J Mol Microbiol Biotechnol 2006;11: 256–264
DOI: 10.1159/000094059
Cytoskeleton of Mollicutes
Makoto Miyata a, b Hiroshi Ogaki a
a
b
Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka,
PRESTO, JST, Sumiyoshi-ku, Osaka, Japan
Key Words
Cell wall-less Mycoplasma Polarity Binary division Characteristic cell shapes
Abstract
Mollicutes are a class of bacteria that lack a peptidoglycan
layer but have various cell shapes. They perform chromosome segregation and binary fission in a well-organized
manner. Especially, species with polarized cell morphology
duplicate their membrane protrusion at a position adjacent
to the original one and move the new protrusion laterally to
the opposite end pole before cell division. The featured various cell shapes of Mollicutes are supported by cytoskeletal
structures composed of proteins. Recent progress in the
study of cytoskeletons of walled bacteria and genome sequencing has revealed that the cytoskeletons of Mollicutes
are not common with those of other bacteria. Mollicutes
have special cytoskeletal proteins and structures that are
sometimes not shared even by other mollicute species.
ma and others, and are often referred to as ‘mycoplasmas’.
Phylogenetically, they belong to the high-AT branch of
Gram-positive bacteria, which also includes Clostridium
and Bacillus [Weisburg et al., 1989]. Unlike the cells of
other bacterial groups, mollicute cells totally lack the
peptidoglycan layer and are covered with membrane-anchored proteins, including antigenic variants [Razin et
al., 1998]. They are also characterized by plasticity, since
they lack a peptidoglycan layer [Miyata, 2002; Miyata and
Seto, 1999]. However, mollicute cells have a wide variety
of shapes. For example, Spiroplasmas are helical, some
Mycoplasma species are polarized with a membrane protrusion at a cell pole, and others have coccoid, rod-like,
or filamentous shapes [Trachtenberg, 1998]. This diversity suggests the existence of different structures to support these various morphologies, and it has guided many
researchers to the study of mollicute ‘cytoskeletons’.
Modes of Cell Division
Copyright © 2006 S. Karger AG, Basel
Mollicutes
Mollicutes are a class of generally parasitic or commensal bacteria featuring reduced genome sizes (560–
2,300 kbp) [Razin et al., 1998]. Mollicutes include the genera Mycoplasma, Ureaplasma, Spiroplasma, Achoreplas-
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Several modes of cell division have been proposed for
Mollicutes, including binary fission, fragmentation of an
elongated cell, and budding [Boatman and Kenny, 1970;
Freundt, 1969; Miyata, 2002; Miyata and Seto, 1999].
However, not all of these modes have been well substantiated. Binary fission was demonstrated for Mycoplasma
species with polarized cell morphology, including M.
pneumoniae, M. gallisepticum, and M. mobile, as dis-
Makoto Miyata
Department of Biology, Graduate School of Science
Osaka City University, Sumiyoshi-ku
Osaka 558-8585 (Japan)
Tel. +81 6 6605 3157, Fax +81 6 6605 3158, E-Mail [email protected]
cussed below [Bredt, 1968; Morowitz and Maniloff, 1966;
Rosengarten and Kirchhoff, 1989; Seto et al., 2001], and
also for mycoplasmas without apparent cell polarity [Garnier et al., 1981, 1984; Robertson et al., 1975; Seto and
Miyata, 1998, 1999]. Mycoplasma capricolum has rodshaped cell morphology without apparent polarity [Seto
and Miyata, 1998, 1999]. In larger M. capricolum cells
observed in a fast-growing culture, the center of the cell
was constricted, suggesting reproduction by binary fission. This assumption was supported by observations
that both DNA mass and cell length rarely exceeded twice
their minimal values. Spiroplasma citri, featuring filamentous helical cell morphology, can be monitored for
cell elongation according to the number of helical turns
[Garnier et al., 1981, 1984]. Cells with two helical turns
were the major fraction of a cell population in a fastgrowing culture, and only occasionally were cells with
more than four turns observed. Constriction was seen
with a periodicity of two helical turns. Pulse labeling of
the membrane revealed that elongation occurs at a cell
pole and at the center where constriction would occur.
These observations lead to the conclusion that S. citri reproduces through binary fission after cell elongation at
one cell pole.
