JOURNAL OF BACTERIOLOGY, Dec. 1996, p. 6994–6997
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology
Vol. 178, No. 23
N-Acetylputrescine as a Characteristic Constituent of Cyanelle
Peptidoglycan in Glaucocystophyte Algae
BEATRIX PFANZAGL,1 GÜNTER ALLMAIER,2 ERICH R. SCHMID,2 MIGUEL A. DE PEDRO,3
1
AND WOLFGANG LÖFFELHARDT *
Institut für Biochemie und Molekulare Zellbiologie der Universität Wien und Ludwig Boltzmann-Forschungsstelle für
Biochemie, A-1030 Vienna,1 and Institut für Analytische Chemie der Universität Wien, A-1090 Vienna,2 Austria,
and Centro de Biologı́a Molecular “Severo Ochoa,” Universidad Autónoma de Madrid, E-28049 Madrid, Spain3
Received 24 June 1996/Accepted 24 September 1996
myces griseus (both purchased from Boehringer, Mannheim,
Germany). Digestion with cellulase (from Trichoderma viride,
purchased from Sigma, St. Louis, Mo.) did not reduce the
amount of insoluble material. To obtain muropeptides, peptidoglycan was digested 24 (C. gloeocystis) or 48 (G. nostochinearum) h at 378C with Chalaropsis muramidase (40 mg/ml) in 50
mM potassium phosphate buffer, pH 4.9 (6). For complete
digestion, fresh muramidase was added to G. nostochinearum
peptidoglycan after 24 h of muramidase digestion. Unsolubilized material contained less than 5% of total diaminopimelic
acid (G. nostochinearum) or putrescine (C. gloeocystis) as determined by two-dimensional chromatography on silica-coated
thin-layer plates (Silicagel 60; Merck). For the first dimension,
butanol-acetic acid-water (2/1/1, by volume) was used; for the
second dimension, isopropanol–25% ammonia–methanol (2/
2/1, by volume) was used and was followed by detection with
ninhydrin. Muropeptides were separated from insoluble material by centrifugation and subjected to reduction with NaBH4.
Reduced muropeptides were injected into an octadecylsilane
high-performance liquid chromatography (HPLC) column
(250 by 4 mm) and eluted at room temperature with a linear
gradient from 0 to 20% methanol in 50 mM potassium phosphate buffer (pH 5.1 or 4.65) at a flow rate of 0.5 ml/min as
described previously (12). The gradient was started 7 min after
injection and reached final conditions 157 min later. Isocratic
conditions were kept for another 35 min.
The muropeptide pattern of G. nostochinearum was very
similar to that of C. paradoxa (Fig. 1 and Table 1). All major
peaks could be identified by coelution of the muropeptides
obtained from G. nostochinearum and C. paradoxa at two different pH values of the elution buffer (pH 4.65 and 5.1, respectively). Because of the high sensitivities of retention times
to variations of pH, muropeptides with the same retention time
at different pH values are most likely identical (5, 12). The
identity of G. nostochinearum muropeptides with muropeptides from C. paradoxa was also corroborated by size exclusion
chromatography on Biogel P6 and subsequent examination of
the different fractions by HPLC as previously described (12).
