Heterocyst Differentiation in the Cyanobacterium …HETEROCYST DIFFERENTIATION IN M. LAMINOSUS 515 terns, however, and such an investigation will be required before the mechanism for

Vol. 157, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1984, p. 514-5250021-9193/84/020514-12$02.00/0Copyright ©3 1984, American Society for Microbiology

Heterocyst Differentiation in the Cyanobacterium Mastigocladuslaminosust

S. A. NIERZWICKI-BAUER,'t D. L. BALKWILL,2 AND S. E. STEVEN§, JR.3*Department of Microbiology, University ofNew Hampshire, Durham, New Hampshire 038241; Department ofBiologicalScience, Florida State University, Tallahassee, Florida 323062 and Department of Biochemistry, Microbiology, Molecular

and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 168023

Received 1 July 1983/Accepted 7 November 1983

The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentouscyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliestdifferentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii)degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or proteinmaterial (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer,development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmicmembranes. Late differentiation stages included further development of necks and continued elaboration ofmembranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained largenumbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocystslacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number ofunusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments werecapable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments- thenumber of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10.Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority ofthem were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets ofadjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetativecells. Groups offour or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments,and in some of these filaments virtually all of the cells appeared to be capable of differentiating intoheterocysts.

Cyanobacteria (blue-green algae) are phototrophic micro-organisms that carry out oxygenic photosynthesis to generatethe energy and reducing power required to assimilate (or"fix") atmospheric CO2 and, in some species, N2. Nitrogenfixation in filamentous cyanobacteria usually occurs in spe-cialized cells called heterocysts (1, 11, 39). These hetero-cysts differentiate from certain vegetative cells within afilament by undergoing a complex series of ultrastructuraland biochemical changes. The differentiation process ap-pears to be controlled by an intricate and precise mechanismthat permits the formation of heterocysts only at specificlocations within a filament, thereby producing a regularheterocyst "spacing pattern" (36).Almost all of the detailed data currently available on the

ultrastructural changes that take place during heterocystdifferentiation, on the ultrastructure of mature heterocysts,and on the mechanism by which heterocyst differentiation iscontrolled have come from studies on a few species of thegenus Anabaena (12-14, 18-20, 34-36). Many other cyano-bacterial genera can produce heterocysts, but the differentia-tion process has not been investigated thoroughly in any ofthem. Consequently, it is not known whether the mecha-nisms of heterocyst differentiation and its control in thesegenera differ from those of Anabaena spp. Adams and Carr(1) recently speculated that significant deviations from thepicture seen in Anabaena spp. probably would be detected

* Corresponding author.t Paper no 6715 in the journal series of the Pennsylvania Agricul-

ture Experiment Station.t Present address: Department of Biophysics and Theoretical

Biology, University of Chicago, Chicago, IL 60637.

when other heterocystous genera were examined in detail.Such deviations could be very important because they mightpoint to significant variations ip the mechanisms by whichdifferent heterocysts and heterocystous cyanobacteria func-tion.The branching, filamentous (pr stigonematacean) cyano-

bacteria probably have received the least attention in regardto heterocyst differentiation and ultrastructure. Light micro-scopic studies (15-17, 23, 25, 27) have shown that theseorganisms possess complex morphological characteristics.Mature cultures consist of dense mats of intertwined narrowfilaments (that contain cylindrical, actively dividing cells)and wide filaments (that contain spherical, nondividingcells). The wide filaments produce numerous lateralbranches, thereby contributing to the overall morphologicalcomplexity and pleomorphism of a mature culture. Perhapsbecause of this morphological complexity, only a few inves-tigators have examined the ultrastructure of stigonemata-cean cyanobacteria (2, 21, 24, 33, 37). The most detailed ofthese studies (21, 33) involved nitrate-grown cultures thatdid not form heterocysts. Only recently was the ultrastruc-ture of a stigonematacean species (Mastigocladus lamino-sus) growing under nitrogen-fixing conditions examined indetail (Nierzwicki-Bauer, Balkwill, and Stevens, Arch. Mi-crobiol., in press), and that study did not include a thoroughanalysis of heterocyst differentiation. Furthermore, almostnothing is known about control of heterocyst differentiationin stigonematacean species. Rippka et al. (23) used lightmicroscopy to identify various types of heterocysts thatwere produced by M. laminosus and to note some of thelocations at which heterocysts could form within branchingfilaments. They did not investigate heterocyst spacing pat-

514

on June 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

HETEROCYST DIFFERENTIATION IN M. LAMINOSUS 515

terns, however, and such an investigation will be requiredbefore the mechanism for controlling heterocyst differentia-tion in M. laminosus or other stigonematacean species canbe compared with that used by Anabaean spp.The present study was undertaken to examine in detail the

ultrastructural and morphological aspects of heterocyst dif-ferentiation in M. laminosus. The primary purpose of thisinvestigation was to obtain data on differentiation and con-

trol of differentiation in this organism that would facilitatecomparison of these phenomena with those occurring inAnabaena spp. Specific issues that were of particular inter-est included (i) the detailed sequence of ultrastructuralchanges taking place as vegetative cells differentiate intoheterocysts, (ii) the detailed ultrastructural characteristics ofmature heterocysts, (iii) the range of morphological celltypes capable of differentiation, (iv) the positions of hetero-cysts in narrow and wide filaments, especially with respectto the occurrence of any regular spacing patterns like thoseseen in Anabaena spp., and (v) the formation of double or

multiple adjacent heterocysts or proheterocysts (or both).We describe here results indicating that heterocyst differen-tiation in M. laminosus proceeds by a somewhat differentsequence of events than that described for Anabaena spp.

and, more importantly, that differentiation in M. laminosuseither is not controlled at all or is controlled by a mechanismquite different from that used in Anabaena spp.

