Biophysics

R - plasmid influence on the Salmonella derby Сells

Membrane Structure

Astghik Z. Pepoyan, Hasmik R. Yepiskoposyan, Natella S. Mirzoyan, Marina O. Sahakyan

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: membrane; Salmonella derby; phospholipid; X-ray diffraction

It is well known that R-plasmids can regulate the synthesis of bacterial cell wall’s components. On the other hand, the electronically-microscope method allows revealing geometrical parameters of bacterial membranes.

By the method of electronically-microscope we were revealed an essential difference in Salmonella derby bacterial cell membranes morphology and ultra structure depending on the absence of R-plasmid.

The goal of this work was to investigate S. derby cells membrane structure by the roentgenografic method.

Salmonella derby cells membranes structure is investigated by the method of X-ray diffraction under small and large angles, for the membrane suspensions both plasmid and plasmid-free Salmonella derby cells. Reflexes are discovered with midline 8 Å and 11 Å distances that are typical only for plasmid cells of Salmonella derby.

According to the supposition indicated reflexes are conditioned by the equidistance well-regulated position of polar groups of phosphatidyletanolamines and phosphatydilcholines molecules on the membrane surface of Salmonella derby cells.

It is established that the reason of formation of discovered structure are most probably considered peculiarities of hydrophilic-hydrophobic reactions of phospholipid molecule in membranes.

R - plasmid influence on the Salmonella derby Сells

Membrane Structure

Astghik Z. Pepoyan, Hasmik R. Yepiskoposyan, Natella S. Mirzoyan, Marina O. Sahakyan

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: membrane; Salmonella derby; phospholipid; X-ray diffraction

It is well known that R-plasmids can regulate the synthesis of bacterial cell wall’s components. On the other hand, the electronically-microscope method allows revealing geometrical parameters of bacterial membranes.

By the method of electronically-microscope we were revealed an essential difference in Salmonella derby bacterial cell membranes morphology and ultrastructure depending on the absence of R-plasmid.

The goal of this work was to investigate S. derby cells membrane structure by the roentgenografic method.

Salmonella derby cells membranes structure is investigated by the method of X-ray diffraction under small and large angles, for the membrane suspensions both plasmid and plasmid-free Salmonella derby cells. Reflexes are discovered with midline 8 Å and 11 Å distances that are typical only for plasmid cells of Salmonella derby.

According to the supposition indicated reflexes are conditioned by the equidistance well-regulated position of polar groups of phosphatidyletanolamines and phosphatydilcholines molecules on the membrane surface of Salmonella derby cells.

It is established that the reason of formation of discovered structure are most probably considered peculiarities of hydrophilic-hydrophobic reactions of phospholipid molecule in membranes.

Material and Methods

In this work we have used conditionally pathogenic plasmid carrying strain S.derby K89 and it’s plasmid-free S.derby K82 variant.

The isolation of S.derby cell membranes was carrying out by Inoye method/5/.

The main method of membrane suspensions investigation was the method of X-ray diffraction. Samples were prepared and researched by method /6/. Membrane suspension of definite concentration was introduced into a capillary and restored in hermetically closed state at room temperature /t°=25C/.

Samples had cylindrical or plane form with a diameter or thickness 0,4-1,0 mm.

X-ray equipments made filming YPC-60, YPC-2 with modified cameras such as KPOH, PCKO, intended for research of small- angle dispersion and enabling to make filming on a plane film. The simple – film distance is 100-150 mm. In our research we have used X-ray tubes BSV-23, BSV -24Â (Russian), hanging radiation in 1,54 Å wavelength with tension of anode 40 KB, when the anode current is 20 mA. The exposure time is 10-14 hours. For preparation of samples the quartz thin-walled capillaries / production of Germany/ with thickness of walls of 0,01 mm and diameter of 0,4-1 mm were used. The background from capillaries and cells practically are absent. In the mentioned field of waves they don’t absorb.

The diffraction patterns of cell membranes water suspensions coincide with diffractogrammes of liquid – crystal lamellar phase of phospholipid-water system /6,7,8,9,10,11/. Thus, midplanar distances calculated by small-angle reflexes and appropriate distance between regularly alternating membranes in space can be present as /see fig.1/:

d =d_M + d_W (1)

Where d_M and d_W are thick nesses of membrane and intermembrane water layer.

For dependence of midplanar distance on concentration of phospholipid /C_M/ and water /C_W/ for the phospholipid water system, the formula of Luzatti /7/ is used:

d = dì (1 + r_MC_W /r_WC_M), (2)

where r_{M and} r_W are the membrane and water’s densities

From (2) equation it is obvious, that d, in case of phospholipid-water system is directly proportional to C_W/C_M, and, that by declination of dependence of d on C_W/C_M it is possible to definite the ratio r_M/r_W.

For experimental definition of d the formula (3) is used:

tg2q = l/D

This formula involves the radius of roentgenogram me – l, the diffraction angle-q and d is measured in Å.

Definition the diffraction angle from (3) and using conditions of Bregg for diffraction 2dsinq = l, we definite d, where l is the wavelength (in our case =1,54 Å).

