ORIGINAL PAPER

Insights into the ultrastructural morphology of novelPlanctomycetes

Olga Maria Lage • Joana Bondoso •

Alexandre Lobo-da-Cunha

Received: 3 May 2013 / Accepted: 3 July 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Knowledge of the interesting phylum of

Planctomycetes has increased in the last decades both

due to cultural and molecular methods. Although a

restricted number of species have been described to

date, this group presents a much larger diversity that

has been mainly revealed by molecular ecology

studies. Isolation experiments allowed us to get a

number of new Planctomycetes taxa that extend the

already described ones. In this work we present the

ultrastructural morphological characterization of these

new taxa as well as we give new details of Aquisph-

aera giovannonii ultrastructure. Furthermore, our

interpretation on Planctomycetes cell envelope is

provided.

Keywords Planctomycetes � Ultrastructure � TEM �Cell envelope

The Planctomycetes are part of the PVC superphylum

that also includes the Verrucomicrobia, Chlamydiae

and Lentisphaerae and the candidate phyla Poribac-

teria and OP3 (Wagner and Horn 2006). Their first

observation (Gimesi 1924) and isolation in pure

culture (Staley 1973) were separated by half a century.

Since then, several reports have expanded our knowl-

edge on this particular group of bacteria. Planctomy-

cetes possess a unique combination of physiological,

morphological and genetic features that sets them

apart from other bacteria (Fuerst and Sagulenko 2011;

Devos and Reynaud 2010). Although normally present

in low abundance, they have been observed in a wide

range of terrestrial and aquatic habitats which reflects

their adaptation to different lifestyles and environ-

ments. Members of this group can be found in marine,

hypersaline, hyperthermal, brackish and fresh water

and in many terrestrial environments including soils

and acidic environments (Fuerst 1995; Neef et al.

1998; Wang et al. 2002; Kulichevskaia et al. 2006;

Schlesner 1994; Lage and Bondoso 2012), as part of

the microbial wall community usually found in caves

from various parts of the world (Borsodi et al. 2012;

Pasic et al. 2010), in macroalgae biofilm (Bengtsson

and Ovreas 2010; Fukunaga et al. 2009; Lage and

Bondoso 2011; Burke et al. 2011; Lachnit et al. 2011;

O. M. Lage (&) � J. Bondoso

Departamento de Biologia, Faculdade de Ciencias,

Universidade do Porto, Rua do Campo Alegre s/n8,4169-007 Porto, Portugal

e-mail: olga.lage@fc.up.pt

O. M. Lage � J. Bondoso � A. Lobo-da-Cunha

CIMAR/CIIMAR—Centro Interdisciplinar de

Investigacao Marinha e Ambiental, Universidade do

Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal

A. Lobo-da-Cunha

Laboratorio de Biologia Celular, Instituto de Ciencias

Biomedicas Abel Salazar (ICBAS), Universidade do

Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto,

Portugal

123

Antonie van Leeuwenhoek

DOI 10.1007/s10482-013-9969-2

Miranda et al. 2013) and in association with several

eukaryotic organisms like prawns and sponges (Fuerst

et al. 1991, 1997; Pimentel-Elardo et al. 2003). In the

last decade, special importance has been given to this

group in the field of evolutionary biology because of

the unusual presence of characteristics that are usually

features mainly found in eukaryotic cells (Devos and

Reynaud 2010; Fuerst and Sagulenko 2012). These

include the presence of membrane-bounded cell

compartments (Lindsay et al. 1997, 2001), the absence

of the common bacterial tubulin like protein FtsZ

(Pilhofer et al. 2008; Bernander and Ettema 2010), that

is also absent in eukaryotes and the archaeal group

Crenachaeota (Vaughan et al. 2004), the ability to

perform endocytosis (Lonhienne et al. 2010), that was

never found in Bacteria or Archaea and the presence

of genes homologous to membrane coat protein genes

(Santarella-Mellwig et al. 2010) that are essential in

the eukaryotic endocytosis. Other unusual features in

this group are the budding reproduction of many of

their members, the presence of crateriform structures

on the cell surface, whose function is still unknown

and the presence of a proteinaceous cell wall that lacks

the characteristic bacterial peptidoglycan with conse-

quent ability to resist to b-lactam antibiotics. Plan-

ctomycetes are considered to challenge the typical

bacterial cell organization by possessing compartmen-

talization of their cells by internal membranes. Since

the works of Lindsay et al. (1997) the Planctomycetes

were considered to possess an intracytoplasmic mem-

brane (ICM). This single bilayer membrane lays inside

the cytoplasmic membrane (CM) and separates the

paryphoplasm from the riboplasm or pirellulosome

forming then a compartment. Furthermore, other

major cell compartments are the anammoxosome in

the anaerobic ammonium oxidation (Anammox)

Planctomycetes (Van Niftrik et al. 2004) and the

nuclear body in Gematta obscuriglobus and related

strains (Fuerst and Sagulenko 2013). Even though the

presence of an ICM in Planctomycetes has been

accepted by the scientific community (Jogler et al.

