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About
Structural Organization of Membranes in UV-resistant and UV-sensitive Salmonella derby
Cells: Role of Phospholipids, Phospholipid FATTY Acids and Lipid Peroxidation
Processes
Astghik Z. Pepoyan
Institute of Molecular Biology of the National Academy of
Sciences of the RA
Hasratian st.7,
Yerevan 375014, Republic of Armenia, e-mail -astghik@mb.sci.am
Key words: UV-sensitivity, phospholipid, plasmid, and fatty
acids
The goal of this work was the investigation of correlation
between some peculiarities of membrane phospholipids' structure and the
UV-sensitivity of plasmid - carrying
and plasmid-free Salmonella derby bacterial cells.
Phospholipids and their fatty acids were determined by the
method described in the work of Pepoyan at all (1993) and dynamics of free
radical processes studied as described by Pepoyan at all (1991).
The effect of R-plasmid on the qualitative and quantitative
composition of phospholipids and their fatty acids has been found.
It has been shown that the comparative UV-sensitivity of
plasmid - carrying S.derby mutants is most likely conditioned by the
structure of DNA, its GC-pair content and by level of DNA methylation.
The result permits a conclusion that it isn't exclusive the
decisive role of phospholipid molecules and its interaction with DNA in
formation of the UV-resistance of plasmid-free S.derby cells.
About Structural Organization of
Membranes in UV-resistant and UV-sensitive Salmonella derby
Cells: Role of Phospholipids, Phospholipid FATTY Acids and Lipid Peroxidation
Processes
Astghik Z. Pepoyan
Institute of Molecular Biology of the National Academy of
Sciences of the RA
Hasratian st.7,
Yerevan 375014, Republic of Armenia, e-mail -astghik@mb.sci.am
Key words: UV-sensitivity, phospholipid, plasmid, and fatty
acids
It have been investigated the peculiarities of membrane
phospholipid structure and the phospholipid fatty acids in the bacterial (S.derby)
cells connected with their plasmid - carrying and UV- sensitivity.
Phospholipids and their fatty acids were determined by the
method described in the work of Pepoyan at all (1993) and dynamics of free
radical processes studied as described by Pepoyan at all (1991).
The effect of R-plasmid on the qualitative and quantitative
composition of phospholipids and their fatty acids has been found.
It has been shown that the comparative UV-sensitivity of
plasmid - carrying S.derby mutants is most likely conditioned by the
structure of DNA, its GC-pair content and by level of DNA methylation.
The result permits a conclusion that it isn't exclusive the
decisive role of phospholipid molecules and its interaction with DNA in
formation of the UV-resistance of plasmid-free S.derby cells.
Key words: UV-sensitivity, phospholipid, plasmid, and fatty
acids
Radiation membranology presents in sketch if the importance
of genetic structure in the radiation damage of organisms is well founded
theoretically and experimentally [1-4]. For the evaluation of contribution of
the radiation damage in biological membranes for the final effect of
irradiation it is important to investigate the causative and effective
connection between the damage of biomembrane and breach of it's structure and
the functions of the genetic apparatus with different UV-resistance classes of
the same species of microorganisms. We have shown in our previous reports [5,
6, 7, 8] that the absence of an R-plasmid changes the several morphologic,
genetic and biochemical properties of S.derby cells. For all these
reasons, we investigated the phospholipids (PL) and fatty acid (FA)
compositions and lipid peroxidation processes (LPP) of S.derby K 89
cells and it's UV-sensitive mutants.
Material and Methods
The following S.derby strains were used in this work:
the quasi-pathogenic K 89 strain carrying the R-plasmid, it's plasmid-free
UV-resistant variant S.derby K 82 and UV-sensitive plasmid carrying
mutant S.derby K 134 and its plasmid-free UV-resistant variant S.derby
K 95 and transformed K 95 strain transfected with the plasmid DNA from S.derby
K 89 cells. The S.derby cells were cultivated on the full-bodied medium [9].
Phospholipids were determined in direct acetone extracts
(acetone powders) of S.derby cells prepared from 350 mg (total solids)
of cell biomass.
Individual phospholipids (PLs) were separated by
one-dimensional ascending thin-layer chromatography on silica-coated plates (KSK
silica gel) using the solvent mixture chloroform- methanol- ammonium hydroxide
(65: 35: 5 v/v).
Comparing their mobility with those of PL standards
identified phospholipid spots. Lipid phosphorus was mineralized in medium
containing H2 SO4 and HNO3; its content was
expressed in µg per mg acetone powder [10, 11]. Fatty acids were converted into
their methyl esters by the Stoffe technique [11].