Results and Discussion

The Lauregrammes obtained for water suspensions of membranes of S.derby plasmid-carrying and plasmid-free cells are shown in fig.2. The appearance of small-angle reflexes points out on arising of system with the alternating regularly arranged membranes and water interlayers, as it is shown in fig 1. Comparing the small-angle reflexes of plasmid-carrying and plasmid-free S.derby strains we can make the following conclusions.

Three cyclic reflexes, rising under small-angles, are not aliquot reflection of a main reflex that points out on presence of three types of structures with different midplanar distances.

Thus, the indicated reflexes in a case of plasmid-free S.derby strain less legible and fuzzy comparing to plasmid-carrying strain.

It is shown on spherical reflexes there are allocated condensing, which suggests the presence of oriented sites with a larger midplanar distances ( fig.2).

The calculations have shown, that in plasmid-carrying and plasmid-free cell membranes 15% water suspensions there are structures with midline distance /d/ accordingly of 42 Å, 78 Å, 103 Å and 44 Å, 92 Å, 136 Å.

Taking into account, that the thickness of natural membranes at a limiting hydration is about 49-51 Å /12/ and that the cell walls of Gramme-negative bacteria involves cell membranes, consisting of an outer membrane and peptidoglican layer, and plasmatic membrane, and taking into account also relation (1) it is possible to suppose, that a reason of arising of three different reflexes probably can be the formation of structures from membranes and cell-walls of plasmid-carrying cells with different thickness and hydration.

The reason of blurring small-angle reflexes can become proteins in membrane periplasmatic space, about which there are no dates in the literature.

From obtained results follows that the midline distances /d/ of small-angle reflexes for plasmid carrying cell membranes are a little less, then the same of plasmid-free cell membranes. Probably it is connected to the fact, detected by electronic microscopy, that the absence of R-plasmid brings to a thickening of S derby bacterial cell walls /1/.

For finding out the legitimacies of changes of midline distances /d/ of small-angle reflexes and the definition of both characteristics of membrane and intermembrane distances for more expressed reflexes, the dependence of /d/ on the ratio of concentration of water and membranes-C_W/C_M were obtained /fig.3/. As it is obvious from the figure with increasing concentration of water, similarly to the system of phospholipid - water /7/, there is a nonlinear increasing of d with achievement of saturation.

In accordance with increase of water it’s accumulation in intermembrane space takes place that reduces in increasing of distance between them. Thus the obtained curve is not linear and therefore does not submit to equation (2).

It’s agreed /8/, that nonlinear character of obtained dependence can be determined by high hydrophoby of membranes causing the origin of separate water phase out of membrane-water regular system. Thus, above definite concentration, water cannot penetrate into intermembrane space. The equation (2) is fulfilled for hydrophilic membranes. In this case at increase of concentration of water it completely accumulated in intermembrane space bringing to linear increasing of intermembrane distance.

From this we can suppose, that the saturation point curve d-C_W/C_M in a definite level characterizes the presence of intermembrane hydrophobic interaction. Thus, obtained results testified, that the membranes of S.derby plasmid-carrying and plasmid-free cells are different by thickness of cell membranes and plasmatic membranes and by hydrophobicity. Thus, for plasmid-free cells intermembrane distances less than the same for plasmid carrying cells.

It is well known, that intermembrane in ordinary arrangement is substantially determined by their lipid composition. We have established, that qualitative and quantitative composition of phospholipids /15,16,17/ and fatty acids of phospholipids /12,15/, lipid peroxidation and status of antioxidative system /12,13,14,17/ of S.derby cells must effect on a formation of liquid state of membranes of S.derby cells /especially for plasmid-free strain/, that should have an effect on X-ray diffraction under big angles.

For clarifying of structure of separate membranes the X-ray diffractions under big angles of the same systems was researched.

It is known, that the reflex with midplanar distance 4,3 Å characterizes a status of hydrocarbonaceous chains of phospholipid molecules in membranes /7/. The absence of diffraction at 4,3 Å points on liquid state of membrane, and the presence of galog points on amorph state, and the presence of circle- on mutual parallel equidistant occurrence of phospholipid molecules in membrane.

As we supposed, the reflexes at 4,3 Å on roentgenograms are missed /fig.2/, that points out that in membranes of both types of cells S.derby phospholipids are in liquid state.

However, in case of plasmid-free cell membranes at 8 Å and 11 Å there are two legibly expressed reflexes, shaped circle. For membranes of plasmid-free cells the indicated reflexes are absent.

On the value, the midplanar distance of the detected reflexes coincide with length of polar groups of molecules of phospatidyletanolamines /PEs/ - 8 Å and phosphatidylcholines /PCs/ - 11 Å /8/.

Earlier we have shown /15,16,17/, that main components of S.derby cell membranes phospholipids are PCs and PEs, and that the absence of R-plasmid in S.derby cells reduces in sharp decrease of a quantitative contents of phospholipids, particularly PCs and PEs.