2011; van Teeseling et al. 2013; Fuerst and Sagulenko

2013) the clear observation of the CM has been always

doubtful. A different model of the Planctomycetes cell

envelope considers the presence of an asymmetrical

outer membrane like the one typical of Gram negative

bacteria and considers that the ICM is in fact the CM

and the paryphoplasm an enlarged, potentially spe-

cialized, periplasm (Speth et al. 2012).

Recently, several Planctomycetes belonging to new

genera or new Rhodopirellula species were isolated

from the microbial community of macroalgae (Lage

and Bondoso 2011). Aiming at describing these novel

taxa, several of these Planctomycetes are being

characterized by a polyphasic approach. In this work

we present several aspects of the ultrastructural study

performed. Furthermore, new aspects of the ultra-

structure of Aquisphaera giovannonii isolated from

the sediments of a freshwater aquarium (Bondoso et al.

2011) will be also included.

Materials and methods

Organisms and growth conditions

Strains and cultivation

The strains under study are part of the OJF culture

collection. These include several marine strains isolated

from the biofilm of macroalgae, strains UC17, Cor3,

LF2, UC9, UC8 and LF1 (Lage and Bondoso 2011) and

the freshwater Planctomycete, Aquisphaera giovanno-

nii isolated from the sediment of a freshwater aquarium.

A. giovannonii was cultivated in medium PYGV in the

dark at 35 �C. Marine planctomycetes strains were

cultivated in medium M13 in the dark at 26 �C.

Chemical fixation (CF) for transmission electron

microscopy (TEM)

For conventional TEM, cells from one, two or seven

days old solid or liquid cultures were harvested and fixed

for 2 h in 2.5 % (w/v) glutaraldehyde in marine buffer

(Watson et al. 1986), pH 7.0, post-fixed for 4–16 h in

1 % (v/v) osmium tetroxide in the same buffer followed

by 1 h 1 % (w/v) uranyl acetate. The treated specimens

were subsequently dehydrated through a graded ethanol

series and embedded in Epon resin. Ultrathin sections

were stained for 10 min in 1 % (w/v) uranyl acetate and

10 min in Reynolds lead citrate. The sections were

examined in a JEOL 100CXII.

For the enzymatic protein digestion studies, a

Pirellula-like Planctomycete isolated from cultures

of the marine dinoflagellate Prorocentrum micans was

used. Cells were isolated and cultivated in Zobell agar

medium (0.5 % peptone, 0.8 % agar in 1 L of 90 %

seawater). Ultrathin sections were collected in gold

Antonie van Leeuwenhoek

123

grids covered with 1 % parlodium and treated with

1 % protease E, type VIII, bacterial, for 1 h at 37 �C,

after oxidation with 3 % H2O2 for 10 min. Control

sections were treated with a similar protease solution

that was heat denaturated.

Cryofixation and cryosubstitution (CC) for TEM

Cells from exponentially growing cultures in agar

medium were transferred to 1.5 mm diameter and

200 lm depth planchettes and immediately cryoimmo-

bilized using a Leica EMPact high-pressure freezer

(Leica, Vienna, Austria) and then stored in liquid nitrogen

until further use. They were freeze-substituted over three

days at -90 �C in anhydrous acetone containing 2 %

osmium tetroxide and 0.1 % uranyl acetate at -90 �C for

72 h and warmed to room temperature, 5 �C per hour

(EM AFS, Leica, Vienna, Austria). After several acetone

rinses, samples were infiltrated with Epon resin during

2 days and resin was polymerised at 60 �C during 48 h.

Ultrathin sections were obtained using a Leica Ultracut

UCT ultramicrotome and mounting on Formvar-coated

copper grids. They were staining with 2 % uranyl acetate

in water and lead citrate. Then, sections were observed in

a Tecnai Spirit electron microscope (FEI Company,

Eindhoven) or in a JEOL 100CXII.