MATERIALS AND METHODS

Organism, medium, and growth. M. laminosus was pro-

vided by C. 0. Patterson (Texas A&M University), whoprocured it from the former Indiana University CultureCollection as IUCC 1931 (now UTEX 1931). The strain was

originally isolated from the hot spring in Hoeragerd, Iceland,by R. Lewin in August 1961. The culture used in the presentstudy was purified as described previously (21).

Broth cultures were grown in medium B of Stevens et al.(31), without sodium nitrate. The cultures were incubated at46°C in large, glass growth chambers (working volume, 1,800ml) developed by Mehta and Stevens (unpublished observa-tions). The jncident light intensity at the center of thesegrowth chambers was 540 ILE m-2 s-'.

Heterocyst differentiation. M. laminosus was incubated for27 h under the above conditions, during which the cultureswere bubbled with sterile 5% (vol/vol) CO2 in N2 to providecontinuous agitation and a source of carbon and nitrogen forgrowth. Heterocyst differentiation was then stimulated bybubbling the cultures with sterile 5% (vol/vol) CO2 in Arinstead of C02-N2 (12). Incubation continued for 44 h afterthe switchover from C02-N2 to C02-Ar. The morphologicalcharacteristics of the cultures were monitored by lightmicroscopy (see below) throughout the entire incubationperiod (71 h), to determine appropriate sampling times fordetailed electron microscopical analysis of heterocyst differ-entiation (see below).

Light microscopy. Phase-contrast light microscopy of un-

stained wet mounts was used to monitor cell morphologyduring heterocyst differentiation, to ensure that a repre-

sentative sampling of morphological types was being seen

with electron microscopy (see below), and to determine thepositions of heterocysts in narrow filaments. Each samplewas examined extensively at magnifications from x400 to

x 1,000, and representative fields were photographed on

Kodak Technical Pan film (TP-135-36). Photographic en-

largements of these micrographs were compared with thoseof the electron micrographs (see below) to assess the thor-oughness and accuracy of each electron microscopic assay.

Electron microscopy. The positions of heterocysts in nar-row and wide filaments, as well as the detailed ultrastructureof vegetative cells, heterocysts, and the heterocyst differen-tiation process were determined by transmission electronmicroscopy of thin-sectioned preparations. Two fixationswere used: a ruthenium red fixation (29) and a glutaralde-hyde-Os04 fixation (30). Samples from both fixations weredehydrated through a graded ethanol series and embedded inSpurr's low-viscosity epoxy resin (28). Thin sections werecut with a Diatome diamond knife (M. J. 0. Diatome, Inc.,Fort Washington, Pa.) on an LKB Ultratome III ultramicro-tome. The sections were mounted on coated 200-meshspecimen grids and poststained as described previously (21).Thin sections were examined with a JEOL JEM-100S

transmission electron microscope operated at 80 kV. Eachsample was examined extensively to assess the variety ofmorphological and ultrastructural features present. Morethan 2,000 representative cells or fields were then photo-graphed, and the results were analyzed by studying enlarge-ments of these micrographs. Both fixation procedures gaveequivalent results, with only minor variations in the stainingreactions of intracellular inclusion bodies. Micrographs pro-duced with the glutaraldehyde-Os04 fixation were chosenfor the illustrations in this report.

RESULTS

Morphology and ultrastructure of undifferentiated vegeta-tive cells. The morphological and ultrastructural characteris-tics of undifferentiated vegetative cells are illustrated in Fig.1. Narrow filaments contained distinctly cylindrical cellsthat were uniform in width, but varied in length (Fig. la),because they were actively elongating and dividing. Cells inwide filaments (Fig. lb) were comparatively pleomorphic(most were roughly spherical) and divided only to initiateformation of branch filaments. Each vegetative cell wassurrounded by a typical gram-negative cell envelope (7), andeach filament of cells was surrounded by a multilayered,common sheath (21). The cytoplasm of narrow vegetativecells (Fig. lc) was separated into two reasonably distinctunits: a central cytoplasmic region and a peripheral thyla-koid region. The central cytoplasmic region contained nucle-ar material, ribosomes, and carboxysomes (or "polyhedralbodies"; 26, 32, 38). Thp peripheral thylakoid region con-tained the thylakoids themselves, phycobilisomes (9, 10), asmall number of lipid bodies (",-granules", 8, 22, 38), andlarge numbers of electron-transparent polysaccharide gran-ules ("oa-granules"; 4, 22, 38). Cells in wide filaments (Fig.ld) differed from those in narrow filaments in that (i) theywere larger, (ii) their central cytoplasmic and peripheralthylakoid regions were not as distinctly separated, (iii) theycontained fewer ribosomes, and (iv) they contained morecarboxysomes and lipid bodies.

Ultrastructure of the differentiation process. Vegetativecells in both narrow and wide filaments differentiated intoheterocysts. The sequence of ultrastructural changes thattook place during heterocyst differentiation (Fig. 2) was thesame in both cases. Shortly after stimulation of heterocystformation, certain vegetative cells were modified to producethe earliest recognizable proheterocysts. These cells wereclearly proheterocysts because the homogeneous wall layerthat is characteristic of mature heterocysts had started toform along their lateral surfaces (Fig. 2b). The formation ofthis homogenous layer was preceded by a series of intracyto-plasmic changes that included (i) partial degradation ofpolysaccharide granules (co-granules), (ii) complete degrada-

VOL. 157, 1984


.org/D

ownloaded from

http://jb.asm.org/

516 NIERZWICKI-BAUER, BALKWILL, AND STEVENS J. BACTERIOL.

. . T>lg|||||||l|............... ............

...........

