JOURNAL OF BACTERIOLOGY, Apr. 1973, p. 434-438Copyright 0 1973 American Society for Microbiology

Vol. 114, No. 1Printed in U.S.A.

NOTES

Mesosomes in Pseudomonas aeruginosaHANS-PETER HOFFMANN, SAM G. GEFTIC, HANS HEYMANN, AND FRANK W. ADAIR

Department of Biological Sciences, Douglass College, Rutgers University, New Brunswick, New Jersey 08902,and Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit,

New Jersey 07901

Received for publication 2 January 1973

The use of a combination fixative-staining procedure has allowed a detailedobservation of mesosomes in thin sections of Pseudomonas aeruginosa.

Involutions of the bacterial cytoplasmicmembrane are commonly referred to as meso-

somes (3, 11). Reports on the occurrence ofthese structures in gram-negative bacteria,especially Escherichia coli and Pseudomonasaeruginosa, are rare. Pontefract and co-workers(9), using the standard osmium fixation tech-nique of Kellenberger et al. (7), reported thepresence of mesosomes in thin sections of E.coli. The spatial relationships between themesosomes, cytoplasmic membrane (CM), andthe nucleoid were clearly defined. A recentultrastructural study of P. aeruginosa by Car-rick and Berk (2) described large, membrane-bound, circular inclusions proximal to the CMin thin sections of this organism. The authorsreported that these inclusions did not representinvolutions or direct extensions of the CM. Itwas suggested that the inclusions were storagevacuoles. In addition, Carrick and Berk (2) alsodescribed mesosomal inclusions which were

closely associated with the CM. Visualizationof the inclusions was facilitated by use of a

double fixative.In the present study, we utilized a combina-

tion fixative-staining technique which alloweda detailed observation of mesosomes in P.aeruginosa. These mesosomes differ in appear-ance from any membranous inclusions reportedpreviously for this organism (2). They more

closely resemble the mesosomes found in E.coli by Pontefract et al (9).P. aeruginosa strain CI-3 was cultured in a

glucose-Casamino Acids (Difco)-minimal saltsmedium as described earlier (1). The cells weregrown, without shaking, for 8 to 12 h (exponen-tial growth phase) at 30 C, centrifuged in thecold (4 C), and washed three times in freshmedium of ambient temperature. The washedcell suspension was again centrifuged in the

cold to form a pellet (500 mg wet weight). Thepellet was treated with 2 ml of an ice-coldsolution of 1.5% glutaraldehyde, 1% osmiumtetroxide (reduced with 1.5% potassium fer-rocyanide), and 1% phosphotungstic acid(GOP) prepared in 0.1 M sodium dimethylar-sonate buffer (pH 6.8). This is a modification ofa procedure reported by Schafer-Danneel (13).The pellets were fixed for 45 min at 4 C, washedin ice-cold, distilled water, embedded in 1%agar, stained overnight in 1% aqueous uranylacetate solution, and then dehydrated in gentlyincreasing, graded solutions of ethanol. AnEpon mixture was used for embedding (8).Ultrathin sections, 40 to 90 nm thick, wereprepared on an LKB Ultrotoffe. The sectionswere post-stained in 1% uranyl acetate andlead citrate (10). The thin sections were viewedin an RCA 3G electron microscope with ac-celerating voltages of 50 and 100 kV.

Fixation by the standard Ryter et al. tech-nique (12) did not permit the observation ofmesosomes in thin sections of P. aeruginosa. Incontrast, the GOP technique facilitated theobservation of these structures (Fig. 1). Thethin section shown contains a gently coiledmesosome which is located midway betweenthe poles of the cell and protrudes into thenucleoplasm. The trilaminar mesosomal mem-brane is approximately 5 nm thick and isclosely associated with the trilaminar CM.Next to this mesosome, there appears to beanother mesosome which lies parallel to the cellenvelope. Mesosomes were also positioned at ornear the poles of the cells. Figure 2a depicts anenlarged view of one pole of a cell. The meso-some is immediately adjacent to the pole and isin contact with the nucleoplasm. The point ofinvagination of the CM is clearly visible.Again, as noted in our earlier report (5), the

434

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FIG. 1. Ultrathin, longitudinal section of a cell of P. aeruginosa. Two mesosomes, which are closely asso-ciated with the trilamirwr cytoplasmic membrane, are visible approximately midway between the poles of thecell. The coils of the mesosomes also appear to have a trilaminar structure. Abbreviations: CM, cytoplasmicmembrane; CW, cell wall; IB, inclusion body; M, mesosome; N, nucleoplasm; R, ribosomes. Bar indicates 100nm. Insert shows enlarged view of mesosome. Bar indicates 50 nm.