The composition of the FA fraction was determined by
gas-liquid chromatography on a Chrom-5 chromatograph (Czechoslovakia) with
flame- ionization detector. Individual methyl esters were identified by
comparing the chromatogram with those produced by a standard mixture of
saturated and unsaturated FA methyl esters (14 to 22 carbon atoms chain
length).
Dynamics of free radical processes intensity in
none-enzymatic (ascorbate-dependent) and enzymatic (NADP.H-dependent) systems
of lipid peroxidation in S.derby cells were determined by quantity of
malonic dialdehyde (mg of PL) as described by Pepoyan and Ktsoyan [7, 12].
Results and Discussion
Figure 1 demonstrates that the wild strain S.derby
K 89 is distinguished from mutant S.derby K 134 (as well as the plasmid free
strain S.derby K 82 from mutant strain S.derby K 95) by the content of
DNA blocks, the temperature of melting is 73, 85° C and 72, 5° C,
correspondingly, the GC-content (47, 82 per and 45, 38 per), the interval of
melting (T=8° C and T=11° C correspondingly) and by forms of DNA that one can
see the represented charts.
The sensitivity of bacterium to UV-irradiation determines by
losses the aptitude of the exposed cells for "infinite" division
(formation of macrocolonies). And the methylation of DNA's molecules has a
great importance for the solution of problem about mechanisms of conservation
the DNA during the indefinite numbers of generation. It is known that in a
result of addling the methyl groups to the rest of adenine and cytosine the
site becomes stable for restriction. The level of their DNA methylation
distinguishes the studying strains of S.derby (Table 1).
The UV-sensitivity of bacterium mainly is determined by
characteristic of reconstruction of DNA damaging, too. The reparation of DNA is
closely connected with the other matrix processes - replication and
recombination. We have shown in our previous works; that the S.derby K
89 and S.derby K 134 strains principally are similar by their
peculiarities and the DNA-polymerase activity in the plasmid-free UV-resistant
S.derby cells measured under the conditions optimal for DNA-polymerase 1 from
coli was found to be decrease 10-50 fold [8].
Thus, the above-mentioned results affirm that the
comparative UV-sensitivity of S.derby K 134 mutants is most likely
conditioned by the structure of DNA, it's GC-pair content and by the level of
methylation. Especially it is interesting the fact of the UV-resistancy of S.derby
K 82 and S.derby K 95 plasmid-free cells [13].
It is clear that the biological membranes are the most
important compounds of the cells, from which the primary chemical radiation
processes could be originated.
The physical-chemical organization of bacterial cell
membranes is largely determined by their lipid composition; moreover,
amphophilic PLs are known to be predominant in membranes of bacteria [13]. For
all these reasons, we have investigated the PL composition of S.derby
cells both quantitatively and qualitatively connected with their plasmid
-carrying and UV-sensitivity.
On the other hand it is well known that the R-plasmids of
bacteria can influence on the different metabolic processes of cell-host,
including the metabolism of the lipid components. The plasmids can determine
the UV-resistant characteristics of the bacterial cells [13].
Table 2 lists the data on the qualitative and quantitative
PL composition of following S.derby cells: S.derby K 89 strains
carrying the R-plasmid and it's plasmid-free UV-resistant strains S.derby
K 82; S.derby K 134 plasmid carrying UV-sensitive mutants and it's
plasmid-free UV-resistant strains S.derby K 95.
It is evident that the content of PLs is related to the
presence or absence of the R-plasmid. In the latter case the PL contents are
dramatically decreased (Table 2).
The transformed cells S.derby K 95 were
quantitatively and qualitatively similar in PL composition to their
plasmid-carrying S.derby K 134 counterparts (Table 2). Our observations
clearly indicate that the PL content of S.derby cell walls is associated
to the presence of the R-plasmid (in our previous works we showed the PL
content of transformed strains S.derby K 82 [7, 11]).
From Table 2 follows the quantitative prevalence of
phosphatidylcholines (PCs) in all studying cells of S.derby. It is
peculiar to all S.derby cells [11]. In addition to lowered PC content,
the UV-resistant plasmid-free strains are characterized by an increase in the
percentage of lysophosphatidylcholines (LPCs). As it is obvious from Table 2,
the percentage of phosphatidylserines (PSs) and phosphatidylinositides (Fls)
decreases in S.derby cells during the time of the reduction of their
UV-resistancy. These PLs mainly consist of saturated fatty acids (FA) and
participate in the intermembrane organization, membrane viscosity.
The participation of plasmids in genetic processes of
bacterium is in part of problem of products` interaction encoded by the
chromosomal and plasmid genes and determining the sum phenotype of plasmid
-carrying cells what may be explain the displacements in quantitative
composition of individual PLs of S.derby cells.