C PS ;'';* *";< " i

t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~NUM;s&4~~~~~~~~~~~

-b,\,V. ,-......*

AN~~~~~~~~*~

A /F

*.'rf#1sai~;;f.;

FIG. 1. Morphological and ultrastructural characteristics of undifferentiated M. laminosus vegetative cells. (a) Light micrograph of youngfilaments, including cylindrical vegetative cells and heterocysts/proheterocysts (arrows). Note the lack of regular heterocyst spacing pattern.Bar, 10.0 ,um. (b) Light micrograph of typical old filament, illustrating roughly spherical, somewhat pleomorphic vegetative cells. Bar, 10.0,um. (c, d) Electron micrographs of thin sections through typical undifferentiated cells. C, Carboxysome; L, lipid body; N, nuclear material;PS, group of polysaccharide granules; T, thylakoid. Bars, 1.0 p.m. (c) Cell in narrow filament. (d) Cell in wide filament.


.org/D

ownloaded from

http://jb.asm.org/


A

--@^> NK

HLSt,%. CM,L -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-

FIG. 2. Electron micrographs of thin sections through proheterocysts, illustrating stages of heterocyst differentiation. CM, Cytoplasmicmembrane; HL, homogeneous extra wall layer; LM, bundles of stacked, lamellar membranes; NK, neck leading to adjacent cell; PG,peptidoglycan layer of cell wall; R, electron-dense ribosomal or protein material (or both). (a) Very early proheterocyst. Note homogeneouswall layer along lateral surfaces of cell, presence of ribosomal or protein material (or both), and the tendency of thylakoids to form parallel ar-rays. Bar, 2.0 ,um. (b) More advanced early proheterocyst. Note extension of homogeneous wall layer around ends of cell, development ofneck, early formation of lamellar membrane bundles, and increased electron density of cytoplasm owing to elaboration of thylakoidmembrane. Bar, 2.0 ,um. (c) Typical intermediate proheterocyst with bundles of stacked, lamellar membranes leading into neck area. Bar, 2.0p.m. (d) Detail of features in a bundle of stacked, lamellar membranes. Bar, 100 nm. (e) Typical late proheterocyst. Note relatively highelectron density of cytoplasm, owing to accumulation of membranous material. Bar, 2.0 p.m. (f) Detail of late proheterocyst neck leading toadjacent vegetative cell. Cells are separated by the cytoplasmic membranes of the proheterocyst (CM1) and the adjacent cell (CM2), and by alayer of peptidoglycan material. Bar, 0.3 p.m.

VOL. 157, 1984


.org/D

ownloaded from

http://jb.asm.org/

518 NIERZWICKI-BAUER, BALKWILL, AND STEVENS

tion of carboxysomes, (iii) accumulation of centrally located,electron-dense ribosomal or protein material (or both), and(iv) alteration of the thylakoids. The thylakoids in early pro-heterocysts tended to straighten out and elongate, some-times forming small parallel arrays (Fig. 2a). As these earlyproheterocysts continued to differentiate, their overall elec-tron density increased. This was due to (i) continued deple-tion of polysaccharide, (ii) continued accumulation of ribo-somal or protein material (or both), and (iii) an increase inthe amount of thylakoid membrane. Thylakoid elaborationproduced longer or more numerous thylakoids and smallbundles of stacked, lamellar membranes (Fig. 2b). At thesame time, the poles of many proheterocysts began todevelop "necks" or "pore channels" (3, 13, 14) leading toadjacent cells. The homogeneous wall layer extended aroundthe ends of the cell as these necks developed (Fig. 2b).The intermediate stages of proheterocyst differentiation

are illustrated in Fig. 2c and d. Considerable amounts ofmembrane appeared during this period, resulting in theformation of larger and more numerous bundles of stacked,lamellar membranes (Fig. 2c). Most of these lamellar mem-brane bundles were located near (and appeared to lead into)the developed necks at each end of the cell, although some ofthem were located in other parts of the cell as well. Thebundles comprised a series of regularly spaced (periodicity,approximately 32 nm) membrane pairs (Fig. 2d). The twomembranes in each pair were closely apposed to one anoth-er. The areas between the membrane pairs were approxi-mately 16 nm in width and were filled with a granularsubstance. Alteration and elaboration of the thylakoid sys-tem continued throughout the intermediate stages of differ-entiation, thereby producing a marked increase in overallelectron density. It appeared that unusually large numbers ofsmall thylakoid segments were present and were coalescingto form a complex, rather unthylakoid-like network ofmembranes. Lipid bodies were still numerous in the interme-diate proheterocysts, but the number of polysaccharidegranules was markedly reduced.The late stages in proheterocyst differentiation are depict-

ed in Fig. 2e and f. Membrane elaboration continued,producing an increased number of closely packed mem-branes throughout the entire cell (Fig. 2e). Elaboration ofmembranes and disappearance of remaining polysaccharidegranules combined to produce cells of increased and moreuniform electron density. The necks leading to adjacent cellsbecame fully developed during the last stages of prohetero-cyst differentiation, and the homogeneous extra wall layerthickened noticeably around these necks (Fig. 2f). Thecytoplasm of the proheterocyst was separated from that ofthe adjacent vegetative cell by the following three structuralunits (i) the cytoplasmic membrane of the proheterocyst, (ii)the cytoplasmic membrane of the vegetative cell, and (iii) asingle layer of peptidoglycan wall material (Fig. 2f). It wasnot possible to determine with certainty whether the pepti-doglycan layer was penetrated by "microplasmodesmata"(14).Morphology and ultrastructure of mature heterocysts. The

morphological and ultrastructural characteristics of matureheterocysts are illustrated in Fig. la and 3. Mature hetero-cysts (and differentiating proheterocysts) in narrow fila-ments (Fig. la and 3a) were somewhat longer and wider thanundifferentiated vegetative cells, whereas those in widefilaments (Fig. 3b) and intermediate filaments undergoingtransition between the narrow and wide forms (Fig. 3c) werethe same size as vegetative cells. In all filaments, hetero-cysts assumed the basic shape of the cell from which they

