Potato suberin induces differentiation and secondary metabolism in the genus Streptomyces.
ABSTRACT Bacteria of the genus Streptomyces are soil microorganisms with a saprophytic life cycle. Previous studies have revealed that the phytopathogenic agent S. scabiei undergoes metabolic and morphological modifications in the presence of suberin, a complex plant polymer. This paper investigates morphological changes induced by the presence of potato suberin in five species of the genus Streptomyces, with emphasis on S. scabiei. Streptomyces scabiei, S. acidiscabies, S. avermitilis, S. coelicolor and S. melanosporofaciens were grown both in the presence and absence of suberin. In all species tested, the presence of the plant polymer induced the production of aerial hyphae and enhanced resistance to mechanical lysis. The presence of suberin in liquid minimal medium also induced the synthesis of typical secondary metabolites in S. scabiei and S. acidiscabies (thaxtomin A), S. coelicolor (actinorhodin) and S. melanosporofaciens (geldanamycin). In S. scabiei, the presence of suberin modified the fatty acid composition of the bacterial membrane, which translated into higher membrane fluidity. Moreover, suberin also induced thickening of the bacterial cell wall. The present data indicate that suberin hastens cellular differentiation and triggers the onset of secondary metabolism in the genus Streptomyces.
- SourceAvailable from: sciencedirect.com[show abstract] [hide abstract]
ABSTRACT: The inhibition of glycerol 3-phosphate oxidation by oleic acid correlates with changes in membrane microviscosity monitored by the steady-state fluorescence anisotropy of DPH. The dynamic measurements indicate that the changes of both the limiting anisotropy and rotational relaxation time occur in a concentration range where the enzyme activity is strongly inhibited.FEBS Letters 10/1986; 206(1):1-3. · 3.58 Impact Factor
Article: Demystifying suberin[show abstract] [hide abstract]
ABSTRACT: Suberin is a term used to define a specific cell wall component that occurs, for example, in phellem (cork) endodermal and exodermal cells and is characterized by the deposition of both poly(phenolic) and poly(aliphatic) domains. Historically, the poly(phenolic) domain has been likened to lignin, and while there is an element of truth to this comparison, recent evidence supports an alternative view in which the poly(phenolic) domain contains a significant amount of nonlignin precursors (principally hydroxycinnamic acids and their derivatives) that are covalently linked to each other in a manner analogous to the monolignols in lignin. Similarly, the conceptual model in which the poly(aliphatic) domain of suberized tissues is represented as a random network of polyesterified, modified fatty acids and alcohols has been replaced with one comprising a three-dimensional, glycerol-bridged network. Taken together, a new model for suberin is emerging in which a hydroxycinnamic acid monolignol poly(phenolic) domain, embedded in the primary cell wall, is covalently linked to a glycerol-based poly(aliphatic) domain located between the primary cell wall and the plasma membrane. The structural and biochemical evidence supporting this new suberin paradigm are examined in this minireview, along with the presentation of a new structural model encompassing a current view of the structure of suberin.Key words: suberin, lignin, hydroxycinnamic acid, monolignol, poly(aliphatic) domain, poly(phenolic) domain, glycerol polyester.Canadian Journal of Botany 02/2011; 80(3):227-240. · 1.40 Impact Factor
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ABSTRACT: The biocontrol agent Streptomyces melanosporofaciens strain EF-76 produces geldanamycin, an ansamycin antibiotic which displays antagonistic activity towards several Gram-positive bacteria and fungi. Biopolymers [chitin, carboxymethylcellulose (CMC) and xylan] or N-acetylglucosamine were added to S. melanosporofaciens culture media and the effect of these additional carbon sources on geldanamycin production was analysed. Geldanamycin biosynthesis was only stimulated in the presence of 1 g L of chitin. Higher concentrations of chitin, as well as the presence of CMC or xylan, did not improve antibiotic production. N-acetylglucosamine, the final degradation product of chitin, did not stimulate geldanamycin production at all concentrations tested. Nevertheless, the addition of chitin in culture media had no or a negative impact on the antagonistic activity of EF-76 towards the pathogen Streptomyces scabies. Furthermore, the effect of chitin, CMC and xylan on the biocontrol ability of EF-76 against S. scabies was tested on radish seedlings. None of the polymers affected the biocontrol efficiency of EF-76 under gnotobiotic conditions. However, the addition of CMC or xylan to plant growth medium protected radish seedlings against S. scabies infection.Canadian Journal of Plant Pathology 10/2010; 32(4):481-489. · 1.12 Impact Factor
