Staphylococcus aureus growing in a normal NaGI medium
has a specific NaGI tolerance property to grow in the medium contain. ing NaGl in as high a concentration as over 10%. In our comparative study of the cells proliferating in the normal NaGI medium and 10% NaGl medium, we have observed the following differences aside from the changes of lipid composition in the cytoplasmic membrane previously reported. 1. S. aureus grown in high NaGl medium undergoes changes as to increase its size and reduce its surface area. 2. The thickness and weight of cell wall are increased to about 1. 7 times and 1. 32 times, respectively. 3. The protoplast prepared from S. aureus growing in the high NaGI medium shows a weaker resistance to hypotonic condition than that from normal cell.</p

Hayashi, Hideo

Kanemasa, Yasuhiro

Katayama, Takeshi

Takai, Ken-ichi

Takatsu, Tieko

Okayama University Digital Information Repository

Ultrastructural alteration of the cell surface of Staphylococcus aureus cultured in a different salt condition

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 28, Issue 5 1974 Article 2OCTOBER 1974Ultrastructural alteration of the cell surface ofStaphylococcus aureus cultured in a differentsalt conditionYasuhiro Kanemasa∗ Ken-ichi Takai† Tieko Takatsu‡Hideo Hayashi∗∗ Takeshi Katayama††∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Ultrastructural alteration of the cell surface ofStaphylococcus aureus cultured in a differentsalt condition∗Yasuhiro Kanemasa, Ken-ichi Takai, Tieko Takatsu, Hideo Hayashi, and TakeshiKatayamaAbstractStaphylococcus aureus growing in a normal NaGI medium has a specific NaGI toleranceproperty to grow in the medium contain. ing NaGl in as high a concentration as over 10%. In ourcomparative study of the cells proliferating in the normal NaGI medium and 10% NaGl medium,we have observed the following differences aside from the changes of lipid composition in thecytoplasmic membrane previously reported. 1. S. aureus grown in high NaGl medium undergoeschanges as to increase its size and reduce its surface area. 2. The thickness and weight of cell wallare increased to about 1. 7 times and 1. 32 times, respectively. 3. The protoplast prepared from S.aureus growing in the high NaGI medium shows a weaker resistance to hypotonic condition thanthat from normal cell.∗PMID: 4281995 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 28,311-320 (1974)ULTRASTRUCTURAL ALTERATION OF THE CELL SURFACEOF STAPHYLOCOCCUS AUREUS CULTURED INA DIFFERENT SALT CONDlTIONYasuhiro KANEMASA, Ken-ichi T AKAI, Tieko TAKATSU,Hideo HAYASHI and Takeshi KATAYAMADepartment of Microbiology, Okayama University Medical School,Okayama, Japan (Director: Prof. J~ Tawara)Receivedfor publication, May 2, 1974Abstract: Staphylococcus aureus growing in a normal NaGI mediumhas a specific NaGI tolerance property to grow in the medium contain.ing NaGl in as high a concentration as over 10%. In our comparativestudy of the cells proliferating in the normal NaGI medium and 10%NaGl medium, we have observed the following differences aside fromthe changes of lipid composition in the cytoplasmic membrane previ-ously reported. 1. S. aureus grown in high NaGl medium undergoeschanges as to increase its size and reduce its surface area. 2. Thethickness and weight of cell wall are increased to about 1. 7 times and1. 32 times, respectively. 3. The protoplast prepared from S. aureusgrowing in the high NaGI medium shows a weaker resistance tohypotonic condition than that from normal cell.It is well known that Staphylococcus aureus (to be abbreviated S. aureus)shows a strong resistance to a high concentration of NaGl and it cangrow in the medium containing NaCl in as high a concentration as 15%(1). Furthermore, we have recognized that S. aureus, despite proliferating ina high NaGI medium, has maintained constancy of Na+ and K+ concentrationswithin the cell. Consequently, when S. aureus is cultivated in the mediumof a high NaGl concentration, it must adapt itself to acquire some specialdefense mechanism to high salt and high osmotic pressure in order to pro-liferate. Therefore, it