So far, detailed analyses have shown binary fission as
the division mode of Mollicutes. However, alternative division modes cannot be ruled out for almost 200 mollicute species. Moreover, the division modes have been
studied basically in laboratory conditions, which most
likely differ from conditions found in nature.
Segregation of Nucleoids
Although the genomic DNA of bacteria does not, unlike the eukaryotic chromosome, have a nuclear membrane, it takes on a condensed form and is called either a
‘chromosome’ or a ‘nucleoid’. The nucleoid is duplicated
before cytokinesis and is delivered into each daughter cell
by the segregation machinery, as observed in eukaryotic
cells [Gerdes et al., 2004]. In Mollicutes, are the duplicated nucleoids faithfully segregated by a special mechanism, or are they distributed to the daughter cells
randomly? Visualization of the chromosomal DNA by
staining with DAPI (4,6-diamidino-2-phenylindole)
has helped to address this question. DAPI staining of
M. capricolum, M. pneumoniae, M. gallisepticum, and
M. mobile revealed that, in cell populations of normally
growing cultures, no cells lacked a chromosome (fig. 1)
[Seto and Miyata, 1999; Seto et al., 2001]. Also, a small
Cytoskeleton of Mollicutes
Fig. 1. Cell structure of M. mobile. Upper: Putative antigenic vari-
ant proteins, namely MvspN and MvspO, Gli521 protein, and nucleoid are labeled green, magenta, and blue, respectively. Each image was recorded and merged. Bar: 1 m. This image is by courtesy of Dr. Shintaro Seto, Hamamatsu University School of
Medicine, Hamamatsu, Japan. Lower: Schematic of cell structure.
The major part of the nucleoid is found in the cell body. Gliding
proteins occupy the surface of the cell neck, form a collar, and
prevent distribution of other membrane proteins into the neck
region. Antigenic proteins MvspN and MvspO are distributed
only in the head region.
constriction was observed in elongated cells of M. pneumoniae, suggesting that the nucleoid is segregated into
daughter cells faithfully [Seto et al., 2001]. The genomic
DNA is condensed in M. capricolum, whose cells have
greater volume than those of the other species, whereas
such distinct condensation cannot be observed for
M. pneumoniae, M. gallisepticum, or M. mobile (fig. 1)
[Seto and Miyata, 1999; Seto et al., 2001]. The segregation
of the nucleoid was observed in M. capricolum [Seto and
Miyata, 1999]. In a fast-growing culture of M. capricolum,
most cells had one or two nucleoids each, and no cells
without nucleoids were found. The nucleoids were positioned at the center in mononucleoid cells or at one-quarter or three-quarters of the cell length in binucleoid cells.
These observations suggest that M. capricolum has a
method of ensuring that replicated DNA is delivered to
daughter cells.
J Mol Microbiol Biotechnol 2006;11:256–264
257
lipid synthesis, suggesting that nucleoid movement seems
to be linked to the extension of the cytoskeleton rather
than the expansion of the membrane. When protein synthesis was inhibited, the final nucleoid positions were significantly biased toward the cell poles. This suggests that
de novo protein synthesis is required to control nucleoid
movement. In Escherichia coli, a low copy number plasmid, R1, is segregated by a cytoskeletal structure composed of an actin homolog, ParM [Carballido-Lopez and
Errington, 2003]. Recently, the segregation of nucleoid of
Caulobacter crescentous was also shown to depend on an
actin homolog, MreB, suggesting that the segregation of
low-copy-number DNA is generally caused by actin homologs in bacteria [Margolin, 2005]. Mollicutes may have
similar mechanisms. However, an obvious actin homolog
has been identified only in Mesoplasma florum among the
14 mollicute genomes determined so far [Barre et al.,
2004].
Division Scheme of Mycoplasma Species with
Polarized Cell Morphology
Fig. 2. Cell structure of M. pneumoniae. Upper: P65 and P24 pro-
teins are labeled blue and green, respectively, by fusion with fluorescent proteins. The cell marked by a circle is undertaking duplication of the attachment organelle. Bar: 1 m. This image is by
courtesy of Dr. Tsuyoshi Kenri, National Institute of Infectious
Diseases, Musashimurayama, Japan. Lower: Schematic illustration. The architectures and their component proteins are presented.