Microbes Environ. Vol. 27, No. 1, 36–42, 2012
Potato Suberin Induces Differentiation and Secondary Metabolism in the
SYLVAIN LERAT1, MARTIN FOREST1, ANNIE LAUZIER1, GILLES GRONDIN1, SERGE LACELLE2, and CAROLE BEAULIEU1*
1Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec J1K 2R1, Canada; and
2Département de Chimie Université de Sherbrooke, Sherbrooke, Québec J1K 2R1, Canada
(Received May 21, 2011—Accepted August 23, 2011—Published online December 1, 2011)
Bacteria of the genus Streptomyces are soil microorganisms with a saprophytic life cycle. Previous studies have
revealed that the phytopathogenic agent S. scabiei undergoes metabolic and morphological modifications in the presence
of suberin, a complex plant polymer. This paper investigates morphological changes induced by the presence of potato
suberin in five species of the genus Streptomyces, with emphasis on S. scabiei. Streptomyces scabiei, S. acidiscabies,
S. avermitilis, S. coelicolor and S. melanosporofaciens were grown both in the presence and absence of suberin. In
all species tested, the presence of the plant polymer induced the production of aerial hyphae and enhanced resistance
to mechanical lysis. The presence of suberin in liquid minimal medium also induced the synthesis of typical secondary
metabolites in S. scabiei and S. acidiscabies (thaxtomin A), S. coelicolor (actinorhodin) and S. melanosporofaciens
(geldanamycin). In S. scabiei, the presence of suberin modified the fatty acid composition of the bacterial membrane,
which translated into higher membrane fluidity. Moreover, suberin also induced thickening of the bacterial cell wall.
The present data indicate that suberin hastens cellular differentiation and triggers the onset of secondary metabolism
in the genus Streptomyces.
Key words: cell wall, common scab, membrane, secondary metabolites, Streptomyces scabiei.
Suberin is among the most recalcitrant plant molecular
structures in soils (31). Suberin forms a protective barrier in
tissues such as woody stems, roots and underground storage
organs which undergo secondary growth (10). This barrier
controls the flux of water and also protects plant tissues
against biotic diseases (29). Suberin is a biopolymer com-
posed of polyaromatic and polyaliphatic domains linked by
glycerol moieties (3). Microbial degradation of suberin is a
process that is poorly characterized. Suberinases are poly-
esterases that can depolymerize, at least partially, the lipidic
polymer (16). Suberinases have been shown to be produced
by some fungi belonging to the following genera: Armillaria,
Aspergillus, Coprinopsis and Fusarium (16). There is also
evidence that some actinomycetes produce suberin-degrading
esterases. Esterase activity is induced in Thermoactinomyces
vulgaris (10) and the plant pathogen Streptomyces scabiei
(32) in the presence of suberin.
The genus Streptomyces belongs to the order of Actino-
mycetales, a division of Gram-positive bacteria that are
characterized by a genome with a high G+C content. Their
complex life cycle includes soil colonization by mycelial
growth and terminates with sporulation. Streptomyces are
known for producing a wide variety of biologically active
secondary metabolites such as antibiotics; however, among
the numerous species of the genus Streptomyces, a few have
developed phytopathogenic traits, mainly relying on their
ability to produce plant toxic secondary metabolites called
thaxtomins (2). Streptomyces scabiei is the main causal agent
of common scab, a severe disease which affects potato tubers
and tap root crops (18).
The cellular response of S. scabiei exposed to suberin was
investigated using a proteomic differential display technique.
It was revealed that its presence up-regulated proteins related
to the stress response, glycolysis, morphological differentia-
tion and secondary metabolism (17). The effect of suberin
on differentiation was corroborated by cultivating S. scabiei
in the presence or absence of suberin (19). Suberin strongly
stimulates aerial mycelium development in S. scabiei which,
in the presence of cellobiose, can lead to the production of
the secondary metabolites, thaxtomins (19).
