INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 202-206
0020-7713/86/020202-05$02.OO/O
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36. No. 2
Polar Lipid Profiles of the Genus Deinococcus
T. J. COUNSELL AND R. G. E. MURRAY*
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada
The radiation-resistant, red-pigmented bacteria belonging to the genus Deinococcus appear phenotypically
similar to Micrococcus roseus, but the complex envelope profile and biochemical phylogenetic markers show
them to be gram-negative clones of ancient lineage. All the type strains of indubitable Deinococcus species ( 0 .
radiodurans, D. radiophilus, D . proteolyticus and D. radiopugnans) and all the representative strains of D .
radiodurans have unusual numbers of polar lipids (mostly phosphoglycolipids) and do not have
phosphatidylglycerol (PG) or the phospholipids derived from it. This polar lipid profile is distinctive of the
genus, and the patterns are useful for characterization. The lack of PG or di-PG and their derivatives provides
a reasonably easy assignment to the genus Deinococcus and may allow recognition of relatives within the
Deinococcaceae, which may not be radiation resistant. The species incertae sedis, D . erythromyxa, has a normal
form of phospholipid profile, including PG. This, together with the peptidoglycan type L-Lys-L-Ala3+ forces
exclusion from Deinococcus and reassignment to Micrococcus.
incubator shaker (Psychrotherm, New Brunswick Scientific
Co., Inc., Edison, N.J.) at 30°C and 150 rpm for approximately 48 h. Cells were harvested by centrifugation at 13,000
x g for 15 min at 4°C. Cell pellets were stored at -20°C until
extracted.
(iii) Lipid extraction. Lipids were extracted at 4°C by the
procedure of Bligh and Dyer (6). The procedure allows for
many modifications, but the mixture of chloroform, methanol, and water was kept in the proportions of 1:2:0.8 and
2:2:1.8 before and after dilution, respectively. Cell slurry (5
ml) was mixed with 6.25 ml of chloroform and 12.5 ml of
methanol and was stirred on a magnetic stir plate for at least
6 h. The mixture was then acidified with 0.1 ml of 5 N HC1
(to assist the extraction of some phospholipids; R.
Anderson, personal communication) and left stirring overnight. After filtration through Whatman no. 1filter paper, the
filtrate was collected in conical glass tubes and diluted with
chloroform and water, mixed, and allowed to separate. The
lower phase was removed; the upper phase was re-extracted
with 12.5 ml of chloroform and treated as before. The
combined lower phases represent the lipid extract. This
extract was concentrated under nitrogen and stored in
chloroform-methanol (1:1 [vol/vol]) at -20°C.
(iv) Lipid chromatography. Lipid analysis and identification was performed by one- and two-dimensional thin-layer
chromatography (TLC) on 0.25-mm thick silica gel G-25precoated glass plates (20 cm by 20 cm; Brinkmann Instruments [Canada] Ltd., Rexdale, Ont.) in rectangular glass
tanks.
Two different solvent systems were used for onedimensional chromatography: a chloroform-methanol-water
mixture (65:25:4 [vol/vol]) (solvent 1) and a chloroformacetone-methanol-acetic acid-water mixture (10:4:2:2: 1
[vol/vol]) (solvent 2). For two-dimensional chromatography,
chloroform-methanol-28% ammonia (65:35:5 [vol/vol]) was
used in the first dimension, and solvent 1 was used in the
second dimension.
(v) Lipid detection. Lipids were detected through reactions
with general developing reagents. Lipid detection was carried out by charring after spraying with 25% sulfuric acid in
ethanol, immersion in iodine vapor for several minutes, or
visualization of spots under ultraviolet light after spraying
with an aqueous 0.0012% rhodamine 6G solution (14).
(vi) General lipid analysis. Identity was determined by
mobility and specific chemical detection tests in comparison
Routine identification of Deinococcus species is hindered
by their general unreactivity on substrates. The similarity in
phenotype to the red-pigmented Micrococcus roseus resulted in their classification with Micrococcus (3, 4). This
association, however, was based largely on the clustering of
negative characters.