The molecular weight of muropeptide 9, which was expected to
be Tetra(NAP)-Tri, was determined by positive and negative
matrix-assisted laser desorption-ionization mass spectrometry
in the reflector mode as described previously (12). For matrixassisted laser desorption-ionization mass spectrometry sample
preparation, the volume technique (12) with 2,5-dihydroxybenzoic acid was applied. Because of the small amounts obtained
Among eukaryotes, peptidoglycan has been found in cyanelle-containing organisms only. It constitutes part of the envelope of these peculiar plastids and is one of the cyanobacterial features that render cyanelles a living example for an origin
of photosynthetic organelles from endosymbiotic cyanobacteria. The only cyanelle-containing organism studied in detail at
the molecular level is Cyanophora paradoxa, an obligatorily
photoautotrophic protist. The small genome size of C. paradoxa cyanelles defines them as true plastids (8, 11). Because of
its unusual localization in the envelope of the photosynthetic
organelles of a eukaryotic cell, the structure of cyanelle peptidoglycan—which like cyanobacterial peptidoglycan is of the
A1g type (1)—was examined more closely by our group (12,
13). Its distinguishing feature is the amidation of 40 to 60% of
the 1-carboxyl groups of D-glutamic acid with N-acetylputrescine (NAP). To define whether the presence of NAP is a
general feature of the cyanelle wall and possibly related to the
adaptation to the intracellular environment, we examined
other cyanelle-containing algae with respect to this or similar
modifications in cyanelle peptidoglycan. Sufficient quantities
could be obtained only from Glaucocystis nostochinearum and
Cyanoptyche gloeocystis. These two species have been grouped
with C. paradoxa and Gloeochaete wittrockiana under the denomination of glaucocystophytes for morphological reasons
(10). The close relationship of C. paradoxa, G. nostochinearum,
and G. wittrockiana has recently been corroborated by 18S
rRNA-derived phylogenetic analysis (2). Notably, the irregular
wedge-shaped cyanelles of G. nostochinearum differ considerably in morphology from the coccoid cyanelles found in the
other three species. However, in 16S rRNA-derived phylogenetic analysis they clearly group together (7). C. gloeocystis was
not included in that study.
Growth conditions for G. nostochinearum and C. gloeocystis
as well as purification of cyanelle peptidoglycan were as described for C. paradoxa (3, 12). Peptidoglycan isolated from C.
gloeocystis was only moderately contaminated with other sodium dodecyl sulfate (SDS)-insoluble material. In contrast, G.
nostochinearum peptidoglycan was only a minor constituent of
the SDS-insoluble material remaining after digestion with
a-amylase from Bacillus subtilis and pronase E from Strepto* Corresponding author. Mailing address: Institut für Biochemie
und Molekulare Zellbiologie, Biozentrum der Universität Wien, Dr.
Bohrgasse 9, A-1030 Vienna, Austria. Phone: 43-1-79515-5110. Fax:
43-1-7995272. Electronic mail address: [email protected].
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Cyanelle peptidoglycan from the glaucocystophyte algae Glaucocystis nostochinearum and Cyanoptyche gloeocystis was investigated by high-performance liquid chromatography of muropeptides, supported by matrixassisted laser desorption-ionization mass spectrometry. The peptidoglycans of both species are modified with
N-acetylputrescine, as has been demonstrated for cyanelle peptidoglycan of Cyanophora paradoxa.
VOL. 178, 1996
NOTES
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FIG. 2. Positive-ion matrix-assisted laser desorption-ionization mass spectrum of peak 4 from C. gloeocystis (Fig. 1a), the reduced disaccharide-tripeptide modified
with NAP, accumulated from 50 single-shot spectra. [M 1 K]1, potassium adduct.
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FIG. 1. Reverse-phase HPLC at pH 5.1 of reduced cyanelle muropeptides of C. gloeocystis (a), G. nostochinearum (b), and C. paradoxa (c). Peak numbers 1 to 29
refer to C. paradoxa (12) and specify muropeptides of known structure (Table 1). Differences in interpeak distances in the case of some especially pH-sensitive
muropeptides are caused by slight deviations of the actual pH. The rise in baseline at high retention times in chromatogram b is caused by impurity of the G.
nostochinearum muropeptide preparation. Muropeptide pattern and peak areas are not affected, as judged from comparison of variably contaminated preparations.
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NOTES
J. BACTERIOL.
TABLE 1. Comparison of the structural features of peptidoglycan from G. nostochinearum, C. gloeocystis, and C. paradoxa (12)a
C. paradoxa
Feature
Peak
no.b
Monomers
1
2
4
6
Dimers
Trimers
7
12
13
15
22
23
Higher oligomers
28
29
30
31
Cross-linkage
Amidation with NAP
Cyanelle shape
Growth rate
Cyanelle no.