Suberin is not the only biopolymer that influences
differentiation and secondary metabolism in Streptomyces.
For instance, chitin, the main polymer of insect cuticles
and crustacean shells, modulates antibiotic biosynthesis and
development in Streptomyces coelicolor (33). Since soil-
dwelling streptomycetes can hydrolyse complex natural
biopolymers, it was suggested that these polymers play a
determinant role in the production of their bioactive molecules
In the present paper, suberin is shown to affect development
and secondary metabolite biosynthesis not only in S. scabiei,
but also in the plant pathogen S. acidiscabies, as well as in
saprophytic species such as S. avermitilis, S. coelicolor and
S. melanosporofaciens. The molecular mechanisms respon-
sible for the stimulation of differentiation are still unknown,
but we demonstrate that suberin acts as a membrane and cell-
wall perturbant in streptomycetes.
Materials and Methods
Suberin purification, bacterial cultivation and growth conditions
Suberin was obtained from potato peel (Solanum tuberosum
* Corresponding author. E-mail: email@example.com;
Tel: +1–819–821–8000; Fax: +1–819–821–8049.
Streptomyces Differentiation by Suberin37
‘Russet’) and purified (15). Briefly, potato tubers were sliced and
boiled for 20 min. The skin (suberin) was removed and flesh was
roughly scraped away. The peel was then rinsed with tap water and
residual flesh was digested overnight with cellulase (5 g L−1) and
pectinase (1 g L−1) in 50 mM acetate buffer (pH 4.0). The peel was
rinsed again with chloroform:methanol (2:1) and suberin purification
was achieved using a Soxhlet extractor with chloroform as a solvent.
Finally, suberin was dried and ground for 15 s in a coffee blender.
Streptomyces scabiei strain EF-35 (HER1481) was initially
isolated in Quebec (Canada) from scabby potato tubers (9) and was
used in all assays. Strains S. acidiscabies ATCC 49003, S. avermitilis
ATCC 31267, S. coelicolor M145 (ATCC BAA-471) and S.
melanosporofaciens EF-76 (ATCC BAA-668) were also used in the
morphology and mechanical lysis experiments. Unless otherwise
specified, bacteria were cultivated in liquid medium as follows. 107
to 108 spores were inoculated in 50 mL tryptic soy broth (TSB) and
grown in a rotary shaker (250 rpm) at 30°C for 48 h. Bacteria were
then centrifuged (10 min at 3,450×g) and resuspended in 5 volumes
of sterile 0.85% NaCl. Volumes of 5 mL of this suspension were
used to inoculate flasks containing 200 mL minimal medium (0.5
g L−1 asparagine, 0.5 g L−1 K2HPO4, 0.2 g L−1 MgSO4 and 5 mg
L−1 FeSO4–7H2O) supplemented with 1% (w/v) soluble starch and
0% (control medium: CM) or 0.1% (w/v) suberin (suberin medium:
SM). In order to collect suberin-free bacterial samples at the end
of the experiment, suberin was placed in ca. 4×4 cm cotton pouches
(200 mg suberin per pouch). Control flasks contained empty cotton
pouches. Bacteria were grown at 30°C with shaking (250 rpm).
Morphology of bacterial colonies on solid medium
Morphology of the five Streptomyces species tested in this study
was determined as previously described (19). Briefly, 30–50 viable
spores from each species were streaked on Petri dishes containing
solidified (15 g L−1 agar) CM and SM. Petri dishes were incubated
at 30°C for 5 d and representative colonies of each species and each
treatment were photographed.
Production of secondary metabolites
The production of characteristic secondary metabolites, thaxtomin
A for S. scabiei and S. acidiscabies, actinorhodin for S. coelicolor
and geldanamycin for S. melanosporofaciens, was assessed. These
four strains were grown in CM and SM; for the growth of S. scabiei
and S. acidiscabies, a starch/cellobiose combination (0.5% each)
(19) was used instead of 1% starch since cellobiose is required for
the production of thaxtomin A (12, 13). After 4 d, bacterial cultures
were centrifuged (10 min, 3,450×g) and supernatants were decanted
for quantification of metabolites. Pellets were dried (24 h at 50°C)
and weighed to determine bacterial growth.