seems only natural to consider that for the adaptation toenvironments changes in the cell wall as well as in the structures and func.tional changes of the cell membrane should play an important role. In ourobservations of changes in the lipids, the main components of the cell mem-brane, we have observed that cardiolipin contained is only a few per cent ofthat in normal S. aureus, where it is increased to over 50% in the 10% NaClmedium, as we reported elsewhere (1). However, it is difficult to understandwhy the adaptation to such a great change in NaGI concentration as 14 timescan be compensated by this phenomenon alone. In order to elucidate thisproblem we have studied changes in the cell wall of S. aureus mainly fro~3111Kanemasa et al.: Ultrastructural alteration of the cell surface of StaphylococcusProduced by The Berkeley Electronic Press, 1974312 Y. KANEMASA, K. TAKAI, T. TAKATSU, H. HAYASHI and T. KATAYAMAmorphological asPects and give some consideration on its physiological state.MATERIALS AND METHODSMicroorganism and Culture Method: S. aureus 209P was used and the semi-synthetic medium containing a small amount of peptone and yeast extract wasused for cultivation (1), occasionally supplemented with 0.05% NaCl (to makeits final concentration 145 mM Na + equivalent to physiological saline solutionwhen the total medium components are taken into consideration) or 10% NaCl(to make 1,900 mM Na+). The former medium was designated as normalmedium and the latter as 10% NaCl medium. The culture was conducted bygentle shaking with Monod's tube at 37°C, and the checking of bacterial pro-liferation was doneby measuring the absorbance at 650 nm. S. aureus grown innormal medium was called as N-Staph and that grown in 10% NaCl medium aslO-Staph.Preparation of protoplast: S. aureus harvested at late log phase was washedthoroughly with 0.8 M sucrose-5OmM Tris-HCl buffer (pH 7.2), and was treatedwith L-ll enzyme (cell wall lytic enzyme purified from Flabobacterium sp. whichwas offered by Prof. KOTANI of Osaka University) for preparation of protoplast(3,4). The details of procedure are shown in the legend of Fig. 5.Ana(ysis of Cell Wall: Twenty ml of suspension containing 300 mg late logcells/ml in 50 mM Tris-Hel buffer (pH 7.2) were shaken with 40 ml of glassbeads (0.1 mm in diameter) in a Vibrogen Cell Mill (Edmund Buhler) for 30 min.The glass beads and the remaining intact cells were removed by centrifugationat 1, 400xg for 5 min. The cell wall fraction was collected by centrifugationat 1O,OOOxg for 25 'min. Contaminating cytoplasmic debris was digested withtrypsin and RNase. For amino acid analysis of cell wall, 10 mg sample hydro-lyzed with 6 N HCl in sealed tube at llO°C for 16 hrs was analyzed on aNihon Denshi JLC-6AH amino acid analyzer (5). Phosphorus content of cellwall was determined by the method of KATES (6).Morphological Observation: For the observation of bacteria and protoplasts,a phase-contrast microscope was used. In order to stop the brawnian movementof specimens during the observation, the sample was placed on 0.3% agar con-taining salts of a suitable concentration for the purpose. For the observationwith a scanning electron microscope, the specimen was prepared by TAwARA'smethod (7) after OS04 vapor fixation and was observed with a JSM-UIIIscanning electron microscope at 25 KV accelerating voltage. For the obser-vation of thin sections, the sections were prepared by 1% OS04 fixation andobservation was carried out as it was or after uranyl acetate staining in Hitachielectron microscope Hu-ll.RESULTSMorphological Observation: In comparing the growth curves between N-Staph and lO-Staph (Fig. 1), the doubling time of lO-Staph was prolonged and2Acta Medica Okayama, Vol. 28 [1974], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss5/2S. aureus Cultured in High NaCI Medium 31312IO-Stoph8TIME IN HOURS4---N-StoplJ--------,..".-------......,/,/,/ ............,/"///"//"/"/".-.