Table 3 shows the results of our analysis of FA composition
of PLs in the plasmid-carrying and plasmid-free strains of S.derby. It
is seen that the cells contained saturated and unsaturated straight-chain FAs
and saturated FAs with branched chains (Table 3). In a variety of bacteria the
latter species are known performing the functions of unsaturated FAs, such as
the maintenance of membrane fluidity [23, 24].
The decrease in the amount of FAs observed in the
plasmid-free strain was a natural consequence of the lowered amount of PLs. As
is obvious saturated FAs (including the isoforms of saturated FAs) are not
appreciably affected by the presence of the R-plasmid and the S.derby
cell's UV-resistancy. The possibility that unsaturated fulfills similar
functions in the cells under chains study cannot be ruled out.
The transformed cells were quantitatively and qualitatively
similar in FA composition to their wild-type plasmid-carrying S.derby
counterparts, too. Our observations clearly indicate that the PL and FA
contents of S.derby cell walls are associated to the presence of the
R-plasmid.
The level of lipids oxidation and antioxidation systems has
a great role in defining of cell UV-resistancy and the changes allowances to
reveal formatting regularities in organisms under the damaging factors.
We have also demonstrated the role of membrane interactions
in revealing LPP intensity and it appeared to be dependent on the square of the
S.derby cell membrane free surface [7].
Table 4 shows the results of intensity LPP in UV-resistant
and UV-sensitive S.derby cells.
The definition of malonic dialdehyde quantity in S.derby
cells was carried out for the concentration when the intercellular interactions
do not influence on the secretion of malonic dialdehyde to the intercellular
space.
It was established the same level of both ascorbate and
NADPH depending systems of peroxidation for investigated cells of S.derby.
The presence of LPP in bacterial cell suspensions and growth liquid mediums
(after incubational period) was found. By the way it was fixed the existence of
R-plasmid in the S.derby cells leads to the LPP intensity decline.
As follows from Table 3 the absence of R-plasmid in S.derby
K 89 cell suspensions entailed a 12,5 fold increase of LPP intensity and more
than 32-fold increase of malonic dialdehyde decreased quantity in all studying
cells growth medium it was shown the same level of LPP for UV-sensitive mutants
S.derby.
As it is obvious from Table 2 the UV-resistant plasmid-free S.derby
cells are characterized by decrease of PCs and increase in the percentage of
LPs connected with increased LPP intensity for plasmid-free S.derby K 82
and S.derby K 95 strains. It is noteworthy that the increased intensity
LPP occurred in the S.derby K 82 strain (even though the content of
tocopherol in these bacteria) was relatively high [7] which in its turn influenced
on UV-resistancy of these cells. Thus, the above -presented data allow to
consider that it is not exclusive the decisive role of PL molecules and their
interactions with DNA in formation of the radioresistant quality of
plasmid-free S.derby cells.
There are no doubt in structural and metabolic changes
determined the UV-sensitivity of the S.derby cells and deserve to
attentively investigation for discovery of their really participation in the
formation of molecular mechanisms of R-plasmid actions as well as the point
mutations in provision of UV-sensitivity of S.derby cells.
Table 1.
Structural Changes of DNA in UV-resistant Salmonella derby Cells
|
Strains |
Content of 5 mC (methyl cytosine in %) |
Tm |
Content of C (methyl cytosine in %) |
|
Salmonella derby K89 |
2.15 |
73.5 |
47.82 |
|
Salmonella derby K134 |
1.68 |
71.5 |
45.38 |
A TºC
dn/dT B dn/dT dn/dT
TºC
Fig.1. Differential curves of DNA preparation
melting:
A- S.derby K 134 plasmid- carrying UV-sensitive mutant strain, B- S.derby
K 89 plasmid-carrying wild strain.
Table 2.