Thaxtomin A produced by S. scabiei and S. acidiscabies was
purified as previously described (11) and quantified by HPLC
Agilent 1260 Series (Agilent Technologies, Santa Clara, CA, USA)
at 249 nm using a Zorbax SB-C18 column (Agilent Technologies).
Abamectin (B1a) was extracted with ethyl acetate and quantified by
HPLC at 246 nm (20). γ-Actinorhodin produced by S. coelicolor
was quantified by spectrophotometry according to Kieser et al. (14).
Geldanamycin produced by S. melanosporofaciens was purified by
chloroform extraction and quantified by HPLC at 306 nm (4). This
experiment was carried out in triplicate.
Cell wall morphology of Streptomyces scabiei
The cell wall morphology of S. scabiei grown in the absence or
presence of suberin was determined after 7 d of growth. Preparation
of bacterial cells, the microscopy procedure and image analyses
were carried out according to Miguélez et al. (25). Samples were
examined with a Philips EM201 (FEI Company, Hillsboro, OR,
USA) electron microscope at 60 kV and photographed on an Eastman
Fine Grain Positive film 5302 (Eastman Kodak, Rochester, NY,
USA). High-contrast photographic negatives were digitized with a
HP Scanjet 6300C slide scanner and images were analyzed using
the Image-Pro Plus v.4.5 software (Media Cybernetics, Elizabeth,
IN, USA). Bacteria sliced in the middle of the cell, i.e., with a well-
defined cell wall and a clearly visible DNA zone in the centre, were
selected for analysis. Pixel intensity from zones randomly selected
in cell walls (40 zones per treatment) was measured. Cell wall
thickness was also determined (25 walls per treatment).
Bacterial resistance to mechanical lysis
The five Streptomyces strains were grown for 7 d in liquid CM
and SM (using cotton pouches) containing 2% starch. Bacteria were
collected by centrifugation, rinsed with 10 mM Tris-HCl (pH 8.3)
and centrifuged again. Supernatants were thoroughly discarded and
300 mg bacteria (fresh weight) were resuspended in 1 mL Tris-HCl
buffer. Resistance to mechanical stress was assessed using 0.5 mL
of this suspension with 250 mg glass beads (100 μm diameter) using
a bead beater (FastPrep FP-120; Thermo Fisher Scientific, Waltham,
MA, USA) for 45 s (speed 4.5 m s−1). Samples were chilled on ice,
centrifuged and supernatants were filtered. Lysis efficiency was
evaluated by quantifying protein concentration of supernatants using
Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA).
Measurement of bacterial membrane fluidity of Streptomyces
Membrane fluidity of S. scabiei EF-35 bacteria collected from
control and suberin treatments was determined. After 1 d of growth
in CM and SM, bacteria were centrifuged, washed with 0.85% NaCl
and resuspended in 0.85% NaCl to a concentration of 12.5 g L−1
bacteria. Membrane fluidity was assessed by an anisotropy test,
based on the incorporation of 1,6-diphenyl-1,3,5-hexatriene (DPH)
into bacterial membranes. The method described by Shinitzky and
Barenholz (35) was used with minor modifications. Twenty
microliters of 1 mM DPH (prepared in acetone and kept in the dark)
(1) was added to 10 mL bacterial suspension to obtain a final DPH
concentration of 2 μM (30). Suspensions were incubated in the dark
for 2 h at room temperature with mild shaking. Bacteria were then
washed with one volume of 0.85% NaCl, resuspended with exactly
10 mL of 0.85% NaCl and kept on ice. Anisotropy tests were
performed using a spectrofluorimetry system equipped with PTI
polarizers. Fluorescence of the DPH probe was measured with an
excitation wavelength of 355 nm and an emission wavelength of
425 nm (22), from 10°C to 40°C by 5°C increments. Data were
analyzed with Felix 32 v.1.1 software (Photon Technology Interna-
tional, London, ON, Canada).