What mechanism, in turn, propels the nucleoids from
the center of a cell to a position one-quarter or threequarters across the length of the cell? As the distance
between the replicated nucleoids is proportional to the total cell length, the movement of replicated nucleoids in
M. capricolum may be coupled with a mechanism responsible for cell elongation. Generally, the elongation of wallless cells (such as animal cells) is a two-part process. The
first is membrane expansion via the insertion of new
membrane components, and the second is the extension
of cytoskeletal structures. Nucleoid movement (actually,
partitioning) in M. capricolum was observed even after
cell elongation was totally stopped by the inhibition of
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J Mol Microbiol Biotechnol 2006;11:256–264
Some Mycoplasma species, including M. pneumoniae,
M. genitalium, M. gallisepticum, M. mobile, M. penetrans,
and M. pulmonis, have a membrane protrusion at a cell
pole. The membrane protrusion of M. pneumoniae [Krause
and Balish, 2004; Miyata, 2005] and that of M. mobile [Kusumoto et al., 2004; Miyata, 2005; Uenoyama et al., 2004]
are enriched for proteins of cytadherence and used as organelles; they are responsible for cell adhesion and gliding
motility, which is the smooth translocation on a solid surface. This is believed to be the case also for the other three
species. In a population of Mycoplasma cells in culture, no
cells without a protruding membrane were found, indicating that the formation of the organelle is tightly coupled
to the cell reproduction cycle (fig. 1, 2).
How is the membrane protrusion formed? In the
1960s, Bredt recognized the membrane protrusion of M.
pneumoniae as a small knob on the cell surface under
phase-contrast microscopy, and he observed two small
knobs actually adjacent to each other at an early stage of
cell division. This hypothesis was substantiated by microscopic analysis of cells whose membrane protrusion
was fluorescently labeled by an antibody to P1 adhesin,
which was clustering on the organelle [Seto et al., 2001].
Seto et al. classified M. pneumoniae cell images into three
types based on the position of the protrusion and concomitantly measured the DNA content of each cell by the
signal intensity of DAPI. The DNA content differed sigMiyata/Ogaki
nificantly among the cell types. Cells with a single protrusion at one cell pole had a lower DNA content than
cells with two protrusions. Those with one protrusion at
each cell pole had the highest DNA contents. This observation suggests that the nascent protrusion forms next to
the original one and migrates to the opposite cell pole
before binary fission (fig. 3). This scheme may be similar
for all mycoplasmas with a membrane protrusion. This
assumption is supported by the results of electron microscopic (EM) studies for M. gallisepticum [Morowitz and
Maniloff, 1966] and M. mobile [Rosengarten and Kirchhoff, 1989].
Genes Related to Bacterial Cytoskeletons
Fig. 3. Cell division scheme in M. pneumoniae. The original and
nascent attachment organelles are presented as solid circles.
Recently, many proteins have been identified as components of cytoskeletons in bacterial cells [Amos et al., 2004;
Löwe et al., 2004; Moller-Jensen and Löwe, 2005]. Those
proteins are essential for (i) maintenance of cell shape as
represented by MreB, Mbl, and CreS, (ii) segregation of
DNA as represented by ParM and MreB, (iii) control of
cytokinesis as represented by MinC, MinD, and MinE,
and (iv) achievement of cytokinesis as represented by FtsZ.
So far, the genomes of 14 mollicute species have been sequenced and searched for these proteins [Barre et al.,
2004]. However, most cytoskeletal proteins of walled bacteria cannot be found in the annotation of mollicute ORFs.
Only the ftsZ gene is found in the genomes of 10 species.