Although the deinococci are gram positive, they resemble
gram-negative bacteria in biochemical and structural characters (8). The cell envelope of Deinococcus radiodurans
has been characterized in the greatest detail, and it is
complex and unconventional. The peptidoglycan differs in
amino acid composition (Om-Gly2 type) from that of the
micrococci (29); there is an outer membrane (17, 25-27, 30),
and the fatty acid profile is reminiscent of gram-negative
bacteria (8, 10, 13, 15).
The deinococci represent phylogenetically distinct clones
of ancient lineage, based on 16s ribosomal ribonucleic acid
catalog data (8, 23), and might be expected to have some
unique biochemical features. D. radiodurans has been
shown to possess none of the conventional bacterial
phosphatides (phosphatidyl-glycerol [PG], -ethanolamine,
-choline, or -inositol), but does show a number of unidentified polar lipids (21, 24).
We have extended the investigation of the polar lipid
profiles to the remaining Deinococcus species and can now
report on the qualitative examination of polar lipids in these
organisms, including representative strains of D .
radiodurans.
MATERIALS AND METHODS
Organisms, growth conditions and harvesting. (i) Organisms. Cultures used in this investigation were obtained from
the University of Western Ontario (UWO) and the
Czechoslovak (CCM) culture collections (Table 1). For a
review of strain designations and sources, see Brooks et al.
(8).
(ii) Growth conditions. All cultures were grown in the
medium described by Work and Griffiths (30), modified by
the omission of DL-methionhe. The medium consists of
(wthol) 0.5% tryptone, 0.3% yeast extract (both from Difco
Laboratories, Detroit, Mich.), and 0.1% glucose (McArthur
Chem. Co., Montreal). Broth cultures were grown in an
* Corresponding author.
202
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POLAR LIPIDS OF DEZNOCOCCUS
VOL.36, 1986
TABLE 1. Strains used
Species
Culture collection and no."
D.radiodurans ....................
.UWO 288T, UWO 298, UWO
395, UWO 1038, UWO
1048, UWO 1051, UWO
1054, UWO 1071, UWO
1072, UWO 1073
D.radiopugnans. ...................UWO 293T
D.radiophilus .....................
.UWO 10ST
D.proteolyticus ....................UWO
D.erythromyxa.. ...................UWO 706
E. coli strain B
....................
.UWO 301
" UWO, University of Western Ontario culture collection (London,
Ontario, Canada); CCM, Czechoslovak Collection of Microorganisms (Brno,
Czechoslovakia).
with authentic lipid standards. Cardiolipin, PG, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylinositol (PI) (all from Serdary
Research Laboratories, London, Ont.) were used as standards. Lipid properties were determined by spraying with
the following reagents (14): 0.25% ninhydrin in acetone for
amino lipids; 0.5% a-naphthol in methanol and water (1:l
[vol/vol]) followed by spraying with 95% sulfuric acid for
glycolipids; 0.9% ferric chloride and 0.55% orcinol in acidified ethanol (28) (Bial's reagent; Sigma Chemical Co., St.
Louis, Mo.) for glycolipids; molybdenum blue reagent
(Sigma) for lipid phosphate groups, and the Dragendoa
stain for choline-containing lipids (5, 28).
FIG. 1. One-dimensional TLC of whole-cell lipid extracts from
D . radiodurans strains developed in solvent 1 and acid charred.
Lanes: 1, UWO 288; 2, UWO 395; 3, UWO 1038; 4, UWO 1048; 5,
UWO 1051; 6, UWO 1054; 7, UWO, 1071; 8, UWO 1072; 9, UWO
1073. Abbreviations: 0, origin; NL, neutral lipids; F, solvent front.
4, Phosphoglycolipid doublet.
203
RESULTS
Figures 1, 2, and 3 show the lipid profiles seen on
one-dimensional TLC; Fig. 4 and 5 summarize the corresponding reactions of the lipids. Not all lipids gave reactions
with one or more of the reagents used, and only the clearly
distinguishable reactions are shown.
No spots corresponding to the mobility and chemical
behavior of the common bacterial polar lipids were found in
the extracts of D . radiopugnans, D . radiodurans, D .
radiophilus, or D . proteolyticus. D . erythrornyxa showed a
lipid with the mobility and reactions of PG, but this comigrated with a fraction showing an anomalous ninhydrin reaction; the profile in this case was resolved by using twodimensional TLC both alone and mixed with an equal
amount of a lipid extract from Escherichia coli (Fig. 6a
through c).