-//;"1.0ec010U>cici0.1:r:f-~0a::<-'0.010Fig. 1. Comparison of the growth rates in the semi-synthetic media with 0.05% or 10% NaC!. Cultures werecarried out with gentle shaking at 37°C. Optical densityat 650 nm was followed as a function of culture time.the growth maximum was somewhat less than that in case of N-Staph. Inaddition, IO-Staph was somewhat viscous and its orange color was marked ascompared with N-Staph.Fig. 2 shows the results of phase-contrast microscopic observation on thetwo groups of S. aureus just at a late log phase, indicating the difference inFig. 2. Phase-contrast micrographs of N-Staph (a) and lO-Staph (b). Markersindica te 5 pm.3Kanemasa et al.: Ultrastructural alteration of the cell surface of StaphylococcusProduced by The Berkeley Electronic Press, 1974314 Y. KANEMASA, K. TAKAI, T. TAKATSU, H. HAYASHI and T. KATAYAMAtheir size. Scanning electron microscopic observations were also carried outto see details of their structures as shown in Fig. 3. There could be observedFig. 3. Scannig electron micrographs of N·Staph (a) and lO·Staph (b).4Acta Medica Okayama, Vol. 28 [1974], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss5/2S. aureus Cultured in High NaCl Medium 315Fig. 4. Thin section of N-Staph (a) and IO-Staph (b). Markers indicate 100 nm.5Kanemasa et al.: Ultrastructural alteration of the cell surface of StaphylococcusProduced by The Berkeley Electronic Press, 1974316 Y. KANEMASA, K. TAKAI, T. TAKATSU, H. HAYASHI and T. KATAYAMAno difference in the shape, manners of cell division and surface structure.But there was a distinct difference in their size; namely, the diameter of N-Staph was a. 81 pm in average while that of lO-Staph was 1. a4 pm. More-over, in the intercellular space of 1a-Staph, there was a slime substancethought to be the source of their viscocity.Next, intracellular structures of the two groups were compared with thinsections. As shown in Fig. 4, while there was no marked difference in thethickness of cell membrane, the cell wall of N·Staph was about 22 nm thickand that of 1a-Staph about 38 nm. In addition, 1a-Staph had an irregular cellwall with a slime substance on its surface. As for intracellular structure,cytoplasm of 1a·Staph revealed an electron lucent structure on its periphery.Reactivity of S. aureus to L-ll Enzyme: Fig. 5 illustrates the time courseof loss of optical density (aD) by the cell wall lysis of the two groups ofS. aureus when they were exposed to L-ll enzyme. Despite the fact that theEt:o O.10Woo-O--O--o--o----o- __<>---~--,oOL.-._..J.-_-L.._---L__L...-_..J.-_-'--o 30 60INCUBATION TIME (min.)Fig. 5. Change of optical density after incubation with L-II enzyme. 0---0N·Staph without enzyme; 0-0 N-Staph with enzyme; .---. lO-Staph with-out enzyme; .-. lO-Staph with enzyme. Cells and enzyme were added to50 mM Tris-HCI buffer (pH 7.2) containing 0.8 M sucrose, to give 6 mg/ml andI unit/ml at final concentration, respectively. The incubation was carried outat 3JOC in a shaking water bath. Optical density was measured at 650 nm in aSpectronic 20 (Shimadzu).experiments were started at the same weight (6 mg wet weight/ml) there wasa considerable difference in their aD. This finding can be interpreted as6Acta Medica Okayama, Vol. 28 [1974], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss5/2S. aureus Cultured in High NaCl Medium 317due to the difference in their cell sizes. When N-Staph was exposed to L·llenzyme, there occurred a gradual decrease in the OD which reached plateauwithin about 60 minutes. In the case of 10-Staph, the down-slope had ashoulder which rose in OD once at about 1bminutes after incubation. In otherwords, there was a difference between the two groups at the time whenthe cell wall seemed to have undergone half lysis.Analysis of Cell Wall: When the cell wall weight of N-Staph and 10-Staph was compared with the same dry welght of cell, it was found to be 13.6%and 15.8% r~spectively(Table). Phosphorous content in cell wall fraction fromTABLE ANALYSIS OF CELL WALL OBTAINED FROM N-STAPH AND lO-STAPHN-Staph lO-StaphWall % to dry cell (w/w) 13.6 15.8Phosphorous content 1. 31-1. 33 0.94-1. 01(pmoles/mg of dry wall)Glycine'; 4.60 4.28Alanine 2.41 2.26Molar ratio of Lysine 1.05 1. 