in clumps when let to propagate in 2xYT-no
salt medium.
4. Materials and Methods
4.1 Strains and Culture Conditions:
The strains used for the experiment were Streptomyces coelicolor, M145, S. venezuelae, and a
S. coelicolor mutant (dpsA-
) lacking the dpsA gene (Facey et al., 2009). Cloning processes
were made using E. coli JM109 strain. The plasmids used were pDpsA7AprR
(Facey et al.,
2009) and pDpsADCT. Using established protocols, the transfer of the recombinant plasmids
into Streptomyces strains was performed with E. coli ET12567/pUZ8002 (Facey et al., 2009;
Kieser et al., 2000). The cultures of E. coli cells were performed on LB (L-Broth) agar at
370
C incubation temperature. Inoculation of E. coli JM109 cells took place on LB and LB
agar (L-Broth) (Sambrook et al., 2001) and selection using alternated antibiotics
(Hygromycin, Apramycin). All E. coli cells were incubated at 370
C (LB), while Streptomyces
strains at 300
C (on MS). Preparation of 1 litre LB was produced using 0.5% NaCl (5g), 1%

tryptone (10g), 0.5% yeast extract and 1% agar (15g). 1 litre MS medium (mannitol and soya
flour medium) was made using 20g Mannitol, 20g Soy flour and 20g agar in 1000ml tap
water. Additionally 2xYT (no salt) liquid medium was prepared using 1.6% Tryptone and
1% yeast extract (Sambrook et al., 1989; Kieser et al., 2000). Agarose gel (1%) for the
verification of the correct plasmid restriction procedures was made as in protocols using
100ml 1xTBE, 1g of Agarose and 1.5µl of Ethidium Bromide (Metzenberg, 2007; Dale et al.,
2007; Kieser et al., 2000). Common intergeneric conjugation techniques were used for the
incorporation of the plasmids from JM109 cells into Streptomyces strains (Kieser et al.,
2000). The ET E. coli cells were platted on Luria-broth agar Petri dishes (LB agar containing
Kanamycin, Chloramphenicol and Hygromycin) and let incubate at 370
C. Inoculation of ET
E. coli cells containing the plasmid was performed using LB liquid medium and incubation
over night at 370
C temperature. Following centrifugation protocol the re-suspended cells
were mixed with equal volume containing Streptomyces spores (10µl of spores in 500µl of
2xYT (two times yeast tryptone liquid medium) were heat shocked at 500
C for 10 minutes
time and then they allowed cooling. Proceeding, the mixture was transferred onto MS
(mannitol and soya flour agar) containing MgCl2 and Streptomyces recombinants were
selected using overlay of antibiotic mixture containing 1ml of H2O, 50µl Nalidixic acid
(50µg/ml) and 6.25µl of Apramycin (100mg/ml). Similar procedures were followed for all
strains as well as for both plasmids pDpsA7 and the truncated version pDpsADCT. The
collection of Streptomyces spores was made using a solution of 20% v/v glycerol.
Table 1: Strains and Plasmids
Strains Description Source
S. coelicolor M145 Prototrophic SCP1-SCP2- Pgl+ Kieser et al., 2000
S. venezuelae Del Sol, personal communication
S. coelicolor dpsA
-
M145 dpsA-
::Tn5062 Facey et al., 2009
Plasmids
pDpsA7 dpsA::His6, ApramycinR
Facey et al., 2009
pDpsA7H dpsA::His6, HygromycinR
Facey et al., 2009
pDpsADCT dpsA::His6,DCT This study
4.2 DNA manipulation and plasmids:
Plasmid pDpsA7 (Facey et al., 2009) expressing the full DpsA protein fused to a His-tag at
its C-end was used as a template for the production of the truncated version pDpsADCT. This
was achieved using XhoI restriction enzyme that had a restriction site situated near the 3’
region of the dpsA gene. Prior digestion the plasmids were purified via extraction, which was
made using plasmid DNA purification kit (Zyppy™ Plasmid Miniprep Kit). All restriction
enzymes and T4 DNA ligase used were obtained from New England Biolabs. The
electroporation of the plasmid was made using E. coli JM109 electro-competent cells (Facey
et al., 2009; Kieser et al., 2000). To verify that the recombinants were indeed producing
DpsA protein (either truncated or full protein version) a Western blotting was performed
using the 6-histidine tags that were already fused to the proteins [Results shown in Figure 3]
(Facey et al., 2009).

4.3 Osmotic Susceptibility Assay:
The experiment was refined several times to correct the difference regarding the incubation
times of the strains in response to higher concentrations of KCl (>300mM KCl). Initially
using a range of concentrations up to 500mM KCl the strains showed different growth rates
with the most susceptible of all to be S. venezuelae. For the susceptibility testing to be
calibrated the bacterial strains were grown on MS agar in 3 different potassium chloride
concentrations of 0mM, 200mM and 300mM KCl for an incubation time of 3 days. The
experiment proceeded until collecting 4 replicates of each strain in each concentration of KCl
and the colony forming units (C.F.U) were plotted against the concentrations giving rise to
the diagram in Figure 4 (see data treatment section). To have a better possibility of specificity
the spores have also been diluted in a range of spore suspensions (1/104
, 1/105
and 1/106
)
using a 20% v/v glycerol solution. After observing the differences in growth rates under high
salt concentrations it was necessary to further investigate the phenomenon. Using 2xYT
(yeast tryptophan medium – without salt) it has been tried to calculate the growth rates using
absorbance without success. These gram positive bacteria have the tendency to grow in
clumps even in liquid media like 2xYT - no salt and due to limited time provided it was not
further analysed.
4.4 Nucleoid Morphology Studies:
Spores of Streptomyces coelicolor M145, Streptomyces coelicolor dpsA-
and Streptomyces
dpsA-
/pDpsADCT were platted on MS agar and incubated at 300
C overnight. Under aseptic
conditions a sample of each strain were placed onto a microscopy slide and stained using
Syto-9 live stain (Invitrogen) using the protocol described (Facey et al., 2009). The slides
were then covered using cover slips and examined under a fluorescent microscope. Pictures
of the spore compartments of each strain were taken and analysed with free online software
(Image J).

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