There were lipids in the Deinococcus profiles which had Rf
values near the standard phospholipids (Fig. 2 and 3). None
of these lipids, however, showed the necessary Rf, and
qualitative chemistry was needed to confirm that they were
identical with any of the lipid standards.
DISCUSSION
Markers that are desirable for identification are seldom the
same as those best suited for classification. However, in the
case of the Deinococcus species, their general unreactivity
makes generic identification dependent upon such traits as
radiation survival, peptidoglycan typing, and cell envelope
profiles as seen by electron microscopy of a thin section (7,
8). A stable marker for both group and family identification
and classification is needed.
Lipid profiles are not yet used routinely in bacterial
FIG. 2. One-dimensional TLC of lipid extracts developed in
solvent 1 and acid charred. Lanes: 1, lipid standards; 2, M . roseus;
3, D. radiopugnans; 4, D. radiodurans; 5, D . radiophilus; 6, D.
proteolyticus; 7, D . erythromyxa: Abbreviations are as defined in
the legend to Fig. 1. PE, Phosphatidylethanolamine; CDL,
cardiolipin; PC, phosphatidylcholine; PI, phosphatidylenositol; PS,
phosphatidylserine.
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204
INT. J .
COUNSELL AND MURRAY
FIG. 3. One-dimensional TLC of lipid extracts developed in
solvent 2 and acid charred. Lanes: 1, lipid standards; 2, M . roseus;
3 , D . radiopugnans; 4, D . radiodurans; 5 , D . radiophilus; 6 , D .
proteolyticus; 7 , D . erythromyxa. Abbreviations are as defined in
the legend to Fig. 1 and the legend to Fig. 2.
taxonomy, but extensive studies have shown their potential
as taxonomic markers, particularly when, as in this case, we
are dealing with the absence of conventional and the presence of unconventional lipids (18). We have applied the
FIG. 4. Schematic diagram of Fig. 2. Reactions to detection
sprays are indicated by the following signs: 0 , positive for anaphthol and Bial’s reagent; I ~ I ninhydrin
,
positive; @, phosphate
positive.
SYST.
BACTERIOL.
FIG. 5. Schematic diagram of Fig. 3. Reactions to detection
sprays are indicated by: 0 , positive for a-naphthol and Bial’s
reagent; :I], ninhydrin positive, 6 ,phosphate positive.
simplest possible method as an approach to determinatim.
The identity of the lipids is for others to study (2).
The taxonomic utility of these polar lipid profiles requires
reasonable uniformity within a species. This requirement is
somewhat empirical, but for the D . radiodurans strains (nine
representatives are shown in Fig. l),the profile was recognizably conserved. In examining the profile, for overall
pattern iq D. radiodurans we observed that the most consistently conserved and readily identifiable feature was the
major phosphoglycolipid doublet (Fig. 1).The lower lipid in
this doublet migrates with an Rf similar to PG but is
distinguished by its carbohydrate-positive reaction.
The absence of conventional polar lipids in D. radiodurans
(21, 24) required that comparison with extracts from the
remaining members of the genus be made. We found that the
type strains of D . radiopugnans, D . radiophilus, and D .
proteolyticus (see Fig. 2 through 5 ) followed a similar
principle, exhibiting a large number of strange phospholipids
and lacking PG. It appears that the lack of PG is a unique
property of the genus. Not enough strains were available to
test consistency within these species.
D. erythromyxa, however, appeared to contain PG, but
the spot gave an anomalous ninhydrin reaction, which was
caused by the comigration of a component that interfered
with the identification of PG. Furthermore, the peptidoglycan type of D. erythromyxa has already been determined as
L-Lys-L-Ala3-4,corresponding to that of Micrococcus and
distinct from Deinococcus (7). These results show that D.
erythromyxa should be excluded from Deinococcus and
returned incertae sedis to Micrococcus pending further
taxonomic study.
One of the D. radiodurans phosphoglycolipids (lipid 7 in
reference 25), characterized by Anderson (1) as a novel
bacterial alkylamine, has been identified by Anderson and
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VOL.36, 1986
POLAR LIPIDS OF DEINOCOCCUS
205
than radiation resistance are necessary for assignment to the
group. The polar lipid profile along with the presence of
ornithine in the peptidoglycan of the wall fits that need.