Generally, in walled bacteria, FtsZ protein polymerizes
into filaments, thereby forming a ring structure called a
Z-ring at the future constriction site before the cell division. Like the contractile ring in eukaryotic cells, the Zring performs cytokinesis. As we discussed above, many
Mycoplasma species also exhibit binary fission. This suggests that the FtsZ proteins have similar roles in mycoplasmas. However, this assumption is still on the argument
from the following observations. (i) No other fts gene homologs can be found in the mollicute genomes, whereas in
walled bacteria, fts gene homologs are coded around the
ftsZ gene and participate in connecting the Z-ring and the
peripheral structures that involve the cell membrane and
peptidoglycan layers. (ii) The ftsZ gene from M. pulmonis
did not rescue ftsZ mutants of E. coli [Wang and Lutkenhaus, 1996]. (iii) The ftsZ gene is not found in the genome
of M. mobile, whose cell shape suggests well-controlled cell
division [Jaffe et al., 2004b]. These observations may suggest that FtsZ is not involved in the cell division mechanism of mycoplasmas. If this suggestion is correct, how
does cytokinesis occur in Mollicutes? An observation
Then, what structures support the featured cell shapes
of Mollicutes? The cytoskeletal structures of M. pneumoniae have attracted researchers since the 1970s [Göbel
et al., 1981; Meng and Pfister, 1980; Wilson and Collier,
1976]. At one pole of an M. pneumoniae cell, a cone-shaped
membrane protrusion, known as the attachment organelle, is formed (fig. 2). When the interaction between the
organism and the host tissue is examined, this structure
is always observed to bind to the tissue [Razin and Jacobs,
1992; Wilson and Collier, 1976]. Negative-staining EM reveals a surface structure called a ‘nap’ at the tip [Kirchhoff, 1992; Kirchhoff et al., 1984]. After extraction with
Triton X-100, an insoluble Triton shell, which appears to
be a cytoskeletal structure, remains. It is composed of a
relatively thick ‘rod’ and a filamentous network forming
a basket-like structure. It is thought that the rod supports
the attachment organelle and the basket supports the remainder of the cell [Göbel et al., 1981; Meng and Pfister,
Cytoskeleton of Mollicutes
J Mol Microbiol Biotechnol 2006;11:256–264
about eukaryotic cells may provide some clues. The ‘contractile ring’ formed in eukaryotic cells prior to cytokinesis is mainly composed of actin rather than tubulin, which
is the ortholog of FtsZ. When the contractile ring is depleted, motile animal cells can perform cytokinesis if the
cell can bind to a solid surface [Uyeda and Nagasaki, 2004].
As well, the study of subcellular localization of FtsZ in a
mollicute cell may yield a clue about the roles of the FtsZ
protein in a mollicute cell.
Cytoskeletal Architectures of M. pneumoniae
259
Fig. 4. Isolated rod of M. pneumoniae [pers. unpubl. data]. Upper: EM image of negative staining. Lower: Sche-
matic with dimensions. Mean values were obtained from 10 isolated images.
1980; Regula et al., 2001]. When examined by negative
staining EM, the isolated rods each seem to consist of a
striated bundle of filaments, suggesting a structure organized from many protein subunits [pers. unpubl. data; Hegermann et al., 2002; Regula et al., 2001] (fig. 4). The inside
of the attachment organelle, as observed by thin sectioning, has a low electron density and an electron-dense core
(EDC) [Biberfeld and Biberfeld, 1970; Shimizu and Miyata, 2002; Wilson and Collier, 1976], which is believed to
be identical to the rod observed by negative staining. The
EDC can be observed also in negatively stained images of
intact cells, if the cells have been washed several times by
water [pers. unpubl. data]. Hegermann et al. [2002] suggested a striking model of cell construction based on EM
observations of cryosections, with the rod attached at its
proximal end to a wheel-like complex of spikes that connect the rod and the periphery of the cell (fig. 2). Subcellular localization of proteins P41 and P24 suggests the existence of unknown structures [Kenri et al., 2004].
260
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Protein Components of Cytoskeletons of
M. pneumoniae
In M. pneumoniae, the protein components of the cytoskeletons are essential for cytadherence, which is required for parasitism. Therefore, several cytoskeletal proteins have been identified as pathogenicity determinants.
They can be classified into five groups, as follows (fig. 2).
(i) P1 adhesin: a 170-kDa protein with 3–7 putative transmembrane segments, responsible for binding to host cells
and solid surfaces such as glass and plastics via sialic acid
[Krivan et al., 1989; Razin and Jacobs, 1992; Roberts et al.,
1989], and also for gliding motility [Seto et al., 2005a].
(ii) Proteins that support P1 adhesin with physical interactions: P90, P40, etc. [Layh-Schmitt and Herrmann,
1994; Layh-Schmitt et al., 2000; Seto and Miyata, 2003].