Organization
37.5 6 3.1
16.3 6 2.6
11.6 6 1.7
54.4 6 2.1
17.5 6 2.1
40 6 1.7
—d
6.4 6 1.3
—
—
—
—
61.8 6 0.9
31.7 6 0.5
14.8 6 1.9
NDf
—
—
—
ND
ND
7.6 6 0.4
ND
ND
ND
ND
35.8 6 1.8
85.5 6 2.7
Round
Several
days
Many
Palmelloid
G. nostochinearum
40.9 6 1.7
76.5 6 4.2
4.35 6 1.75
16.1 6 1.4
3.0 6 1.0
58.1 6 7.6
35.1 6 3.6
6.1 6 0.0
10.5 6 1.8
19.8 6 0.2
2.7 6 1.6
6.3 6 0.5
14.8 6 0.6
6.8 6 1.3
11.6 6 1.1
ND
ND
ND
ND
ND
ND
—
—
—
—
33.2 6 1.0
31.5 6 3.0
Exponential
growth
Stationary
growth
12.7 6 1.3
51.0 6 7.1
11.1 6 3.8
13.4 6 0.6
24.2 6 4.0
44.8 6 0.1
20.1 6 0.1
2.2 6 0.6
3.3 6 0.1
17.9 6 1.2
4.6 6 0.1
2.4 6 0.5
16.8 6 0.1
32.7 6 0.4
23.0 6 0.5
ND
ND
ND
ND
ND
ND
19.5 6 1.3
ND
ND
ND
ND
52.4 6 1.7
60 6 6
14.2 6 0.7
68.6 6 5.9
3.5 6 1.7
11.7 6 0.9
15.9 6 3.1
35.0 6 0.8
26.9 6 1.8
2.8 6 0.1
4.5 6 0.3
21.4 6 0.1
5.9 6 0.9
2.2 6 0.1
14.9 6 1.1
21.3 6 0.9
28.0 6 0.4
ND
ND
ND
ND
ND
ND
22.7 6 0.1
ND
ND
ND
ND
53.2 6 0.8
40 6 3
Irregular
24 h
Round
12 h
Round
No growth
Many
Coccoid
1–2
Monadoid
1–2
Monadoid
Proposed structurec
Tri
Tet
TriNAP
TetNAP
Tet-Tri
Tet-Tet
Tet-TriNAPe
TetNAP-Trie
Tet-TetNAPe
TetNAP-Tete
TetNAP-TriNAP
TetNAP-TetNAP
Tet-Tet-Tri
Tet-TetNAP-Trie
TetNAP-Tet-Trie
TetNAP-TetNAP-Trie
TetNAP-TetNAP-TriNAP
TetNAP-TetNAP-TetNAP
TetNAP-TetNAP-TetNAP-TriNAP
TetNAP-TetNAP-TetNAP-TetNAP
TetNAP-TetNAP-TetNAP-TetNAP-TriNAPg
TetNAP-TetNAP-TetNAP-TetNAP-TetNAPg
a
The percentages of the major muropeptide subunits (disaccharide and peptide side chain) participating in the different kinds of muropeptides were calculated from
the peak area percentages (A214) of the muropeptides. Since the individual extinction coefficients are unknown, the approximation developed by Glauner (5) for the
muropeptides of E. coli was used for the conversion of the UV data into molar percentages. For this calculation, it was assumed that the contribution of one NAP
residue equals that of two amide bonds. Data are averages from at least two individually grown cultures. All results are expressed as percentages of the whole murein
(boldface) or as percentages of the molecular weight group under consideration. Cross-linkage and extent of amidation with NAP were calculated from peak areas for
C. gloeocystis and G. nostochinearum.
b
Peak numbers correspond to the numbers given in Fig. 1.