Cocci with suggestive properties, including moderate radiation resistance, have been isolated from extreme, dry
environments exposed to a considerable flux of solar ultraviolet radiation. For example, strains from the “desert
varnish” found on rocks in the Mojave desert proved to have
a wall structure like Micrococcus and polar lipids of normal
bacterial type (20). On the other hand, one of four such
strains isolated from specimens of weathered granite collected in the Antarctic dry valleys (kindly provided by P.
Hirsch, University of Kiel) proved to be D . radiopugnuns on
the basis of phenotype, ultraviolet resistance, and cell-wall
and polar-lipid profiles (unpublished data).
It is possible that these unusual polar lipids may confer
properties on the membranes of Deinococcus species thatare important to survival after exposure to extremes of
radiation and dessication, but experimental approaches to
these questions have not yet been designed. Further study of
the unusual polar lipid should facilitate identification, allow
recognition of familial relatives, and stimulate efforts to
discover the natural environment of these bacteria.
ACKNOWLEDGMENTS
We are indebted for cultures and information to J. T. Staley,
Department of Microbiology and Immunology, University of
Washington, Seattle and P. Hirsch, Institut fur Algemeine
Mikrobiologie, Universitat Kiel, Federal Republic of Germany. The
expertise provided by R. Anderson, University of Calgary, Alberta,
and the technical assistance at the University of Western Ontario of
D. Moyles is greatly appreciated.
We acknowledge the continuing support of our research by the
Medical Research Council of Canada.
LITERATURE CITED
1. Anderson, R. 1983. Alkylamines: novel lipid constituents in
Deinococcus radiodurans. Biochim. Biophys, Acta 753:26&
268.
2. Anderson, R., and K. Hansen. 1985. Structure of a novel
phosphoglycolipid from Deinococcus radiodurans. J. Biol.
Chem. 260:12219-12223.
FIG. 6. Two-dimensional TLC of whole-cell lipid extracts from
3. Baird-Parker, A. C. 1963. A classification of micrococci and
E. coli (a), D. erythromyxa (b), and a mixture of lipid extracts from
staphylococci based on physiological and biochemical tests. J.
E. coli and D . erythromyxa (c). 1, First dimension. Solvent used was
Gen. Microbiol. 30:409-427.
chloroform-methanol-ammonia (65:35:5 [vol/vol]). 2, Second dimen4. Baird-Parker, A. C. 1965. The classification of staphylococci
sion. Solvent 1 was used.
and micrococci from world-wide sources. J. Gen. Microbiol.
38:363-387.
5. Beiss, U. 1964. 2ur Papierchromatographischen auftrennung
von Pflanzenlipiden. J. Chromatogr. 13:104-110.
Hansen (2) as 2’-0-(1,2-diacyl-sn-glycero-3-phospho)-3’-06. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid
(galactosy1)-N-D-glyceroylalkylamine. It seems likely that
extraction and purification. Can. J. Biochem. Physiol.
other polar lipid components from Deinococcus species will
37:911-917.
be equally unusual, but their identification will take time and
7 . Brooks, B. W., and R. G. E. Murray. 1981. Nomenclature for
expert analysis.
“Micrococcus radiodurans” and other radiation-resistant
The natural habitat of the members of the genus
cocci: Deinococcaceae fam. nov. and Deinococcus gen. nov.,
Deinococcus is unknown, largely because recognition is not
including five species. Int. J. Syst. Bacteriol. 31:353-360.
easy. They have been isolated a number of times from
8 . Brooks, B. W., R. G. E. Murray, J . L. Johnson, E.
Stackebrandt, C. R. Woese, and G. E. Fox. 1980. Red pigmented
diverse environments (9, 11, 12, 16) usually selected and
Micrococci: a basis for taxonomy. Int. J. Syst. Bacteriol.
characterized by survival after high-dose irradiation. How30:627-646.
ever, radiation resistance may not be completely reliable as
9. Christensen, E. A., and H. Kristensen. 1981. Radiationan indicator for Deinococcaceae because Moseley (19)
resistance of micro-organisms from air in clean premises. Acta
showed it to be a mutable character. An interesting addiPathol. Microbiol. Scand. Sect. B 89:293-301.
tional trait of these bacteria is their apparent extreme resist10. Girard, A. E. 1971. A comparative study of the fatty acids of
ance to desiccation (22; unpublished data; R. B. Maxcy,
some micrococci. Can. J. Microbiol. 17:1503-1508.
personal communication). Whether or not this ability is
11. Ito, H. 1977. Isolation of Micrococcus radiodurans occurring in
important to their continued survival and selection for reradurized sawdust culture media of mushroom. Agric. Biol.