(iii) Proteins functioning from the early stage in the formation of the attachment organelle, including the EDC.
These include HMW1 and HMW2 [Hahn et al., 1998;
Miyata/Ogaki
Popham et al., 1997; Seto and Miyata, 2003; Seto et al.,
2001; Willby et al., 2004]. (iv) Protein localizing at the
proximal end of the EDC, i.e., the position where the
wheel-like structure has been predicted. These include
P24, P41, etc. [Kenri et al., 2004]. (v) Other proteins localizing at the attachment organelle, such as HMW3, P65,
and P30 [Jordan et al., 2001; Seto and Miyata, 2003; Seto
et al., 2001]. All of the proteins in these groups are known
to be coded in three loci in the genome [Krause, 1996].
The cytoskeletal structure of M. genitalium has been
studied less extensively than that of M. pneumoniae.
However, M. genitalium is phylogenetically closely related to M. pneumoniae, and they have similar cell morphology. Moreover, all M. pneumoniae [Himmelreich et al.,
1996] genes described here can be found in the genome
of M. genitalium [Fraser et al., 1995]. Therefore, the architectures for cell morphology, cytadherence, and gliding motility are believed to be common between these
two species.
Possible Scaffold Supporting Force for Gliding of
M. mobile
M. mobile, isolated from the gills of a freshwater fish
in the early 1980s, is the fastest-gliding Mycoplasma
known [Kirchhoff and Rosengarten, 1984; Kirchhoff et
al., 1987]. It glides smoothly and continuously on glass at
an average speed of 2.0–4.5 m/s, or 3–7 times the length
of the cell per second [Rosengarten and Kirchhoff, 1987],
exerting a force of up to 27 piconewtons (pN) [Hiratsuka
et al., 2005; Miyata et al., 2002]. Recently, we identified
three large proteins, Gli349 (a 349-kD protein) [Kusumoto et al., 2004; Miyata et al., 2000; Uenoyama et al.,
2004], Gli521 (a 521-kD protein) [Miyata et al., 2000; Seto
et al., 2005b], and Gli123 (a 123-kD protein) [Miyata et
al., 2000; Uenoyama and Miyata, 2005a], involved in the
gliding mechanism of M. mobile. Analysis of the inhibitory effects of the anti-Gli349 and anti-Gli521 antibodies
on gliding mycoplasmas suggested that Gli349 and Gli521
are responsible for hemadsorption and glass binding, and
for force generation and/or transmission, respectively.
The characters of a mutant suggest that Gli123 is responsible for localization of other gliding proteins. These proteins localize exclusively at the base of a head-like structure named a ‘neck’, which is specialized for gliding and
binding (fig. 1) [Kusumoto et al., 2004; Miyata and Uenoyama, 2002]. Rapid-freeze- and freeze-fracture rotaryshadow EM showed many spike-like structures, approximately 50 nm in length, sticking out around the neck and
Cytoskeleton of Mollicutes
binding to the glass surface with their distal ends [Miyata and Petersen, 2004]. The spikes seem to be composed
of Gli349 molecule and function as ‘legs’ in the gliding
mechanism, because the subcellular localization and appearance agree with those of Gli349 [Adan-Kubo et al.,
2006; Metsugi et al., 2005], and because no spikes were
found in a non-binding mutant. ‘Cell ghosts’ permeabilized and killed by Triton-X100 treatment were reactivated to glide at the speeds very similar to those of living
cells, showing that the gliding is driven by the energy of
ATP [Jaffe et al., 2004a; Uenoyama and Miyata, 2005b].
These observations lead us to assume that the spikes composed of Gli349 repetitively bind with and release from
glass, propelling the cell by the force exerted from or
through the Gli521 molecule, based on the energy of ATP
hydrolysis [Charon, 2005; Miyata, 2005; Seto et al., 2005b;
Uenoyama et al., 2004]. If this is correct, what structure
supports the force transmitted from the gliding machinery to the cell body? So far, no cytoskeletal structure like
that of M. pneumoniae has been found in M. mobile. We
counted the Gli349 and Gli521 molecules on the M. mobile cell surface and concluded that around 450 molecules
of each exist on a cell [Seto et al., 2005b; Uenoyama and
Miyata, 2005a; Uenoyama et al., 2004]. Considering the
molecular weight of Gli521 and the small space of the cell
neck, Gli521 should be packed into a structure similar to
a two-dimensional sheet. This sheet would form a ‘collar’
that functions as an outer frame for gliding [Kusumoto
et al., 2004; Seto et al., 2005b]. This assumption is supported by observations that surface proteins anchored to
the cell membrane do not distribute into the neck region
[Kusumoto et al., 2004]. M. pulmonis, which is phylogenetically closely related to M. mobile, also has a membrane protrusion at a cell pole, and has orthologs of Gli349
and Gli521 [Chambaud et al., 2001; Uenoyama and Miyata, 2005a]. These facts may suggest that M. pulmonis
has architectures similar to those of M. mobile.