Qualitative and
Quantitative Phospholipid Composition of Salmonella
derby Cells
|
|
Strains |
||||
|
Phospholipid Fractions |
S.derby K 89 wild (plasmid- carrying) |
S.derby K 134 (plasmid-carrying
UV-sensitive mutant strain) |
S.derby K 82 (plasmid-free
UV-resistant strain) |
S.derby K 95 (plasmid -free
UV-resistant mutant strain) |
S.derby Tr 95 |
|
Lysophosphatidylcholines |
A.21.65±1.25 B.1.52±0.09 |
A.23.4±1.4 B.1.57±0.1 |
A.8.34±0.91 B.9.97±0.1 |
A.7.13±0.24 B.8.85±0.2 |
A.23.01±1.1 B.1.56±0.2 |
|
Sphingo- myelines |
A.28.97±1.48 B.2.03±0.1 |
A.32.0±1.5 B.2.14±0.01 |
A.2.84±0.11 B.3.43±0.28 |
A.2.0±0.1 B.2.48±0.3 |
A.32.45±4.5 B.2.2±0.11 |
|
Phosphatiyl-seines |
A.64.94±0.37 B.4.55±0.03 |
A.34.5±2.04 B.2.31±0.07 |
A.4.7±0.53 B.5.62±0.1 |
A.4.43±0.03 B.5.5±0.01 |
A.32.6±0.24 B.2.21±0.07 |
|
Phospho- Inositides |
A.34.64±0.23 B.2.42±0.02 |
A.17.32±1.4 B.1.16±0.08 |
A.4.38±0.63 B.5.24±0.39 |
A.4.62±0.2 B.5.74±0.7 |
A.15.93±0.3 B.1.08±0.04 |
|
Phosphatidyl cholines |
A.692.64±2.8 B.48.48±0.12 |
A.710±64.1 B.47.5±2.04 |
A.14.86±0.2 B.17.76±0.64 |
A.10.1±0.12 B.12.5±0.21 |
A.685.88±4.5 B.46.5±0.2 |
|
Phosphatidyl ethanol amines |
A.179.65±1.5 B.12.58±1.0 |
A.112.6±10.1 B.7.53±0.7 |
A.11.57±0.33 B.13.83±0.89 |
A.8.22±0.08 B.10.2±0.1 |
A.115.05 ± 10.2 B.7.8±0.2 |
|
Phosphatidyl glicerols |
A.168.8±11.6 B.11.82±0.11 |
A.274.5±10.1 B.18.4±0.74 |
A.12.03±0.34 B.14.38±1.48 |
A.24.67±0.7 B.17.5±0.65 |
A.274.5±1.72 B.18.61±0.5 |
|
Total Phospholipid
Content |
1191.3 |
1204.32 |
58.72 |
61.17 |
1179.4 |
|
Sum of the above
individual phospho-lipids |
1428.6 |
1493.68 |
83.65 |
80.56 |
1475 |
A.
Expresses as mg
phosphorus per mg total solids
B. Percentage of an individual
phospholipid relative to total phospholipid content
Table 3.
Fatty Acid Composition of Phospholipids in Salmonella
derby Cells
|
Strains |
Percentage of phospholipid - associated fatty acid
relative to the total amount of esterited fatty acid |
||||||
|
16: 0 |
16: 1 |
I 18: 0 |
18: 0 |
18: 1 |
18: 2 |
18: 3 |
|
|
S.derby K 89 wild (plasmid- carrying) |
32,35 ± 8,65 |
11,26 ± 0, 97 |
25,33 ± 4,81 |
0,73 ± 0,15 |
22,17 ± 2.29 |
0,53 ± 0,16 |
6,47 ± 0,99 |
|
S.derby K 134 (plasmid-carrying UV-sensitive mutant
strain) |
32,4 ± 9,6 |
12,9 ± 1,21 |
25,1 ± 3,02 |
0,4 ± 0,04 |
21,01 ± 2,1 |
0,21 ± 0,1 |
4,05 ± 0,8 |
|
S.derby K 82 (plasmid-free UV-resistant strain) |
51,6 ± 6,76 |
9.46 ± 4, 97 |
10,8 ± 1,25 |
1,53 ± 0,82 |
9,66 ± 2,75 |
3,72 ± 1,82 |
11,3 ± 2,16 |
|
S.derby K 95 (plasmid -free UV-resistant mutant strain) |
48,5 ± 3,5 |
10,1 ± 1,01 |
10,2 ±2,1 |
0,99 ± 0,07 |
10,12 ± 1,8 |
2,4 ± 1,05 |
11,4 ± 0,7 |
Table 4.
Shifts of Malonic Dialdehyde (in nM/mg of PL)
Contents of Salmonella derby Cells in Enzymatic and Non-enzymatic
Systems
|
Malonic dialdehyde |
Strains |
|||
|
S.derby K 89 (plasmid -
carrying wild strain) |
S.derby K 134 (plasmid
carrying UV-sensitive mutant) |
S.derby K 82
(plasmid-free UV-resistant strain) |
S.derby K 95 (plasmid- free UV-resistant mutant
strain) |
|
|
Bacterial suspensions |
33,23 ± 1,2 |
12,78 ± 1,4 |
10,5 ± 1,8 |
407 ± 56,1 |
|
Growth medium |
9,1 ± 1,07 |
10,5 ± 1,8 |
278,4 ± 14 |
381 ± 17,44 |