12amino acids Glutamic acid 1.00 1.00Serine 0.29 0.22NH 3 24.2 2.40N-Staph was slightly more that from 10-Staph. The results of amino acidanalysis of wall are also shown in Table. Here hardly any significant differ-ence due to culture condition could be seen.Resistance of Protoplast to Osmotic Shock: The resistance of protoplastprepared with L~ll enzyme to osmotic shock was observed under the sucrosesystem (Fig. 6). The protoplasts of N-Staph maintained their shape intact in0.25 M sucrose but they burst below the osmotic pressure of 0.2 M as thelimit, while the protoplasts of 10-Staph burst at O. 3 M as the limit, showingthe latter to have the cell membrane of a weaker resistance to hypotonicity.DISCUSSIONIn view of the fact that S. aureus can proliferate in a high NaCl containingmedium (1) and cation content in the growing cell is kept constant (2), barriermechanism on the cell surface or an accelerated active transport system seems tobe involved. In addition, S. aureus would require adaptability to maintain itsshape in normal state under an abnormally high osmotic pressure. S. aureus,cultured under a high NaCl condition, sho~ed a marked change in the memb-rane lipid, especially an extreme increase of cardiolipin, which would play7Kanemasa et al.: Ultrastructural alteration of the cell surface of StaphylococcusProduced by The Berkeley Electronic Press, 1974318 Y. KANEMASA, K. TAKAI, T. TAKATSU, H. HAYASHI and T. KATAYAMA10 pmFig. 6. Osmotical fragility of protoplasts derived from N-Staph (a, b, c, d) and lO-Staph(a', b', c'). The resistance of protoplasts on hypotonicity was checked under sucrose systemin phase-contrast microscopy. a and a' show protoplasts in 0.8 M sucrose: band b' in0.4 M sucrose: c and c' in 0.25 M sucrose: d in 0.2 M sucrose.8Acta Medica Okayama, Vol. 28 [1974], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss5/2S. aureus Cultured in High NaCl Medium 319a role in the NaGI tolerance mechanism (1). However, the adaptability to anenormously different environment cannot be met with one phenomenon alonefrom the general aspect of biological phenomena.In the present experiment, it has been demonstrated by both the phase-contrast and scanning electron microscopy that S. aureus cultured in 10% NaGImedium revealed an increase to 1.28 times in its cell size. Moreover, themicrographs revealed only an increase in the cell size but no distortion of itsspherical shape nor any abnormal interruption of cell division. When thebacterial diameter reaches 1. 28 times, the volume increases to 2. 09 timesthe original and the relative surface area (the surface area/its volume) reaches1/1. 28. This phenomenon seems to represent a defense mechanism to escapeas much from the high NaGI condition as possible. In the gravimetric analy-sis of the cell wall, the wall quantity of la-Staph proved to be I. 16 times(15.8%/13.6%) that of N-Staph. This increase coincides approximately withthe wall increase to 1. 32 times as calculated from the sizes of cell and wallin micrographs. This thickening of the cell wall seems to be adaptivechanges as a non-specific barrier to preserve the cell structure -against highosmotic pressure. As to the difference in the intracellular structures, therecould be observed numerous electron transparent structures in the peripheryof the cytoplasm of la-Staph, but it was impossible to clarify whether thesewere mesosomes or other structures (8). As for the membrane itself, therewas no difference in its thickness by electron microscopic observation.GHO et al. stated that H alobacterium halobium growing in high NaGImedium revealed some electron-dense strands inside the cytoplasma runningparallel to the cell envelope (9), and KIRK et al. reported that H alobacteriumfrom the Dead Sea showed hexagonal patterns within layers of the cell en-velope (10). But in our experiments of freeze etching, we have observed nopictures comparable to theirs with N-Staph and la-Staph.Finally, we present some physiological considerations on the protoplast.We have observed that the protoplast from IO-Staph grown in the hypertonicmedium as 1, 900 mM of NaGI has a property much weaker to resist hypo-tonicity than normal cell. The question whether