differentiated. Since they retained that shape even if theadjacent vegetative cells later widened, heterocysts withmorphological characteristics of narrow or intermediate cellswere sometimes observed in wide filaments (Fig. 3d and e).The internal ultrastructural characteristics of all mature

heterocysts were similar, despite wide variations (Fig. 1 and3a through c) in their external morphology. Mature hetero-cysts (Fig. 3f) typically possessed an electron-dense interiorthat contained large numbers of closely packed membranes.The dense interior of the heterocyst was unusually homoge-neous, except for a few ribosomes and light gray patchessituated between the membranes. If nuclear material waspresent, it was obscured by the high electron density of thecytoplasm. Polysaccharide granules, carboxysomes, and lip-id bodies were absent from mature heterocysts. The onlyinclusions visible in these cells were small, spherical bodies(Fig. 3g) of varied electron density (usually not as dense asthe surrounding cell material). These spherical inclusionsaccumulated late in proheterocyst development, about thesame time that the lipid bodies disappeared. They wereabout the same size as lipid bodies (50 to 100 nm indiameter), but had distinctly different ultrastructural charac-teristics. A small minority of mature heterocysts containedcyanophycin-like "plugs" (13, 14, 35) near the necks leadingto adjacent cells (Fig. 3g), but the necks of most heterocystswere filled with stacked, lamellar membranes (see above)instead. All mature heterocysts possessed a W'ell-developedhomogeneous extra wall layer, but other extra layers (14, 35)were not detected with either of the fixations used in thisstudy (see above).

Positions of heterocysts in narrow filaments. The positionsof heterocysts and developing proheterocysts in hundreds ofnarrow filaments were examined with both light and electronmicroscopy, but a reproducible heterocyst spacing patternlike that described for Anabaena spp. (18, 20, 34-36) wasnever observed (Fig. la). The number of vegetative cellsbetween consecutive heterocysts varied by a factor of 10,even within a single filament. In cultures examined 10 h afterstimulation of heterocyst formation (see above), intercalaryheterocysts in single filaments were separated from eachother by any number of vegetative cells between 2 and 20.The number of vegetative cells between heterocysts de-creased between 10 and 44 h after stimulation of heterocystformation, but still ranged from 0 to 9 at the end of thatperiod. Thus, a marked increase in the number of hetero-cysts present in narrow filaments did not lead to the forma-tion of a regular spacing pattern. Furthermore, heterocystsdid not appear to differentiate as a result of asymmetric celldivision (18, 34). Asymmetric septum formation was notobserved in any vegetative cells, with either light or electronmicroscopy.

Positions of heterocysts in wide filaments. Heterocystsdifferentiated at a variety of locations within the branchingportions of wide filaments (Fig. 4). In the main filamentitself, proheterocysts and mature heterocysts were some-times separated from branch points by one or more intercala-ry vegetative cells. Mature heterocysts in these positionswere of two types: those that had terminal necks leading toboth adjacent cells ("intercalary heterocysts"; 23) and thosethat had only a single neck ("terminal heterocysts"; 23).Heterocysts of the second type sometimes became theterminal cell of the main filament (Fig. 4a). The majority ofthe heterocysts and proheterocysts in the main filament weresituated immediately adjacent to branch filaments. Hetero-cysts differentiated either on one (Fig. 4b) or both (Fig. 4c)sides of the branches in these cases. Heterocysts adjacent to

J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/


'K&,,,,-s,0wiy1 , ,.~-.toSR; Vke 0

f

FIG. 3. Electron micrographs of thin sections (a through c, f, g) and light micrographs (d, e) illustrating morphological and ultrastructuralcharacteristics of M. laminosus heterocysts. (a through c) Morphological characteristics of het/pros (arrows) in various types of filaments. (a)Proheterocyst and adjacent vegetative cells in narrow filaments. Note that proheterocyst is longer and wider than vegetative cells. Bar, 3.0,um. (b) Heterocyst and adjacent vegetative cell in wide filament. Bar, 1.0 ,um. (c) Heterocyst and adjacent vegetative cell in filamentundergoing transition from narrow to wide form. Bar, 2.0 p.m. (d, e) Examples of wide filaments that contain heterocysts with morphologicalcharacteristics of cells in narrow filaments. Bars, 10.0 p.m. (f) Typical mature heterocyst. Note uniform electron density of cytoplasm. Bar,1.0 p.m. (g) Detail of neck area in mature heterocyst. Cyanophycin-like plugs (CP) were seen only rarely in mature heterocysts. Note sphericalinclusion bodies (SB). Bar, 0.5 p.m.

VOL. 157, 1984

18Amllil SW


.org/D

ownloaded from

http://jb.asm.org/


C -

1*~~~~~~~.\'i*.41'

;|>*s- 1. 4.

........ ; . 'w

A4..'*\ *

.

p..

'St

E'4% -

FIG. 4. Electron micrographs of thin sections through wide M. laminosus filaments, illustrating various positions at which heterocysts anddifferentiating proheterocysts (H) formed in these filaments. (a) Example of terminal proheterocyst that has become the terminal cell of thefilament itself. Bar, 2.0 p.m. (b) Branch point in wide filament at which heterocyst (Hi) has formed only on one side of the branch.Proheterocysts (H2 and H3) have also formed on both daughter cells produced by division parallel to long axis of the main filament at branchpoint. Serial sections confirmed that both H2 and H3 were terminal cells. Bar, 4.0 p.m. (c) Branch point in wide filament at which heterocysts(Hi and H2) have formed on both sides of the branch. Bar, 4.0 p.m. (d) Portion of wide filament in which two cells have undergone divisionparallel to long axis of the filament. Branch formation appears to be following parallel division in one case (arrow). In other case, one of the re-sulting daughter cells is differentiating into a heterocyst (H). Bar, 4.0 p.m.

