JOURNAL OF BACrERIOLOGY, May 1974. p. 717-724Copyright 0 1974 American Society for Microbiology

Vol. 118. No. 2Printed in U.S.A.

Mesosome Structure in Chromobacterium violaceumT. E. RUCINSKY AND E. H. COTA-ROBLES1

Department of Microbiology, The Pennsylvania State University, University Park, Pennsvlvania 16802

Received for publication 21 November 1973

Exponentially growing cells of the gram-negative bacterium Chromobacteriumviolaceum demonstrate invaginations of the cytoplasmic membrane with a highfrequency. These invaginations conform to the ultrastructural appearance ofmesosomes of gram-positive bacteria. As many as four mesosomes are observedper cell, each of which may increase the total membrane surface of the cell by30%. Washing of cells in dilute tris(hydroxymethyl)aminomethane buffer effectsa distension of the mesosome "neck" and/or cytoplasmic membrane clarifyingthe association of the mesosome to the cytoplasmic membrane. Plasmolysiseffects an eversion of the mesosome into the plasmolysis vacuole.

Fitz-James (9) described mesosomes asunique membranous structures in gram-posi-tive bacilli. Subsequently, mesosomes havebeen described in bacilli and other gram-posi-tive bacteria (3, 4, 10-12, 14, 24, 27, 28). Reavelyand Burge (20) have recently refined the mean-ing of the term "mesosome" when discussinggram-positive bacterial cells as: "sac-like in-vaginations of the cytoplasmic membrane to-gether with the tubular, vesicular, and lamellarmembranous contents of these sacs." Rogers(24) had earlier suggested that the membranouscontents of the enveloping sac are probablyattached to the sac at one or two points thatwould make these tubules or vesicles extensionsof the cytoplasmic membrane. As Burdett andRogers (4) have recently pointed out, neitherthe genesis nor the role of mesosomes is fullyunderstood. The contribution of a mesosome tothe overall structural organization of a bacte-rium is also not understood, although it isrecognized that the nucleoid is in immediatephysical association with a mesosome (27).The term mesosome is frequently used to

describe membranous structures in gram-nega-tive bacteria (5, 13, 18). These structures,however, have not always met criteria acceptedfor gram-positive mesosomes. The reports ofCohen-Bazire et al. (6) and Pate and Ordal (17)describing mesosomes in Caulobactercrescentus and Chondrococcus columnaris sug-gest that certain gram-negative bacteria mightindeed possess mesosomes as classically de-fined. In the report below, we present an ultra-structural study of mesosomes in the gram-negative bacterium Chromobacterium viola-ceum.

' Present address: University of California, Santa Cruz,Calif. 95064.

MATERIALS AND METHODSCultural conditions. The strain of C. violaceum

that produces defective bacteriophage (25, 26) used inthis study has been designated CHRPP in our labora-tory. The organism was cultured in 0.8% (wt/vol)tryptone broth, pH 7.5, at 28 C in a gyrorotaryincubator shaker. The doubling time of C. violaceumstrain CHRPP under these conditions is 100 min.After a 1% (vol/vol) inoculum from cells frozen inexponential growth (25), optical density at 600 nm of0.05, cultures were incubated 5 to 6 h, to an opticaldensity of 0.2 to 0.3, at which time samples ofexponentially growing cells were removed for furthertreatment and electron microscopy.

Plasmolysis. After a single wash in 0.01 M tris(hy-droxymethyl)aminomethane-hydrochloride buffer,pH 8.0, exponentially growing cells of CHRPP wereplasmolyzed by suspension in the above buffer sup-plemented with sucrose to a final sucrose concentra-tion of 0.3 M.

Electron microscopy. Samples to be examined byelectron microscopy were fixed with OS04 accordingto the Ryter-Kellenberger method (29), dehydrated inacetone, and embedded in a low-viscosity epoxy resinby the method of Spurr (30). Thin sections were cutwith a diamond knife and mounted on uncoated400-mesh grids. These sections were routinely stainedwith uranyl acetate and lead acetate (23). Some thinsections were stained solely with bismuth subnitratefor 30 min according to Ainsworth and Karnofsky (1,2) to improve the contrast of mesosomal membranes.Serial sections were mounted on Formvar-carbon-coated slotted grids and stained as above. All micro-graphs were made with a Phillips EM 300 electronmicroscope at accelerating voltages of 60 or 80 kV.