Cytoskeletal Structures of Other Mollicutes
Spiroplasmas have helical cell shapes and swim in viscous conditions. A flat cytoskeletal ribbon of parallel fibrils is attached to the cellular tube and follows the shortest helical line on the inner surface of the cellular tube.
Contraction of the fibrils may cause the changes in the
handedness of helical cells and the resulting kinks may
propel the cell, as discussed elsewhere [Berg, 2002; Kurner et al., 2005; Shaevitz et al., 2005; Trachtenberg, 1998,
2004].
J Mol Microbiol Biotechnol 2006;11:256–264
261
bleb
infrableb
tubular structure
Fig. 5. Cell structure of M. gallisepticum.
This illustration is modified from Korolev
et al. [1994].
M. gallisepticum, a poultry pathogen, also has a membrane protrusion, called a bleb that is attached to another
protruding part, an infrableb (fig. 5). The functions of
blebs in gliding and cytadherence have not been well examined. However, the bleb is expected to have such functions, because gapA and the following crmA of M. gallisepticum code for orthologs of P1 adhesin and ORF6,
which is the precursor of P90 and P40 of M. pneumoniae,
respectively. The bleb also has been suggested to function
as an apparatus for chromosome segregation, because a
small part of the chromosomal DNA was found to be attached to the bleb membrane [Maniloff and Quinlan,
1974; Quinlan and Maniloff, 1972, 1973]. A tubular structure 40 nm in diameter has been found in ultrathin sections under EM: several tubes start near the infrableb,
form loops in the cell body, and return near the infrableb
[Korolev et al., 1994]. The appearance of this tubular
structure is similar to that of a microtubule, although
with a diameter of more than 25 nm, it is wider than a
microtubule. The component protein of this tubular
structure should be identified.
ly on walled bacteria, for which genetic studies are fully
available, and not on Mollicutes. Studies on bacterial cytoskeletons and genome information have shown that the
cytoskeletons of Mollicutes and walled bacteria have few
parts in common. What did the cytoskeletons of Mollicutes originate from? As the component proteins of
mollicute cytoskeletons do not show similarities with
those of eukaryotic cytoskeletons either, horizontal transfer should not be considered. Mollicute cytoskeletons
may have evolved rapidly from those found in walled bacteria under special evolutional pressure generated by the
absence of a peptidoglycan layer and by parasitic environments.
As discussed above, the role of Mollicutes as a cutting
edge in the study of bacterial cytoskeletons may be lost,
but the well-organized architectures and the dynamics of
mollicute cytoskeletons would still attract subjects if we
consider protein functions and evolutional strategies of
microorganisms.
Acknowledgments
I am very grateful to my collaborators who shared in the exciting times, and also colleagues who provided valuable comments
for and encouragement of our studies. Our studies have been supported by grants-in-aid for Scientific Research (C) from the Japan
Society for the Promotion of Science, and for Scientific Research
on a Priority Area (‘Applied Genomics’ and ‘Structures of biological macromolecular assemblies’) from the Ministry of Education, Science, Sports, Culture, and Technology of Japan, and by a
grant from the Institution for Fermentation, Osaka.
Concluding Remarks
Researchers of Mollicutes began to focus on the cytoskeletal structures of mollicute cells well before researchers of walled bacteria did on such structures of walled
bacteria, because Mollicutes lack a peptidoglycan layer
and yet possess diverse cell morphologies. Unfortunately,
images of bacterial cytoskeletons have been based main262
J Mol Microbiol Biotechnol 2006;11:256–264
Miyata/Ogaki
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