J. BACTERIOL.

'I


.org/D

ownloaded from

http://jb.asm.org/


branches were also of the two types described above, andthe terminal variety occasionally became the terminal cell ofthe main filament. It did not appear that branch filamentsarose from heterocysts themselves or that cells giving rise tobranches ever differentiated into heterocysts, since branchfilaments connected to heterocysts were never observed. Itwas noted, however, that some cells in the wide filamentsunderwent a division parallel to the long axis of the mainfilament (as if to form a branch), after which one or both ofthe two daughter cells became heterocysts (Fig. 4d).

Heterocysts also developed in the lateral branches of thewide filaments. These heterocysts usually became terminalcells of those branch filaments (Fig. Sa), possibly preventingtheir further elongation. Terminal heterocysts frequentlyformed in place of branch filaments in M. laminosus, appear-ing directly adjacent to one or (less frequently) both of thedaughter cells formed by division parallel to the long axis ofthe main filament (Fig. 4b).

Multiple adjacent het/pro formation. M. laminosus dis-played a marked tendency to form sets of multiple adjacentheterocysts developing proheterocysts or both (het/pros)that were not separated from each other by interveningvegetative cells. In narrow filaments, this tendency waslimited to the formation of double het/pros. These doublehet/pros were intercalary, having necks on both ends of thecell in both cases. Thus, the two cells themselves wereconnected by a double neck (one neck belonging to each cellin the pair; Fig. 5b). In narrow filaments, both of the cells inpaired het/pros always appeared to be at the same stage ofdifferentiation. The double het/pros represented approxi-mately 5% of the total heterocyst and proheterocyst popula-tion in narrow filaments. Triple or other multiple het/proswere not observed in narrow filaments.Wide filaments produced sets of adjacent het/pros more

frequently than did narrow filaments. Throughout the entiregrowth period after stimulation of heterocyst formation, atleast 14 to 20% of the heterocysts and proheterocysts in widefilaments were adjacent to other heterocysts or prohetero-cysts. Moreover, the wide filaments were not limited to theformation of double het/pros. Triple het/pros were seenfrequently (Fig. 5c), and some filaments contained sets offour or more adjacent het/pros. Most of the multiple het/prosin wide filaments were of the intercalary type, havingdifferentiated necks on both ends of the cell (Fig. 5d). Theydiffered from those seen in the narrow filaments (see above)in that the individual cells within a multiple set were usuallynot all at the same stage of heterocyst differentiation. It waspossible that all of the cells in wide filaments were at leastcapable of becoming heterocysts, since wide filaments werefound in which all of the cells appeared to be in the earlystages of heterocyst differentiation.

DISCUSSIONThis study has involved the first detailed analysis of

heterocyst differentiation in M. laminosus, a branching,filamentous (stigonematacean) cyanobacterium. The exten-sive ultrastructural changes that took place during differenti-ation are summarized in Table 1, where they are comparedwith those reported (12, 13, 35) to occur in the nonbranching,filamentous genus Anabaena. The earliest ultrastructuralchanges in M. laminosus were cytoplasmic, and the mostnoticeable of these was a rapid degradation of the carboxy-somes normally present in vegetative cells. Carboxysomeswere also degraded during heterocyst formation in Ana-baena spp. (12, 13), but not nearly as quickly as in the

present study. Rapid breakdown of carboxysomes duringheterocyst differentiation would be sensible because matureheterocysts have little or no ribulose biphosphate carboxyl-ase activity (5, 6, 32). Since carboxysomes appear to becomposed primarily of ribulose biphosphate carboxylase(26, 32), there would be no need to retain them for any lengthof time in a cell that was going to become a heterocyst.

Contrary to the present observations, the earliest ultra-structural changes seen during heterocyst differentiation inAnabaena spp. were all external (Table 1). Synthesis ofextra wall layers and preliminary reshaping of the celloccurred before any detectable cytoplasmic changes (12, 13,35). The fibrous wall layer appeared first, followed rapidlyby the homogeneous layer. The homogeneous layer formedsomewhat later in M. laminosus, although the final resultwas essentially the same. On the other hand, M. laminosusdid not produce a fibrous layer at any time during heterocystdifferentiation. The chemical composition of this layer inAnabaena spp. is unknown (1), although it has been suggest-ed that it is composed of uncompacted strands of thepolysaccharides used to form the homogeneous layer (39).Perhaps the fibrous layer did not form in M. laminosusbecause the homogeneous layer was already surrounded bya well-defined extracellular sheath. Mature M. laminosusheterocysts also may have lacked the laminated wall layerthat is formed late in the Anabaena spp. differentiationprocess, although this is uncertain because Lang and Fay(14) reported that the laminated layer is best stained andpreserved by a glutaraldehyde-permanganate fixation thatwas not used in the present study.

Heterocyst differentiation in M. laminosus involved ex-tensive elaboration of thylakoids to produce a complexnetwork of closely packed membranes that filled much of themature heterocyst cytoplasm. In contrast, mature Anabaenaspp. heterocysts always contained large membrane-free re-gions because thylakoids tended to vesiculate and undergopartial degradation during differentiation in that organism(12, 13). Large bundles of stacked, lamellar membranesdeveloped near the poles of all M. laminosus proheterocysts,whereas the thylakoid system elaborated. These lamellarbundles were probably equivalent to the arrays of intracyto-plasmic membranes that formed near the poles of Anabaenaspp. proheterocysts (12, 13, 35), but their detailed ultrastruc-tural characteristics were quite different. In addition, thetotal amount of membrane surface area produced near thepoles of M. laminosus proheterocysts was considerablygreater than that in Anabaena spp. It seems likely that thedistinct lamellar membrane bundles and other intracytoplas-mic membranes that formed in M. laminosus heterocystswould perform an important function. Lang and Fay (14)suggested that the extra intracytoplasmic membranesformed in Anabaena spp. heterocysts might be directlyinvolved in nitrogen fixation. This has never been demon-strated conclusively, but if it is true, M. laminosus hetero-cysts probably have higher nitrogenase activities than doAnabaena spp. heterocysts (Mehta and Stevens, unpub-lished data), because they have more extensive and morehighly organized intracytoplasmic membranes.As did those ofAnabaena spp. (12-14), mature M. lamino-

sus heterocysts lacked all of the inclusion bodies that arenormally present in vegetative cells. However, they didcontain a number of spherical inclusions that were notpresent in Anabaena spp. heterocysts and that accumulatedabout the same time that lipid bodies disappeared. Thesemay have been a modified form of lipid body that wasproduced during the later stages of heterocyst differentia-