c
Tri, reduced N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-g-D-Glu-m-Dap; Tet, reduced N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-g-D-Glu-m-Dap-D-Ala;
Tet-Tri, reduced N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-g-D-Glu-m-Dap-D-Ala3m-Dap-g-D-Glu-L-Ala-N-acetylmuramyl-N-acetylglucosamine.
d
—, not detected.
e
For this muropeptide, it is not known which of the subunits carries the NAP residue. Its assignment to a particular subunit is based on the assumption that NAP
in C. paradoxa is concentrated on tetrapeptide side chains in cross-linked muropeptides, as it is in muropeptide monomers of this organism.
f
ND, percentage not determined.
g
The structure of this muropeptide is inferred only from its retention behavior.
for these muropeptides, the second HPLC step (13) leading to
the removal of alkali ions was omitted. The results ([M 1 K]1
found: m/z 1,947.0; and [M 2 H]2 found: m/z 1,906.4) were in
good agreement with the molecular weight of the proposed
structure ([M 1 K]1 calculated: m/z 1,946.1, Dm 5 10.9; and
[M 2 H]2 calculated: m/z, 1,905.8, Dm 5 10.6).
The muropeptide pattern of C. gloeocystis was the simplest
of the three glaucocystophytes (Fig. 1 and Table 1). Identification of C. gloeocystis muropeptides by coelution with those of
C. paradoxa at pH 4.65 and 5.1, respectively, showed that in
most muropeptides all D-glutamic acid residues were modified
with NAP. This was confirmed by the determination through
matrix-assisted laser desorption-ionization mass spectrometry
of the molecular weights of muropeptides 4 ([M 1 K]1 found:
m/z 1,022.4) and 14 ([M 2 H]2 found: m/z 2,018.0), corresponding to Tri(NAP) ([M 1 K]1 calculated: m/z 1,022.1,
Dm 5 10.3) (Fig. 2) and Tet(NAP)-Tri(NAP) ([M 2 H]2
calculated: m/z 2,018.1, Dm 5 20.1), respectively. While a
small percentage of muropeptides were completely unmodified, partially modified muropeptides were not present in
amounts high enough to allow their identification by HPLC.
This unusual feature suggests a difference in peptidoglycan
metabolism between C. gloeocystis and the other algae investigated. An enzyme activity cleaving off NAP from peptidoglycan, which is present in C. paradoxa (12), might be missing in
C. gloeocystis. The apparently exclusive cross-linkage of unmodified and modified muropeptides with muropeptides of
their kind might result from high specificity of cross-linking
enzymes or from the existence of NAP-free zones. A comparison of cyanelle shape, growth rate, and peptidoglycan structure of the three investigated species showed no relationship
between these parameters (Table 1).
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3
5
8
9
10
11
16
18
C. gloeocystis
VOL. 178, 1996
NOTES
This work was supported by grants P10860-MOB (to W.L.) and
P11183-CHE (to G.A.) from the Fonds zur Förderung der wissenschaftlichen Forschung. We thank professor L. Kies (Hamburg) for
providing cultures of G. nostochinearum and C. gloeocystis.
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In conclusion, the modification of D-glutamic acid residues
present in cyanelle peptidoglycan with NAP is not a unique
feature of C. paradoxa. The presence of the same modification
in the peptidoglycan of all investigated glaucocystophyte algae
might point to a general function in cyanelle peptidoglycan.
Experiments with isolated sacculi of Escherichia coli and B.
subtilis indicate that globular proteins with molecular masses of
up to 50 kDa might be able to cross the peptidoglycan network
by simple diffusion (4). NAP reduces the polarity of cyanelle
peptidoglycan, and this might facilitate protein diffusion. It
should be emphasized that cyanelles like higher plant chloroplasts continuously have to import around a thousand precursor proteins from the cytoplasm. For both plastid types, the
protein translocation machinery appears to function in an analogous way (9). Our findings are also in accordance with the
phylogenies of glaucocystophytes and their cyanelles based on
rRNA sequences (2, 7) and confirm the affiliation of C. gloeocystis with this algal group, as has been proposed for morphological reasons (10).
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