Results and discussion

Aquisphaera giovannonii

A. giovannonii is a freshwater Planctomycete phylo-

genetic related to genera Isosphaera and Singulisph-

aera (Fig. 1) that forms small light pink colonies in

PYGV medium which can turn red if the cells are

Fig. 1 Optimal maximum-

likelihood tree showing the

phylogenetic relationship of

the strains under study (in

bold) with other

representatives of the

phylum Planctomycetes

based on 16S rDNA

sequences. The numbers

beside nodes are the

percentages for bootstrap

analyses; only values above

50 % are shown. Scale

bar = 0.05 substitutions per

100 nucleotides. Candidatus

Anammox 16S rRNA gene

sequences were used as an

outgroup

Antonie van Leeuwenhoek

123

grown in media containing N-acetylglucosamine. Old

colonies become strongly compact and attached to the

solid medium. In liquid media, cells in late exponen-

tial phase form very large aggregates that resemble

snowflakes. Cells, spherical in shape with 1.6–2 lm in

diameter and non-motile, present the common Plan-

ctomycetes cell morphology and organization. A.

giovannonii has uniformly distributed crateriform pits

on their surface as in Isosphaera pallida and Singu-

lisphaera acidiphila and an intricate paryphoplasm

and a riboplasm with a prominent nucleoid, ribosomes

as well as lipid or glycogen-like granules (Figs. 2, 3a).

The fibrillar extracellular matrix surrounding the cells

was also easily observed under transmission electron

microscopy (Fig. 2). This ultrastructural morphology

is observed when A. giovannonii is grown in PYGV

medium [0.025 % peptone, 0.025 % yeast extract,

0.025 % glucose supplemented with 20 ml.L-1 Hut-

ner’s basal salts and 10 ml.L-1 vitamin solution

Fig. 3 Ultrathin sections of A. giovannonii by CC – TEM (a) or CF-TEM (b, c, d). Cells grown in PYGV medium (a), R2A medium

(b) and PYGV4 (c, d). Pa paryphoplasm, Pi pirellulosome, L lipids, Gl glycocalix

Fig. 2 Ultrathin sections of A. giovannonii by conventional

fixation (CF-TEM). Pa paryphoplasm, Pi pirellulosome, L lip-

ids, M fibrillar extracellular matrix, arrow budding cell

Antonie van Leeuwenhoek

123

(Staley 1968)]. However, if grown in a much more

organic medium (fourfold the concentration of pep-

tone, yeast extract and glucose—PYGV4), A. giovan-

nonii growth is inhibited and huge amounts of lipid

reserves that can reach up to 72 % of the area of the

cells in thin sections are formed (Fig. 3c, d). A.

giovannonii can also form huge glycocalyx if grown in

DSMZ medium R2A (Fig. 3b).

A characteristic feature of A. giovannonii is its

budding reproduction (Figs. 2, 4a, b). A narrow

passage connects the mother cell and the bud (Fig. 4a)

but this connection is quite wide. Furthermore the buds

are surrounded by electron dense fibrillar material that

disappears as the bud maturates (Fig. 4).

Ultrastructure of new Planctomycetes

Novel Rhodopirellula spp.

Isolates LF2 and UC17 for which the designations of,

respectively, R. rubra and R. lusitanica are being

proposed are strains affiliated to R. baltica with about

98 % of similarity in the 16S rRNA gene (Fig. 1).

Both strains form the characteristic rosettes especially

with a huge number of cells in LF2 (Fig. 5).

Strains LF2 and UC9 (Figs. 5, 6), both isolates of

R. rubra, seems to have a wide and very electron

transparent paryphoplam. The pirellulosome, largely

situated in the reproductive pole, is often divided in

small compartment-like sections and contains the

condensed DNA, ribosomes and several inclusion

of unknown nature. At the reproductive pole, besides

thinner pili, fimbriae are prominent and present, at

their base, a ring-like structure. Figure 6 points toward

a connection of the fimbriae to the CM that might

require further verification.

Strains UC17 and Cor3 (Fig. 7), both isolates of

R. lusitanica, have a smaller electron dense parypho-

plasm when compared to LF2 and a well-developed

pirellulosome. When budding, the cells form an

electron dense structure near the budding cell pole of

unknown nature and function in the paryphoplasm or

Fig. 4 Ultrathin sections of two budding cells of A. giovannonii by CF-TEM. Arrow narrow passage connects the bud and the mother

cell; arrowhead electron dense fibrillar material that surrounds the bud

Fig. 5 Ultrathin sections of a rosette of strain LF2 by CC-TEM.

The cells are oriented with the reproductive pole to the outside

of the rosette

Antonie van Leeuwenhoek

123

in the pirellulosome (Fig. 7b, d). van Niftrik et al.

(2009) also reported the presence of a bracket-shaped,

electron dense structure in the paryphoplasm of

dividing cells of the anammox bacterium ‘‘Candidatus

Kuenenia stuttgartiensis’’. This structure is the divi-

sion ring. Older UC17 and Cor3 cells are generally

bigger, present often several internal membranes and

can be transversely crossed by a bunch of paralleled

microtubules-like structures that in cross section are

arranged hexagonally (Fig. 7c). The function of these

parallel fibrils with about 20 nm wide and reaching

about 2 lm long is unknown. Microtubule-like struc-

tures have been referred in prokaryotes (Bermudes

et al. 1994; Pilhofer et al. 2011). Virus-like particles

seems to be associated with these strains (Fig. 7c).