VOL. 157, 1984


.org/D

ownloaded from

http://jb.asm.org/


k.~~~~~~~~~~~~~4.

C%2 4

a.~~~~~3'E .,, ....i3p::*!;;' l̂ 4p

*4 ~ ~0''oi

C~~~~

.4~~~~~~~~4;jj|

H2

'+ i VfIWi

ANN:

* S 7' "'~

d

t~~~~~~~~~~~~~~~~~~~~~

Hlt

:1'i

I-'

,.. ,t .

I I '*

FIG. 5. Electron micrographs of thin sections, illustrating positions and spacing of het/pros in M. laminosus filaments. (a) Branch filamentwith developing proheterocyst (H) as terminal cell of the branch. Bar, 4.0 ,um. (b) Detail of double neck between two cells of a doubleheterocyst in narrow filament. Barrier between two heterocyst cytoplasms consists of two layers of cytoplasmic membrane (Ci and C2) and a

thin layer of wall material (W). Bar, 0.25 p.m. (c) Triple het/pro (see text) set in wide filament, including two heterocysts (Hi and H2) and onecell in early stages of proheterocyst differentiation (P). Bar, 2.0 pum. (d) Multiple het/pro set in wide filament with three mature heterocysts(Hi through H3) and one cell in very early stages of proheterocyst differentiation (P). Note that heterocysts are intercalary, having necks on

both ends of their cells. Note double neck between Hi and H2. Bar, 3.0 p.m.

J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/


TABLE 1. Comparison of ultrastructural changes taking place during heterocyst differentiation in M. laminosus and Anabaena spp.

Time of M. laminosus Anabaena spp.changesEarly Degradation of polysaccharide granules begins Fibrous wall layer forms and is completed rapidly

Accumulation/aggregation of ribosomal/protein material be- Ends of cells begin to reshape in order to form necks lead-gins ing to adjacent cells

Degradation of carboxysomes occurs rapidly Homogeneous wall layer begins to formThylakoid system begins to elaborate Degradation of polyphosphate granules beginsHomogeneous wall layer begins to formNecks leading to adjacent cells begin to developBundles of stacked, lamellar memnbranes begin to develop inneck areas

Intermediate Homogeneous wall layer continues to develop Degradation of polysaccharide granules and carboxysomesbegins

Necks leading to adjacent cells continue to develop Homogeneous wall layer completedThylakoid system elaborates to produce complex network Thylakoid system vesiculates

of membranes throughout cytoplasmBundles of stacked, lamellar membranes enlarge and stop Thylakoid system deteriorates to some extent, leaving cen-

developing ter of cell mostly devoid of membranesNecks leading to adjacent cells completed

Late Membrane elaboration continues, eventually filling much of Laminated wall layer formsbcytoplasm

All remaining inclusion bodies degraded Cell enlarges and rounds upNeck development completed Cyanophycin-like plugs form in neckscSpherical inclusions formed in cytoplasm All remaining inclusion bodies degradedHomogeneous wall layer completed Membranes proliferate at poles of cell, near necks

a Data compiled from references 12, 13, and 35.b Status of laminated layer in M. laminosus not certain (see the text).c Cyanophycin-like plugs were rare in M. laminosus, perhaps because cultures were starved for nitrogen by the end of the differentiation

period.

tion. Further investigation will be required to determine thechemical composition and possible functions of these previ-ously unreported heterocyst inclusions.

All of the various morphological types of cells that occurin mature M. laminosus cultures were found in the presentstudy to be capable of differentiating into heterocysts. This issignificant because these cells probably differed as muchphysiologically as they did morphologically. Narrow vegeta-tive cells were actively growing and dividing when hetero-cyst differentiation was initated, whereas the wide cells hadneither grown nor divided for some time. Wilcox et al. (34)found evidence for a complex interrelation between celldivision and heterocyst differentiation in two species ofAnabaena. Their results implied that a vegetative cell mustbe at a specific stage of the cell cycle to become a candidatefor heterocyst differentiation (1). Apparently, this was notthe case in M. laminosus.The detailed mechanism by which heterocyst differentia-

tion in cyanobacteria is controlled has been studied almostexclusively in Anabaena spp. (see above). The most exten-sive work on this topic was performed by Wilcox et al., whodescribed the occurrence of a regular heterocyst "spacingpattern" in the genus Anabaena (18, 34-36). This term refersto the fact that het/pro within a single filament are almostalways separated by a reasonably uniform number of vegeta-tive cells. It was suggested that heterocysts and differentiat-ing proheterocysts produce an inhibitory compound (not yetidentified) that diffuses down the filament and creates adistinct inhibitory zone on both sides of the cells producingthis compound. Vegetative cells situated within such aninhibitory zone usually cannot differentiate into prohetero-cysts. It is admittedly somewhat difficult to compare M.laminosus with Anabaena spp. because the overall mor-phologies of these two organisms are so different. Neverthe-less, a realistic comparison can be made between Anabaenaspp. and the narrow, unbranched M. laminosus filaments

