JOURNAL OF BACrERIOLOGY, Feb. 1975, p. 721-725Copyright i 1975 American Society for Microbiology

Vol. 121, No. 2Printed in U.S.A.

Demonstration of Cell Division by Septation in a Variety ofGram-Negative Rods

H. E. GILLELAND, JR.,* AND R. G. E. MURRAY

Department of Bacteriology and Immunology, University of Western Ontario, London, Ontario, Canada

Received for publication 19 November 1974

Through use of an initial fixative employing a combination of crotonaldehydeand glutaraldehyde, septa were preserved in thin sections of dividing cells ofstrains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei,and Escherichia coli when grown at 30 C in a dilute basal medium. The same

procedures, however, revealed only a constrictive division process in Proteusvulgaris and Erwinia sp. This adds to the evidence that septation, althoughdifficult to demonstrate, is the process of cell division in the enteric gram-nega-

tive rods and the pseudomonads and that constriction is a fixation artifact inthese organisms.

Septa are readily preserved in dividing cellsof gram-positive bacteria by standard fixationprocedures, but they are extremely difficult topreserve in the gram-negative enteric rods andthe pseudomonads, in which a constrictive or"pinching off" type of division process is usuallyseen. This created doubt as to whether constric-tion represents an alternative type of division inthe gram-negative rods or an artifact producedupon fixation (15, 17).Septa are readily seen in certain of the

gram-negative rods, e.g., Beggiatoa (8, 9).Wiebe and Chapman (17) demonstrated septa-tion in the achromobacters, although they couldfind only constriction in the marine pseudomo-nads examined. Conti and Gettner (3) showedone picture of a septum in Escherichia coli, butall the initial stages of division were constrictivein their study. Steed and Murray (16) showedsepta in Spirillum serpens and E. coli byemploying the well-known Ryter-Kellenbergerfixative (14) under the unusual conditions offixing cells at 45 C or by lowering the osmoticpressure of the fixative solution by diluting thebuffer used 1:6. Septa were later demonstratedin certain mutant strains of E. coli (7, 11, 12).Recently, a high proportion of dividing wild-type cells of E. coli B and B/r were shown todivide by septation by Burdett and Murray (1,2), who utilized a combination of 5% acroleinand 0.25% glutaraldehyde as the initial fixative.These authors concluded that constriction was afixation artifact in E. coli. However, constric-tion remained the only form of division everseen in most of the gram-negative rods.

This paper extends the observation of septumformation to several genera which previouslyhad only exhibited a constrictive division proc-ess under standard Ryter-Kellenberger fixationprocedures and provides a fixation procedurewhich may assist studies of the process ofseptation in gram-negative bacteria.

MATERIALS AND METHODSBacteria examined. The bacteria used in this

study and the source from which they were obtainedare given in Table 1.Development of procedure. This study began as

an attempt to study septation in Pseudomonas

TABLE 1. Bacterial strains examined

U.W.O.Strain collec- Original source andOrganism designation tion year obtained

no."

P. aeruginosa 726 ATCC 15692,1970

P. aeruginosa PAO 1 1013 B. W. Holloway,1974

P. aeruginosa PAO 1654 1014 B. W. Holloway,1974

Escherichia coli B 301 Laboratory collec-tion, 1951

E. coli K-12 649 G. S. Stent, 1969S. typhi- 342 Laboratory collec-murium tion, 1965

Shigella sonnei 362 Laboratory collec-tion, 1966

P. vulgaris 492 Laboratory collec-tion, 1968

Erwinia sp. J 309 E. A. Grula, 1966

a U.W.O., University of Western Ontario.

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GILLELAND AND MURRAY

aeruginosa. Therefore, all initial experiments to de-velop a suitable fixation method for the preservationof septa were performed with strains of P. aeruginosaonly. Because the acrolein-glutaraldehyde fixationeffective for preserving septa in E. coli B and B/r (1, 2)provided only constrictive division profiles in P.aeruginosa, a variety of other fixation procedures weretried. These included growth in two different basalmedia, M9 (10) and that of Helmstetter and Cooper(6), growth of the cells at 30, 37, and 45 C, and fixationwith a variety of combinations of fixatives, includingacrolein, glutaraldehyde, crotonaldehyde, and acetal-dehyde, used in varying concentrations. In view of theobservation that the tonicity of the fixing solution wasimportant (16), the M9 medium was diluted 1:10 and1:20 to reduce the osmotic pressure of the fixingenvironment in some experiments. From these variousexperiments the procedure described below for growthand fixation of the cells was developed.