Probably the presence of more intensive reflex of 11 Å on the roentgenogrammes is connected to major contents of PC in the membranes of S.derby plasmid-free cells. Thus, we can suppose, that in case of plasmid-carrying S.derby cells, we deal with equidistant layout of PC and PE molecules’ polar groups on a surface of membranes, that can become a reason of arising of reflexes 8 Å and 11 Å from the membrane suspensions of S.derby plasmid cells.

It is interesting to compare the results of roentgen graphic researches with dates of investigations the influence of medium pH on growth and reproduction processes of S.derby plasmid-carrying and plasmid-free cells.

It is established, that for S.derby cells the log phase of growth corresponding the

pH _optimum = 7,0-7,5. After transition of cells in a stationary phase of growth the intensity of growth is changing at dependence on medium pH with shift in the acidic side (max growth for plasmid-free night- culture obtained at pH = 6,5, and for plasmid-carrying cells at pH = 5,02). In cell growth process it is detected the legible shift of medium pH from acidic to neutral, however for plasmid carrying cells this transition inappreciable and slow, that points on a slow metabolism of plasmid-free cells.

Thus, at neutral pH plasmid-free cells, which have more ordered inner membrane structure, the accelerated growth is detected (Table).

As appears from experiments, the direction of medium pH effect in both types of S.derby cells is identical. Thus, the violation of metabolism observed for plasmid-free cells, can correlate with unregulated structures of membranes.

On the other hand, as mentioned above and also in /6/, the increase of hydrophicity of phospholipid molecules or membrane must result in increase of membrane water interaction, that eventually will reduce in increase of an amount of molecules of water in intermembrane space, causing the increase of midplanar distance of small-angle reflexes takes place, that according to dependence (1) and fig.1, points out on increase of thickness of intermembrane water interlayer.

Thus, the correlation between presence of R-plasmid and structure of membranes of S.derby cells is detected.

On the bases of all available results we can suggest that the reason of the obtained legitimacies probably are the hydrophobic interactions of molecules of phospholipids in membranes.

Literature cited

1. Ktsoyan J.A., Kostantinova N.D., Sarkisian N.N., Chernov V.I.// Dokl. Arm.SSR, v.89, pp.216-219 /in Russian/.

2. Ktsoyan J.A., Sarkisian N.N., Chernova V.I., Minasian A.S.// Dokl Arm. SSR, 1989, v.88, pp.26-29 /in Russian/.

3. Bogoslovskaya O.A., Burlakova E.B., Glushenko N.N., Konyukhov V.F., Likhard L.Y.// Bacterial Metabolic Plasmids, M. 1982, pp. 33-38.

4. Likhachova N.A., Malchanova E.S., Kim A.A., Ilyashenko B.N.// Mol.Gen.Microb.Virus.1983, pp.15-18 /in Russian/.

5. In: Membrane and Transport. Inoye O. M. N. York Plenum Press, 1982, v. 1, p. 289 - 298.

6. Shahinian A.A., Badalyan H.H., Minasyan M.K.// Izvest.Arm.SSR, 1989, v.23, pp.72-77 /in Russian/.

7. Luzzati V., Huzzon F. // Cell Biol. 1962, v. 12, p. 207, Luzzati V. Biological Membrsnes / Ed. D. Chapman / London Acad. Press. 1965, p. 71.

8. Shahinian A.A., Shahinian Ar.Ar.// Crystallografics.1990, v.35, p.1511 /in Russian/.

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12. Pepoyan A.Z., Haroutyunian A.A., Ktsoyan J.A., Karageuzian K.G.// Dokl A.S. of RA, 1992,v.93,pp.227-230. /in Russian/

13. Pepoyan A.Z., Ktsoyan J.A., Karageuzian K.G.// Dokl. AS of RA, 1991, v.91, pp.219-226. /in Russian/

14. Pepoyan A.Z., Ktsoyan G.A., Karageuzian K.G.// FEBS. Budapest, 1990, p. 503.

15. Pepoyan A.Z., Ktsoyan J.A., Karageuzian K.G.// X-Symp.Biochem.Soc.USSR-DRG, 1989,p.59.

16. Pepoyan A.Z., Ktsoyan J.A., Shahinian A.A., Hovsepyan L.M., Karageuzian K.G.// Biophysics, 1991, v.36, pp.475-479 /in Russian/.

17. Phillips M.G., Finer E.G., Hauser A.J.// BBA, 1972, v. 290, pp. 397-402.

18. Boldirev A.A. Introduction to Biochemistry. M., 1989,p.112.

19. Shahinian A.A.// Colloidal Journal.1978, v.40, p. 297

Table

Influence of Salmonella derby plasmid cell 15% membrane suspensions acidity on the d sum of bacterial membrane thickness and intermembrane distance d

Medium pH	2,5	7,0	10,9
d general (Å)	43,95	42,40	53,07

Figures 1. Frame of lamellar phase of water-phospholipid system.

a.

b.

Figures 2. Lauer patterns (reflexes under large and small angles) of 15% water suspensions of walls of the following cells:

a. Plasmid-free S.derby cells.

b. Plasmid -carrying S.derby cells.

Figures 3. Dependence of d midplanare distance from C_W/C_M.