(with their actively growing and dividing vegetative cells),because the morphological characteristics of the two areactually quite similar. In a limited comparison of this type,one would expect to see distinctly similar heterocyst spacingpatterns if differentiation in the two genera were controlledby the same mechanism. Yet, narrow M. laminosus fila-ments had no regular heterocyst spacing pattern at all. Theseresults demonstrate that control of heterocyst differentiationin M. laminosus is not nearly as precise or as uniform as inAnabaena spp. In addition, the highly variable spacing ofheterocysts in narrow M. laminosus filaments indicates thatheterocyst differentiation in this organism may not be con-trolled by production of an inhibitory compound.Wilcox et al. have reported that asymmetric division of

vegetative cells plays an important role in the control ofheterocyst differentiation in Anabaena spp. (18, 34). Vegeta-tive cells in Anabaena spp. filaments always divide byasymmetric septum formation, thereby producing one largeand one small daughter cell. As a rule, only the smalldaughter cells are capable of later becoming heterocysts.This mechanism automatically limits the number of potentialcandidates for heterocyst differentiation, a process that isconsidered to be the first prerequisite for the formation ofregular spacing patterns (1). In the present study, however,asymmetric septum formation did not occur in M. lamino-sus; daughter cells produced by division of a vegetative cellwere always equal in size. In M. laminosus, then, thereprobably was no preliminary reduction in the number ofcandidates for subsequent heterocyst differentiation. Thismight explain why approximately 5% of the heterocysts innarrow filaments were double het/pros (see above 2), where-as double het/pros are almost never seen in Anabaena spp.under normal growth conditions (1).

Heterocysts formed at a variety of locations in the wide,branching filaments of M. laminosus. There appeared to beno obvious logic behind this variable and irreproducible

VOL. 157, 1984


.org/D

ownloaded from

http://jb.asm.org/


process. This would seem to indicate that there was evenless control of heterocyst differentiation in the wide fila-ments than in the narrow filaments, a conjecture that wasborne out by the fact that groups of multiple adjacenthet/pros occurred frequently in wide filaments. The occur-rence of these multiple het/pros is also an indication thatneither asymmetric cell division nor production of an inhibi-tory substance was involved with the control of heterocystdifferentiation in M. laminosus. The occurrence of multiplehet/pros also raises questions as to what advantage might begained by producing these structures. Adams and Carr (1)have pointed out that formation of adjacent heterocystswould be illogical because it would only serve to maketransport of nutrients less efficient. It is possible that thenumerous heterocysts in wide M. laminosus filaments serveprimarily to provide combined nitrogen to the branch fila-ments, since the few remaining vegetative celis in widefilaments are no longer growing and may not require verymuch nitrogen. However, this would not explain why multi-ple adjacent het/pros should form between branch points inthe main filaments. An investigation of the possible func-tions of multiple het/pros and of the problems involved withnutrient exchange between these structures and vegetativecells would be a worthwhile subject for further study.Adams and Carr (1) recently pointed out that virtually all

of the present information on heterocyst differentiation hascome from studies involving only a few species of Ana-baena, and that significant deviations might be seen whenother heterocystous genera are examined thoroughly. Thepresent study has served to justify that speculation bydemonstrating that (i) the sequence of ultrastructuralchanges taking place during heterocyst differentiation in M.laminosus was clearly different from that of Anabaena spp.(thereby producing mature heterocysts that were also dis-tinctly different in certain respects), and (ii) heterocystdifferentiation in M. laminosus either is not controlled or iscontrolled by a mechanism quite different from that whichhas been characterized so thoroughly in the genus Anabaena(1, 18, 34-36). It is apparent from these results that conclu-sions regarding heterocyst differentiation and its control inAnabaena spp. do not apply to all heterocystous cyanobac-terial genera. Further investigations on heterocyst differenti-ation in organisms like M. laminosus are justified becausethey would serve to provide a more complete picture of thevarious mechanisms by which this important process may beregulated in cyanobacteria.

ACKNOWLEDGMENTSWe express our thanks to R. W. Bernlohr, V. B. Mehta, and

A. T. Phillips for their critical reviews of this work.This study was supported by grant 79000238 from the U.S.

Department of Agriculture.

LITERATURE CITED1. Adams, D. G., and N. G. Carr. 1981. The developmental biolo-

gy of heterocyst and akinete formation in cyanobacteria. CRCCrit. Rev. Microbiol. 9:45-100.

2. Butler, R. D., and A. Allsopp. 1972. Ultrastructural investiga-tions in the Stigonemataceae (Cyanophyta). Arch. Mikrobiol.82:283-299.

3. Carr, N. G. 1979. Differentiation in filamentous cyanobacteria,p. 167-201. In J. Parish (ed.), Developmental biology of pro-karyotes. Blackwell Scientific Publications, Oxford.

4. Chao, L., and C. C. Bowen. 1971. Purification and properties ofglycogen isolated from a blue-green alga, Nostoc muscorum. J.Bacteriol. 105:331-338.

5. Codd, G. A., K. Okabe, and W. D. P. Stewart. 1980. Cellularcompartmentation of photosynthetic and photorespiratory en-

zymes in the heterocystous cyanobacterium Anabaena cylin-drica. Arch. Microbiol. 124:149-154.

6. Codd, G. A., and W. D. P. Stewart. 1977. Ribulose-1,5-diphos-phate carboxylase in heterocysts and vegetative cells of Ana-baena cylindrica. FEMS Microbiol. Lett. 2:247-249.

7. Costerton, J. W., J. M. Ingram, and K.-J. Cheng. 1974. Struc-ture and function of the cell envelope of gram-negative bacteria.Bacteriol. Rev. 38:87-110.

8. Edwards, M. R., D. S. Berns, W. C. Ghiorse, and S. C. Holt.1968. Ultrastructure of the thermophilic blue-green alga, Syne-chococcus lividus Copeland. J. Phycol. 4:283-298.