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FIG. 2. Locations of polar mesosomes of P. aeruginosa. a, An enlarged view of one pole of a cell is shown.The mesosome is located immediately adjacent to the pole. The point of invagination of the cytoplasmicmembrane is visible (arrow). b, The mesosome is located directly at the pole of the cell. Abbreviations: CM,cytoplasmic membrane; M, mesosome; P, periplasm. Bar indicates 100 nm.

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VOL. 114, 1973

periplasmic space contains stainable materialwhich is not usually seen after application ofstandard staining procedures (12). This peri-plasmic material appears to partially occupythe mesosomal sac. The mesosome depicted in

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Fig. 2b is situated directly at one pole of thecell.

In addition to mesosomes, other cytoplasmicinclusion bodies were observed (Fig. 1). Thesestructures were nonmembranous and were cir-

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FIG. 3. a, Thin section of P. aeruginosa showing inclusion bodies (IB) occurring in longitudinal sequencewithin the nucleoplasm. Note the attachment (arrow) of filamentous nuclear material. A polar mesosome (M)is also present. Bar indicates 100 nm. b, The highly electron-dense appearance of inclusion bodies (IB) isshown when overnight exposure to uranyl acetate was deleted from the GOP procedure. Undispersed nuclearmaterial surrounds the inclusion bodies. Bar indicates 100 nm.

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J. BACTERIOL.

cular in shape. They were randomly dis-tributed within the nucleoplasm. Occasionally,the inclusion bodies were found to be arrangedin longitudinal sequence, as seen in Fig. 3a.Filamentous nuclear material appeared to bein direct contact with the inclusion bodies. Ifthe overnight treatment with uranyl acetatewas deleted from the staining procedure, theinclusion bodies were less well define,d. In-stead, highly electron-dense, round bodies,which are usually considered to be diagnostic ofpolyphosphate granules in P. aeruginosa (2, 6)and other bacteria (4), were found in associa-tion with undispersed nuclear material (Fig.3b). Probably, the uranyl acetate treatmentcaused dispersion of the nuclear material andthus permitted resolution of some of the struc-tural characteristics of the inclusion bodies.

LITERATURE CITED

1. Adair, F. W., S. G. G,eftic, and J. Gelzer. 1971. Resist-ance of Pseudomonas to quaternary ammonium com-pounds. II. Cross-resistance characteristics of a mu-tant of Pseudomonas aeruginosa. Appl. Microbiol.21:1058-1063.

2. Carrick, L., and R. S. Berk. 1971. Membranous inclu-sions of Pseudomonas aeruginosa. J. Bacteriol.106:250-256.

3. Fitz-James, P. C. 1960. Participation of the cytoplasmic

membrane in the growth and spore formation ofbacilli. J. Biophys. Biochem. Cytol. 9:507-528.

4. Harold, F. M. 1966. Inorganic polyphosphates in biology:structure, metabolism, and function. Bacteriol. Rev.30:772-794.

5. Hoffmann, H-P., S. G. Geftic, J. Gelzer, H. Heymann,and F. W. Adair. 1973. Ultrastructural alterationsassociated with the growth of resistant Pseudomonasaeruginosa in the presence of benzalkonium chloride.113:409-432.

6.Hubert, E. G., C. S. Potter, T. J. Hensley, M. Cohen, G.M. Kalmanson, and L. B. Guze. 1971. L-forms ofPseudomonas aeruginosa. Infect. Immunity 4:60-72.

7. Kellenberger, E., A. Ryter, and J. S6chaud. 1958.Electron microscope studies at DNA-containingplasms. II. Vegetative and mature DNA as comparedwith normal bacterial nucleoids in differing physi6log-ical states. J. Biophys. Biochem. Cytol. 4:671-676.

8. Luft, J. H. 1961. Improvements in epoxy resin embed-ding methods. J. Biophys. Biochem. Cytol. 9:409-414.

9. Pontefract, R. D., G. Bergeron, and F. S. Thatcher. 1969.Mesosomes in Escherichia coli. J. Bacteriol.97:367-375.

10. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron opaque stain in electron microscopy. J.Cell Biol. 17:208-212.

11. Rogers, H. J. 1970. Bacterial growth and the cellenvelope. Bacteriol. Rev. 34:194-214.

12. Ryter, A., E. Kellenberger, A. Birch-Andersen, and 0.Maaloe. 1958. Etude au microscope electronique deplasmas contenant de l'acide deoxyribonucleique. I.Les nucleosides des bacteries en croissance active. Z.Naturforsch. 13B:597-605.

13. Schafer-Danneel, S. 1967. Strukturelle und funktionelleVoraussetzungen fur die Bewegung von Amoeba pro-teus. Z. Zellforsch. Mikrosk. Anat. 78:441-462.

438 NOTES

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