Cultural conditions. All cells were grown in amedium derived from M9 medium (10), modified bydiluting the M9 medium 1 to 20 with distilled waterand then restoring the original concentration of glu-cose and MgSO4. This modified medium thus con-tained: NH4Cl (0.05 g/liter), Na2HPO4 (0.35 g/liter),KH2PO4 (0.15 g/liter), MgSO47H2O (5 x 10-4 M),and glucose (0.1%c).

All cells were grown in this medium at 30 C withshaking in a reciprocal water bath in 125-ml Erlen-meyer flasks containing 30 ml of medium. Culturesgrown for 16 to 18 h were used to inoculate new flasksof medium to an initial absorbance at 660 nm (A6,60) of0.04 to 0.06. These flasks were incubated 4 to 6 h untilthe cells were in the logarithmic phase of growth (A66,= 0.10 to 0.15).Fixation procedure. A special fixative which con-

tained 2% crotonaldehyde, 2% glutaraldehyde, and0.05 M sodium cacodylate buffer, pH 7.5, was utilizedfor initial fixation. The crotonaldehyde was obtainedfrom BDH Chemicals, Ltd. (Poole, England), and theglutaraldehyde was obtained as EM grade, sealedunder nitrogen from Polysciences, Inc. (Warrington,Pa.). A 1/100 volume of the fixative was addeddirectly to the shaking culture, and the culture wasallowed to continue shaking for 3 to 5 min. The cellswere then harvested by centrifugation at 5,000 x g for5 min at room temperature (RT, 22 C), suspended in 1to 2 ml of the full-strength fixative solution, and heldfor 3 h at RT. After being washed three times in 0.05M sodium cacodylate buffer, the cells were leftovernight at RT in this buffer. After enrobement inagar, the cells were postfixed with 1% OSO4 in 0.05 Msodium cacodylate buffer at RT for 1 h, stained with1% uranyl acetate for 2 h at RT, and dehydratedthrough a graded series of ethanol, followed by em-bedding in Epon.

Electron microscopy. Sections were cut with glassknives on a Reichert ultramicrotome, picked up on200-mesh copper grids coated with Formvar andcarbon, and poststained with 1% uranyl acetate andthen lead citrate (13), each for 2 to 3 min. The sectionswere then examined with a Philips 200 electronmicroscope operated at 60 kV. Micrographs weretaken on 35-mm, fine-grain positive film.

RESULTS

All the initial variations of the growth me-dium, growth temperature, and initial fixativeyielded only constrictive division in P.aeruginosa until, finally, septa were preservedupon growth in diluted M9 medium at 30 C withinitial fixation by crotonaldehyde-glutaralde-hyde. Once these conditions of growth andfixation were discovered to successfully preservesepta in P. aeruginosa PAO 1 and PAO 1654,they were applied to a variety of gram-negativerods to survey for division by septation in theseorganisms.The f ixation procedure preserved septa in

dividing cells of Salmonella typhimurium (Fig.lA-C), Shigella sonnei (Fig. 1D), E. coli K-12(Fig. 1E), E. coli B (Fig. 1F), and P. aeruginosaPAO 1 and PAO 1654. However, only late stageswere preserved in S. sonnei and the two strainsof E. coli.We were unable to preserve any stage of

septation in Proteus vulgaris, Erwinia sp., or P.aeruginosa U.W.O. (University of Western On-tario) 726 by this procedure. These organismsall showed a constrictive division process. Thesame species of Erwinia had previously beenshown by Grula and Smith (5) to reveal con-strictive division when fixed by standardmethods.Even in our best preparations some dividing

cells showed constriction. S. typhimurium ex-hibited septation in 70 to 75% of the dividingcells in better preparations, whereas P.aeruginosa PAO 1654 showed septa in 55 to 65%and both P. aeruginosa PAO 1 and E. coli B hadapproximately 40% of the dividing cells exhibit-ing septa. S. sonnei and E. coli K-12 possessedsepta in only about 10% of the cells undergoingdivision.The formation of the septum in all cases