RESULTSOur earlier ultrastructural studies of the

gram negative bacterium C. violaceum strainCHRPP sampled during stationary phase ofgrowth for particle production have revealed

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718 RUCINSKY AN[

the occasional presence of mesosome-like struc-tures (25, 26). Intracytoplasmic membranestructures that conform to the definition ofmesosomes given above for gram-positive orga-nisms are seen in exponentially growing cells ofC. violaceum strain CHRPP (Fig. 1) as tubular

COTA-ROBLES J. BACTERIOL.

or lamellar-like elements within a membraneenvelope, which may or may not be connectedto the cytoplasmic membrane. The lack ofvisualization of the association of the meso-somes and cytoplasmic membrane may simplybe related to the plane of sectioning. Figure 2; ........ K .

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FIG. 1. Unwashed C. violaceum strain CHRPP. Note that each cell has at least one mesosome visible, butonly those mesosomes marked with the arrow suggest attachment to cytoplasmic membrane. Section stainedwith alkaline bismuth subnitrate. Marker represents 100 nm in all micrographs.

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FIG. 2. Serial sections of mesosome in unwashed C. violaceum strain CHRPP. Arrows in (b) and (c) indicatepossible tubule origin from mesosome sac. Only (d) clearly shows cytoplasmic membrane invagination. Sectionthickness was estimated to be 40 to 45 nm. These and all subsequent sections are stained with uranyl acetateand lead citrate.

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RUCINSKY AND COTA-ROBLES

presents a series of eight serial sections througha chromobacterium mesosome, which substan-tiates this interpretation. The diverse morphol-ogy of the membrane sac and its internaltubular components, as well as the very limited

area of attachment with the cytoplasmic mem-brane, is clear in this series of micrographs. Theorigin of the tubules from the mesosomal sac issuggested in Fig. 2b and c. This is clearly shownin Fig. 3a and b in which one observes branching

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FIG. 3. Mesosomes in unwashed cells of C. violaceum strain CHRPP. (a) Note tubule extending trommembrane sac invagination (0). (b) Cytoplasmic membrane invagination, origin of mesosomal tubules (0),and branching of tubules (B).

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C. VIOLACEUM MESOSOME STRUCTURE

tubules as invaginations of the mesosome sac.

A three-dimensional representation of C.violaceum strain CHRPP mesosome (Fig. 4) hasbeen constructed from examination of serialsections. This artist's reconstruction depicts themesosome as an invagination of the cytoplasmicmembrane to forrn the mesosome sac fromwhich arise numerous multibranched tubules.

Weibull (31) reported that the mesosomes ofBacillus megatherium were dislocated uponplasmolysis and were everted into the spacebetween the cell wall and the protoplast. Theeversion of mesosomes upon plasmolysis hasnever been reported for any of the gram-nega-tive bacteria described as possessing meso-

somes. Figure 5 presents an example of plasmo-lyzed C. violaceum CHRPP in which tubularand vesicular structures are clearly resolvedwithin the periplasmic space. Several of thesevesicles also appear to remain connected to thecytoplasmic membrane.Highton (12) reported that when Bacillus

licheniformis cells are removed from the growthmedium and resuspended in buffer, the meso-

somes fragment into numerous smaller meso-

somes filled with vesicular elements rather thantubules. Resuspending strain CHRPP cells in

FIG. 4. Artist's composite drawing of C. violaceumstrain CHRPP mesosome as multiple branching tubu-lar invaginations of the mesosomal sac, which is itselfan invagination of the cytoplasmic membrane. Thisdrawing was made from analysis of serial thin sec-

tions.

buffer does not increase the number of meso-somes per cell, but it does effect an increase inthe area of cytoplasmic membrane contactallowing one to observe nearly all of the meso-somes in longitudinally sectioned cells as in-vaginations of the cytoplasmic membrane. Fig-ure 6 is a series of four serial sections demon-strating this expansion of the mesosome"neck." Although this series demonstrates theexpansion or distention of the neck through onlythree sections, this effect can frequently beobserved through five serial sections, and occa-sionally through as many as seven serial sec-tions. The membrane-mesosome continuity inunwashed cells could not be observed in morethan two consecutive sections. It is also ofinterest that, although mesosomes of washedcells seem less densely packed, the tubularnature of these structures is retained.The detailed analysis of serial sections of

mesosomes has yielded information other thanthat related to structure. We have ascertainedthat a given cell may contain as many as fourmesosomes, most of which are not associatedwith the cytoplasmic membrane at the point ofcell division. Of more interest, however, is ourcalculation that a single mesosome of the mor-phology shown in Fig. 2 may increase thesurface area of the cytoplasmic membrane of aCHRPP cell by 30%.