this is due to the differencein the substances contained in protoplast or due to the membrane componentshas not been clarified. An 0.8 M sucrose solution was used as a protoplaststabilizer in our experiments, but FUTAI et al. (11) reported that this 0.8 Msucrose is slightly hypotonic when considered from the intracellular pressureof Staphylococcus. As shown in Fig. 4, there is seen a using phenomenon onthe way to decreasing the turbidity of la-Staph by the wall lysis with L-llenzyme. This phenomenon might be interpreted that a slightly hypotonicsolution has affected especially la-Staph, and the cell has swollen, making9Kanemasa et al.: Ultrastructural alteration of the cell surface of StaphylococcusProduced by The Berkeley Electronic Press, 1974320 Y. KANEMASA, K. TAKAI, T. TAKATSU, H. HAYASHI and T. KATAYAMAturbidity temporarily great when the wall has reached half lysis stage (12).These findings suggest that the NaCI tolerance mechanism of S. aureusconsists not only of the inhibition of passive transport of Na + by changes inthe membrane lipid composition but also of the role of a marked thickening ofthe cell wall serving a nonspecific barrier and a guardian of the cell structure.A~knowledgements: This investigation was supported by a grant from the Sanyo Broad-casting Science and Culture Advancement Foundation and by a grant-in-aid for scientificresearch from the Ministry of Education. The authors thank Prof. S. KOTANI of the Depart-ment of Microbiology, Osaka University Dental School for furnishing us with L-ll enzyme.Thanks are also due to Dr. TOMOCHIKA, Dr. OKABE, Dr. SASAI and Miss MIYOSHI for the assist-ance and advices throughout the present investigation.REFERENCES1. KANEMASA, Y., YOSHIOKA, T. and HAYASHI, H.: Alteration of the phospholipid composi-tion of Staphylococcus aureus cultured in medium containing NaCl. Biochim. Biophys. Acta280, 444-450, 19722. KATAYAMA, T., TOMOCHIKA, K., TAKATSU, C., KANEMASA, Y. and TAWARA, j.: Some con-sideration on cation measurem,ent of bacterial cell. Okayama Igakkai Zasshi 85, 481-486,1973 (in japanese)3. KATO, K., KOTANI, S., MATSUBARA, T., KOGAMI, j., HASHIMOTO, S., CHIMORI, M. andKAZEKAWA, I.: Lysis of Staphylococcus aureus cell walls by a lytic enzyme purified fromculture supernatants of Flavobacterium species. Biken J. 5, 155-179, 19624. KATO, K. _and STROMINGER, j. L.: Structure of the cell wall of Staphylococcus aureus. IXMechanism of hydrolysis by the L-ll enzyme. Biochemistry 7, 2754-2761, 19685. KENIG-VAKSHAL, R., ZEHAVI, U. and SHARON, N.: A comparative study of the cell wallsof transformable mutants of Micrococcus lysodeikticus. Biochim. Biophys. Acta 196, 107-109,19706. KATES, M., ALLISON, A. C. and JAMES, A. T.: Phosphat ides of human blood cells andtheir role in spherocytosis. Biochim. Biophys. Acta' 48, 571-582, 19617. TAWARA, j., KANEMASA, Y., OKABE, A., HAYASHI, H., ICHIKAWA, H., and YOSHIOKA, C.:A method for preparing specimen of microorganisms for scanning electron microscopy.Medicine and Biology 83, 25-28, 1971 (in japanese)8. PAPKIN, T. j., THEODORE, T. S. and COLE, R. M.: Electron microscopy during release andpurification of mesosomal vesicles and protoplast membranes from Staphylococcus aureus.J. Bact. 107, 907-917, 19719. CHO, K. Y., Doy, C. H. and MERCER, E. H.: Ultrastructure of the obligate halophilicbacterium Halobacterium halobium. J. Bact. 94, 196-201, 196710. KIRK, R. G. and GINZBURG, M.: Ultrastructure of two species of Halobacterium. J. Ultra-structure Research 41, 30-34, 1972 -11. FUTAI, M., OKABAYASHI, K. and MIZUNO, D.: Electron microscopic studies on the outerlayers of Staphylococcus aureus using a lytic enzyme from Flavobacterium. Japan. J. Microbiol.16, 341-350,197212. WYATT, P.j., BERKMAN, R.M. and PHILIPS, D. T.: Osmotic sensitivity in Staphylococcusaureus induced by streptomycin. j. Bact. 110, 523-528, 197210Acta Medica Okayama, Vol. 28 [1974], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss5/2

Ultrastructural alteration of the cell surface of Staphylococcus aureus cultured in a different salt condition

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