involved the invagination of the cytoplasmicmembrane along with the peptidoglycan layerof the cell wall (Fig. IA and B; Fig. 2C). Theouter membrane of the cell wall did not invagi-nate initially but appeared to slough off mate-rial in the form of blebs (Fig. 1A and E; Fig.2D). After the septum was completed and thetwo daughter cells were divided, the outermembrane grew inwards, resulting in separa-tion of the daughter cells. This septation proce-dure agrees with that seen previously in bacte-ria possessing a gram-negative cell wall archi-tecture (1, 2, 4).Mesosomes have previously been found asso-

ciated with the developing septum in E. coli (1)and in a gram-variable coccus with a gram-neg-ative cell wall and division pattern (4). How-

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FIG. 1. Demonstration of septation in various enteric rods. Abbreviations: CM, cytoplasmic membrane; P,peptidoglycan layer of the cell wall; OM, outer membrane of the cell wall; B, bleb of outer membrane material.The horizontal bar represents 100 nm. (A - C) Various stages of septation are shown in S. typhimurium. Notethat the initial stages of septum formation are well preserved. The developing septur,u is formed by thesymmetrical ingrowth of the cytoplasmic membrane and the peptidoglycan layer of the cell wall. (D)Completed septum in S. sonnei . (E) Late stage of septation in E. coli K-12. (F) Completed septum in E. coli B.In all completed septa shown note the septum consists of two cytoplasmic membrane partitions separated by adouble lamella of peptidoglycan.

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GILLELAND AND MURRAY

GM .^.48 :_FIG. 2. Septation in P. aeruginosa. See legend to Fig. 1 for abbreviations. (A and B) An initial and late stage

of septum formation in the PAO 1 strain. (C and D) An initial and late stage of septation in the PAO 1654 strain.Note the initial stage is poorly preserved, with the two opposing sides of the invaginating septum being widelyseparated. Bar, 100 nm.

ever, we observed no such mesosomes in ourpreparations. This was felt to represent aninability of the fixation procedure employed topreserve these structures.

DISCUSSIONThe acrolein-glutaraldehyde procedure of

Burdett and Murray (1, 2), which worked sowell for E. coli B and B/r, did not preserve septaat all in P. aeruginosa (unpublished observa-tions). After a variety of fixation methods hadbeen tested, the above procedure was developedfor the preservation of septa in P. aeruginosa.Since septa in P. aeruginosa appeared to bemore difficult to preserve than in E. coli, itseemed likely that a procedure which preservedsepta in P. aeruginosa might also preserve themwell in the enteric rods.The fixation procedure given herein is not

offered as a method for the optimum preserva-tion of septa in all the organisms examined.

Whereas the method did preserve septa in allstages quite well in S. typhimurium, the earlyseptal stages seen in P. aeruginosa were poorlypreserved, as evidenced by the wide gap pro-duced between the arms of the invaginatingseptum. Likewise, only late stages of septationwere preserved by this method in E. coli B andK-12 and S. sonnei, whereas early stages havebeen preserved well in E. coli B by acrolein-glu-taraldehyde fixation (1, 2).We believe that the requirements for fixation

of division structures vary for each organism,and fixation procedures need to be developedthat would be applicable to each organism on anindividual basis. Furthermore, we feel thatorganisms which have routinely shown onlyconstrictive division to date, such as P. vulgaris,Erwinia, and P. aeruginosa U.W.O. 726 in thisstudy, would consistently show septation ifsuitable fixation procedures were known. Thedemonstration of septation in the four genera inthis study supports this contention.

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CELL DIVISION BY SEPTATION

The conditions which must be met to success-fully preserve septa in gram-negative rods re-

main entirely unclear. It is difficult to under-stand why the procedure we employed pre-served septa, however poorly, in P. aeruginosaPAO 1 and PAO 1654 but not at all in theU.W.O. 726 strain. Likewise, why the procedureworks for four genera but not for Proteus or

Erwinia remains undetermined. Equally un-

clear is why certain mutant strains of E. colireadily demonstrate septa with only standardfixation procedures. It does appear that the tem-perature at which cells are grown (16), the to-nicity of the fixing environment (16), the me-

dium in which the cells are grown (Gilleland andMurray, unpublished observations), and theinitial fixation conditions employed (1) all couldplay some role.These observations amplify the suggestion of

Steed and Murray (16) that constriction is an

artifact of fixation and not an alternative proc-ess of division. It would be highly unlikely thatan organism would possess both constrictionand septation as methods of division. Further-more, although constriction can readily be envi-sioned as arising from the collapse of a develop-ing septum (1), it is extremely difficult toimagine septation being an artifact from con-

strictive division. Thus, we feel that septationhas been firmly established as the method ofcell division in the gram-negative enteric rodsand pseudomonads. The question of whetherconstriction may be an alternative process ofdivision in these organisms in which septationpreviously had never been found can now beanswered in the negative.