Chem. 41:35-41.
sistance to radiation is unknown. Presumptive tests other
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:05:51
206
COUNSELL AND MURRAY
INT. J.
12. Ito, H., H. Watanabe, M. Takehisa, and H. Iizuka. 1983.
Isolation and identification of radiation-resistant cocci belonging
to the genus Deinococcus from sewage sludges and animal
feeds. Agric. Biol. Chem. 47:1239-1247.
13. Jantzen, E., T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. VI. Fatty acid
composition of strains within Micrococcuceue. Acta Pathol.
Microbiol. Scand. 82:785-798.
14. Kates, M. 1972. Techniques of lipidology, p. 436-477. In T. S .
Work and E. Work (ed.), Laboratory techniques in biochemistry and molecular biology. North-Holland Publishing Co., Amsterdam.
15. Knivett, V. A., J. Cullen, and M. J. Jackson. 1935. Odd
numbered fatty acids in Micrococcus rudioduruns. Biochem. J.
9ik2c-3~.
16. Kristensen, H., and E. A. Christensen. 1981. Radiation-resistant
microorganisms isolated from textiles. Acta Pathol. Microbiol.
Scand. Sect. B 89:303-309.
17. Lancy, P., Jr., and R. G. E. Murray. 1978. The envelope of
Micrococcus rudioduruns: isolation, purification and preliminary analysis of the wall layers. Can. J. Microbiol. 24162-176.
18. Lechevalier, M. P. 1977. Lipids in bacterial taxonomy-a taxonomist's view. Crit. Rev. Microbiol. 5109-210.
19. Moseley, B. E. B. 1976. The isolation and some properties of
radiation-sensitive mutants of Micrococcus rudioduruns. J.
Gen. Microbiol. 49:293-300.
20. Palmer, F. E., J, T. Staley, R. G. E. Murray, T. Counsell, and
J. B. Adams. 1985. Identification of manganese oxidizing bacteria from desert varnish. Geomicrobiology, in press.
SYST.
BACTERIOL.
21. Rebeyrotte, N., P. Rebeyrotte, M. J. Maviel, and D. Montaudon.
1979. Lipides et lipopolyosides de Micrococcus rudioduruns.
Ann. Microbiol. (Paris) 130:407-411.
22. Sanders, S. W., and R. B. Maxcy. 1979. Isolation of radiationresistant bacteria without exposure to irradiation. Appl.
Environ. Microbiol. 38:436439.
23. Stackebrandt, E., and C. R. Woese. 1979. A phylogenetic
dissection of the family Micrococcaceue. Curr. Microbiol.
2:3 17-322.
24. Thompson, B. G., R. Anderson, and R. G. E. Murray. 1980.
Unusual polar lipids of Micrococcus rudiodurans strain Sark.
Can. J. Microbiol. 26:1408-1411.
25. Thompson, B. G., and R. G. E. Murray. 1981. Isolation and
characterization of the plasma membrane and the outer membrane of Deinococcus rudiodurans strain Sark. Can. J. Microbiol. 27:729-734.
26. Thompson, B. G., R. G. E. Murray, and J. F. Boyce. 1982. The
association of the surface array and the outer membrane of
Deinococcus rudf'oduruns.Can. J. Microbiol. 28:1081-1088.
27. Thornley, M. J., R. W. Horne, and A. M. Glauert. 1965. The fine
structure of Micrococcus rudioduruns. Arch. Mikrobiol.
51:267-289.
28. Wagner, H., L. Horhammer, and P. Wolff. 1961. Dunnschichtchromatographie von phosphatiden und glycolipiden. Biochem.
Z. 3M175-184.
29. Work, E. 1964. Amino acids of walls of Micrococcus
rudioduruns. Nature (London) 201:1107-1109.
30. Work, E., and H. Griffiths. 1968. Morphology and chemistry of
cell walls of Micrococcus rudioduruns. J. Bacteriol. 95:641-657.
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