Novel genera of Planctomycetes

Strains LF1 and UC8 are two new genera of Plancto-

mycetes being Rhodopirellula the closest relative with

94.2 and 93.8 % 16S rRNA gene sequence similarity,

respectively (Fig. 1). The proposed designation of

LF1 and UC8 are Rubripirellula obstinata and Rosei-

maritima ulvae, respectively. LF1 was recovered from

the microbial biofilm of the macroalgae Laminaria

and UC8 from Ulva sp.

Cells of strain LF1 (Fig. 8) are ovoid to pear-

shaped, usually organized in rosettes of 3–10 cells

(Fig. 8a). Fimbriae emerge in the apical and repro-

ductive pole and a very robust electron dense holdfast

is present in the opposite pole (Fig. 8a). They appar-

ently possess an electron transparent fibrillar pary-

phoplasm and the pirellulosome, normally close to the

apical pole, presents many ribosomes and the perma-

nently condensed DNA. The cells are surrounded by a

thick cell wall and an evident glycocalyx. Some LF1

cells show hump-like protrusions similarly to Pirellula

staleyi ATCC 35122 (Butler 2002).

Strain UC8 attaches to surfaces when grown in

liquid media as observed for A. giovannonii (Bondoso

et al. 2011). Cells of strains UC8 are circular to ovoid

and organized in rosettes that can reach large numbers

of cells. Division in an electron transparent parypho-

plasm and a pirellulosome with ribosomes, many

storage substances and condensed DNA forming a

nucleoid, is clear (Fig. 9). UC8 presents an extensive

paryphoplasm with granular appearance and a com-

paratively smaller pirellulosome.

New ideas on the structure of the cell envelope

of Planctomycetes

After the observation of hundreds of cell sections of

several different species, some aspects of the Plancto-

mycetes cell envelope seem evident. It should be

noticed that only members of the Planctomycetales

were observed. In all the new taxa studied, the cells are

surrounded by a cell wall having two asymmetric

electron dense layers (the inner thicker than the outer)

separated by an electron transparent one (Figs. 6, 7a, b,

10). This structure is consistent with the structure of the

Gram-negative OM and fits into the model presented

by Speth et al. (2012). This OM should be very rich in

protein content because, after enzymatic digestion with

protease, it appears as a white space (Fig. 11). Of

protein nature are also the fimbriae, the holdfast and

Fig. 6 Ultrathin sections of strains UC9 (a) and LF2 (b) by CC-TEM. Pa paryphoplasm, Pi pirellulosome, I inclusions, F fimbriae,

H holdfast

Antonie van Leeuwenhoek

123

Fig. 7 Ultrathin sections of strains UC17 (a) and Cor 3 (b, c,

d) by CC-TEM (a) and CF-TEM (b, c, d). In b and d cells are

budding. Note the presence of an electron dense structure in the

reproductive pole (arrow). In c the cell presents a bunch of

microtubules in longitudinal view and a virus-like particle can

be seen outside the cell. The inset shows the microtubules in

cross section. Pa paryphoplasm, Pi pirellulosome, F fimbriae,

bMT bacterial microtubules

Fig. 8 Ultrathin sections of strain LF1 by CC-TEM. Pa paryphoplasm, Pi pirellulosome, F fimbriae, H holdfast, arrow glycocalyx

Antonie van Leeuwenhoek

123

some internal inclusions. Speth et al. (2012) consider

that externally to the OM is a proteinaceous cell wall,

fact that is not supported by our observations.

Internally to the cell wall or OM it is clear the

presence of a bilayer structure, the CM (Figs. 6, 7a, b,

10) that normally is referred to be difficult to be

observed (Lindsay et al. 1997). In cell sections the

presence of a second membrane internal to the CM is

not evident. Inside the cell only one membrane is

evident surrounding the cell. Membrane invagination

forming vesicle-like compartments could be viewed in

thin sections (Fig. 10). Our results suggest that

vesicle-like compartments (the pirellulosome or ribo-

plasm) in these novel Planctomycetes are the result of

the CM invagination which is also consistent with

Speth et al. (2012) membrane plasticity theory. The

structure of Planctomycetes cell envelope has been

observed in cell sections prepared by both the

conventional chemical fixation and the cryofixation

and cryosubstitution and results are consistent

between the techniques.

Acknowledgments The authors are grateful to Ana Maria

Parente and Damien Devos for helpful comments and to Carmen

Lopez-Iglesias and Jaume Cambra for the help with the

cryofixation of the samples. This work was supported by

Fundacao para a Ciencia e Tecnologia (FCT, C/MAR/LA0015/

2011). The second author was financed by FCT (PhD grant

SFRH/BD/35933/2007).

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