9. Gantt, E. 1981. Phycobilisomes. Annu. Rev. Plant Physiol.32:327-347.

10. Glazer, A. N. 1982. Phycobilisomes: structure and dynamics.Annu. Rev. Microbiol. 36:173-198.

11. Haselkorn, R. 1978. Heterocysts. Annu. Rev. Plant Physiol.29:319-344.

12. Kulasooriya, S. A., N. J. Lang, and P. Fay. 1972. The hetero-cysts of blue-green algae. III. Differentiation and nitrogenaseactivity. Proc. Roy. Soc. Lond. B 181:199-209.

13. Lang, N. J. 1965. Electron microscopic study of heterocystdevelopment in Anabaena azollae Strasburger. J. Phycol.1:127-134.

14. Lang, N. J., and P. Fay. 1971. The heterocysts of blue-greenalgae. II. Details of ultrastructure. Proc. Roy. Soc. Lond. B178:193-203.

15. Lee, S. 1927. Cytological study of Stigonema mammilosum.Bot. Gaz. 83:420-424.

16. Marcenko, E. 1961. Licht- und elektronenmikroskopische Un-tersuchungen an der Thermalalge Mastigocladus laminosusCohn. Acta Bot. Croatica 20/21:47-74.

17. Martin, T. C., and J. T. Wyatt. 1974. Comparative physiologyand morphology of six strains of stigonematacean blue-greenalgae. J. Phycol. 10:57-65.

18. Mitchison, G. J., and M. Wilcox. 1972. Rule governing celldivision in Anabaena. Nature (London) 239:110-111.

19. Mitchison, G. J., and M. Wilcox. 1973. Alteration of heterocystpattern of Anabaena produced by 7-azatryptophan. Nature(London) 246:229-233.

20. Mitchison, G. J., M. Wilcox, and R. J. Smith. 1976. Measure-ment of an inhibitory zone. Science 191:866-868.

21. Nierzwicki, S. A., D. Maratea, D. L. Balkwill, L. P. Hardie,V. B. Mehta, and S. E. Stevens, Jr. 1982. Ultrastructure of thecyanobacterium, Mastigocladus laminosus. Arch. Microbiol.133:11-19.

22. Pankratz, H. S., and C. C. Bowen. 1963. Cytology of blue-greenalgae. I. The cells of Symploca muscorum. Am. J. Bot. 50:387-399.

23. Rippka, R., J. Deruelles, J. B. Waterbury, M. Herdman, andR. Y. Stanier. 1979. Generic assignments, strain histories andproperties of pure cultures of cyanobacteria. J. Gen. Microbiol.111:1-61.

24. Ris, H., and R. N. Singh. 1961. Electron microscope studies onblue-green algae. J. Biophys. Biochem. Cytol. 9:63-80.

25. Schwabe, G. H. 1960. Uber den thermobionten KosmopolitenMastigocladus laminosus Cohn. Hydrol. 22:759-792.

26. Shively, J. M. 1974. Inclusion bodies of prokaryotes. Annu.Rev. Microbiol. 28:167-187.

27. Spearing, J. K. 1937. Cytological studies of the Myxophyceae.Arch. Protistenk. 89:209-278.

28. Spurr, A. R. 1969. A low-viscosity epoxy resin embeddingmedium for electron microscopy. J. Ultrastruct. Res. 26:31-43.

29. Stevens, S. E., Jr., D. L. Balkwill, and D. A. M. Paone. 1981.The effects of nitrogen limitation on the ultrastructure of thecyanobacterium Agmenellum quadruplicatum. Arch. Micro-biol. 130:204-212.

30. Stevens, S. E., Jr., D. A. M. Paone, and D. L. Balkwill. 1981.Accumulation of cyanophycin granules as a result of phosphatelimitation in Agmenellum quadruplicatum. Plant Physiol.67:716-719.

31. Stevens, S. E., Jr., C. 0. P. Patterson, and J. Myers. 1973. Theproduction of hydrogen peroxide by blue-green algae: a survey.J. Phycol. 9:427-430.

J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/


32. Stewart, W. D. P., and G. A. Codd. 1975. Polyhedral bodies(carboxysomes) of nitrogen-fixing blue-green algae. Br. Phycol.J. 10:273-278.

33. Thurston, E. L., and L. 0. Ingram. 1971. Morphology and finestructure of Fischerella ambigua. J. Phycol. 7:203-210.

34. Wilcox, M., G. J. Mitchison, and R. J. Smith. 1973. Patternformation in the blue-green alga Anabaena. 1. Basic mecha-nisms. J. Cell Sci. 12:707-723.

35. Wilcox, M., G. J. Mitchison, and R. J. Smith. 1973. Patternformation in the blue-green alga Anabaena. II. Controlledproheterocyst regression. J. Cell Sci. 13:637-649.

36. Wilcox, M., G. J. Mitchison, and R. J. Smith. 1975. Mutants ofAnabaena cylindrica altered in heterocyst spacing. Arch. Mi-crobiol. 103:219-223.

37. Wildon, D. C., and F. V. Mercer. 1963. The ultrastructure of theheterocyst and akinete of the blue-green algae. Arch. Mikrobiol.47:19-31.

38. Wolk, C. P. 1973. Physiology and cytological chemistry of blue-green algae. Bacteriol. Rev. 37:21-101.

39. Wolk, C. P. 1982. Heterocysts, p. 359-386. In N. G. Carr andB. A. Whitton (ed.), The biology of cyanobacteria. BlackwellScientific Publications, Oxford.

VOL. 157, 1984


.org/D

ownloaded from

http://jb.asm.org/

Heterocyst Differentiation in the Cyanobacterium …HETEROCYST DIFFERENTIATION IN M. LAMINOSUS 515 terns, however, and such an investigation will be required before the mechanism for

Documents