DISCUSSIONThe occurrence of intracytoplasmic mem-

branes in non-photosynthetic gram-negativebacteria is amply documented (5, 7, 13, 16, 18,21, 27, 32); however, reports of membranes ingram-negative bacteria conforming to the defi-nition of mesosomes in gram-positive bacteria(9, 20) have been few (6, 17). In this report wehave described membranous structures in C.violaceum that conform to the only establishedcriteria for the definition of mesosomes, namelymorphology and physical reaction to environ-mental conditions, in an organism with theultrastructural anatomy and lipid compositioncharacteristic of gram-negative bacteria (19).The morphological similarities and differ-

ences between the gram-positive mesosomesand the C. violaceum mesosome are clearly seenin comparing the reconstruction of a mesosomeby Burdett and Rogers (4) for B. licheniformisand Fig. 5. The only significant difference isthat we depict the mesosomal tubules as multi-branching structures, whereas the B.licheniformis mesosome is presented as singlecoiled tubules. However, Matheson et al. (15)have recently demonstrated extracellularbranching mesosomal tubules after protoplastextrusion in Bacillus subtilis.

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RUCINSKY AND COTA-ROBLES

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FIG. 5. Section of C. violaceum strain CHRPP plasmolyzed in 0.3 M sucrose. Extruded mesosomal tubulesand vesicles are seen between cytoplasmic membrane and peptidoglycan layer (R).

Several studies have described the develop-ment of mesosomes in gram-positive bacteria(8, 10, 12, 14, 24). Analysis of serial sections ofC. violaceum strain CHRPP has not shownmesosomes that could be described as "imma-ture" or "developing." The maximum diameterof mesosomes observed within a single cell isquite varied, but no preferential site within thecell or different morphological form of thebacterium could be detected for any particularmesosome size. The mesosome morphology can

be altered, the area of cytoplasmic membraneattachment increased, and the mesosomal tu-bules "uncoiled" by washing cells in buffer. Wehave further observed (unpublished data) thatC. violaceum strain CHRPP plasmolyzed in thegrowth medium do not extrude mesosomes withthe efficiency exhibited by washed cells.Although any specific mesosome function is

yet to be ascertained, data suggest mesosomeparticipation in septum formation in gram-posi-tive bacteria (20) and in the separation of sisternucleoids (27). Furthermore, differences seemto exist in the chemical and enzymatic composi-tion of the mesosome and cytoplasmic mem-brane (20, 22). Our observations of C. violaceumstrain CHRPP mesosomes show that these

structures are often at or near the point of celldivision. The frequency of this observation,however, is not sufficient to suggest a role in celldivision. Although a similar pattern of deoxyri-bonucleic acid attachment to the cytoplasmicmembrane after plasmolysis of gram-positivesis suggested in C. violaceum strain CHRPP(Fig. 5), neither the site of deoxyribonucleicacid attachment nor role of the mesosome inseparation of sister nucleoids can be resolved. Itis interesting to note that, although contactbetween mesosomes and nuclear material isextensive, this seems to be a random displace-ment except at the point of tubule invagination(Fig. 2). Complete observation of these invagi-nation sites generally requires no more thanthree sections. Nuclear contact at these sites isseldom observed in more than two consecutivesections. These observations, however, neithersupport nor dispute the above suggested meso-somal functions. Why the mesosomes of thisorganism are more similar to those of gram-

positive bacteria than are the membraneousstructures of other well-studied gram-negativebacteria awaits further elucidation of the role ofmesosomes and intracytoplasmic membranes ingeneral.

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C. VIOLACEUM MESOSOME STRUCTURE

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FIG. 6. Serial sections of washed C. violaceum strain CHRPP. Note that the mesosomes presenteddemonstrate continuity with the cytoplasmic membrane through at least three sections. Also note that thetubular nature of the mesosomes is retained.

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