ACKNOWLEDGMENTSWe wish to thank H. Koppenhoefer for her technical

assistance. We express our appreciation to B. W. Holloway,Department of Genetics, Monash University, Clayton, Vic-toria, Australia, for kindly providing us with the PAO 1 andPAO 1654 strains of P. aeruginosa.

The financial support of the Medical Research Council ofCanada in gratefully acknowledged. This investigation was

also supported by a Public Health Service Fellowship (no.1F02 Al 55910-01) from the Institute of Allergy and InfectiousDiseases.

LITERATURE CITED

1. Burdett, I. D. J., and R. G. E. Murray. 1974. Septumformation in Escherichia coli: characterization of septalstructure and the effects of antibiotics on cell division.J. Bacteriol. 119:303-324.

2. Burdett, I. D. J., and R. G. E. Murray. 1974. An electronmicroscope study of septum formation in Escherichiacoli strains B and B/r during synchronous growth. J.Bacteriol. 119:1039-1056.

3. Conti, S. F., and M. E. Gettner. 1962. Electron micros-copy of cellular division in Escherichia coli. J. Bacte-riol. 83:544-550.

4. Gilleland, H. E., Jr., I. L. Roth, and R. G. Eagon. 1971.Ultrastructure of the cell wall and the mechanism ofcellular division of a gram-variable coccus. Can. J.Microbiol. 17:421-424.

5. Grula, E. A., and G. L. Smith. 1965. Cell division in a

species of Erwinia. J. Bacteriol. 90:1054-1058.6. Helmstetter, C. E., and S. Cooper. 1968. DNA synthesis

during the division cycle of rapidly growing Escherichiacoli B/r. J. Mol. Biol. 31:507-518.

7. Kohiyama, M., D. Cousin, A. Ryter, and F. Jacob. 1966.Mutants thermosensibles d'Escherichia coli. Ann. Inst.Pasteur Paris 110:465-486.

8. Maier, S., and R. G. E. Murray. 1965. The fine structureof Thioploca ingrica and a comparison with Beggiatoa.Can. J. Microbiol. 11:654-655.

9. Morita, R. Y., and P. W. Stave. 1963. Electron micro-graph of an ultrathin section of Beggiatoa. J. Bacteriol.85:940-942.

10. Nagata, T. 1963. The molecular synchrony and sequentialreplication of DNA in Escherichia coli. Proc. Nat.Acad. Sci. U.S.A. 49:551-559.

11. Normark, S., H. G. Bowman, and G. D. Bloom. 1971. Celldivision in a chain-forming envA mutant of Escherichiacoli K12. Acta Pathol. Microbiol. Scand. Sect. B79:651-664.

12. Radolakis, A., P. Thomas, and J. Starka. 1973. Morpho-logical mutants of Escherichia coli. Isolation and ultra-structure of chain-forming envC mutant. J. Gen. Mi-crobiol. 75:409-416.

13. Reynolds, E. S. 1963. The use of lead citrate at high pH as

an electron-opaque stain in electron microscopy. J. CellBiol. 17:208-212.

14. Ryter, A., and E. Kellenberger. 1958. Etude au micro-scope electronique de plasmas contenant de l'acidedesoxyribonucleique. I. Les nucleoides des bacteries encroissance active. Z. Naturforsch. Teil B 13:597-605.

15. Slater, M., and M. Schaechter. 1974. Control of celldivision in bacteria. Bacteriol. Rev. 38:199-221.

16. Steed, P., and R. G. E. Murray. 1966. The cell wall andcell division of gram-negative bacteria. Can. J. Micro-biol. 12:263-270.

17. Wiebe, W. J., and G. B. Chapman. 1968. Fine structure ofselected marine pseudomonads and achromobacters. J.Bacteriol. 95:1862-1873.

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