In another experiment, membrane fluidity of S. scabiei EF-35
was determined over a 4-d period. Bacteria were grown in CM and
SM (200 mL, four replicates per treatment) and 20 mL bacterial
suspension was collected every day. Membrane fluidity was readily
measured as described above at a temperature of 25°C.
Determination of bacterial membrane fatty acid composition of
Membrane fatty acid composition of S. scabiei EF-35 bacteria
grown in CM and SM was examined. Bacteria were pelleted by
centrifugation, washed with 0.85% NaCl and lyophilized. Bacterial
membrane fatty acids were extracted and methylated as in Moss
(27), with modifications. Saponification was performed on 150 mg
dried cells with 1 mL of 15% NaOH in 50% ethanol. Suspensions
were incubated for 30 min at 100°C; samples were then cooled and
brought to pH 2.0 with 6N HCl. Methylation of fatty acids was
achieved after adding 3 mL of 14% BF3 to methanol and by
incubating at 80–85°C for 20 min. After cooling, methylated fatty
acids were extracted twice with one volume of petroleum ether/
hexane (1:1) and evaporated to 1 mL with a flow of N2. Extracts
were then washed with 0.3 N NaOH solution (26). The organic
phase was transferred to a new tube and the solvent was completely
evaporated by N2 flow. Residual H2O was eliminated by the addition
of 80–100 mg Na2SO4. Tubes were stored under N2 at −20°C until
analysis. Dried fatty acids were then dissolved in 100 μL hexane
and separated using a gas chromatograph HP6890 (Hewlett-Packard,
Mississauga, ON) equipped with a capillary column RTX-1 of 30 m
LERAT et al.38
× 250 μm × 0.25 μm (Restek, Bellefonte, PA, USA). Fatty acids
were identified by comparing with the commercial standard mixes
Bacterial Acid Methyl Esters Mix (Matreya, Pleasant Gap, PA,
USA) and Supelco 37-component FAME Mix (Sigma, St-Louis,
Suberin promotes secondary growth and production of
secondary metabolites in streptomycetes
The composition of growth media noticeably influenced
the morphology of the five Streptomyces strains tested in this
experiment (Fig. 1). As previously described (19), moderately
hairy colonies were observed when S. scabiei EF-35 was
grown on starch medium while suberin triggered the onset
of secondary metabolism (formation of hairy colonies).
Suberin also strongly stimulated the production of aerial
hyphae in S. acidiscabies ATCC 49003, S. coelicolor M145
and S. melanosporofaciens EF-76 when compared to minimal
starch medium (CM). In S. avermitilis ATCC 31267, suberin
only moderately stimulated aerial growth, while suberin-
deprived colonies were bald (Fig. 1).
In liquid minimal medium, the presence of suberin signif-
icantly stimulated the growth of all Streptomyces spp. tested
(Table 1). Furthermore, the production of secondary metab-
olites typically synthesized by these strains was strongly
promoted by the presence of the plant polymer in S. scabiei,
S. acidiscabies, S. coelicolor and S. melanosporofaciens.
In the absence of suberin, neither thaxtomin A nor
geldanamycin was detected in media inoculated with S.
scabiei and S. melanosporofaciens, respectively, while metab-
olite production was significantly limited in flasks inoculated
with S. acidiscabies and S. coelicolor (Table 1). Substantial
production of unidentified secondary metabolites was also
detected from HPLC chromatograms of S. acidiscabies
and S. melanosporofaciens grown in the presence of suberin
(Fig. S1). Abamectin production by S. avermitilis could not
be detected in the presence or absence of suberin; however,
three-dimensional HPLC profiles showed a strong increase
in the production of various unidentified molecules in the
presence of suberin (Fig. S1).
Suberin alters cell wall morphology
The presence of suberin in growth medium induced
morphological changes in S. scabiei EF-35. These modifica-
tions were clearly visible by electron microscopy after 7 d
of growth. The cell walls of bacteria that had been exposed
to suberin contained a high quantity of electron-dense material
(Fig. 2). Cell-wall density of bacteria grown in the presence
of suberin (90.5±6.7 pixels) thus appeared significantly higher
than cell-wall density of control bacteria (75.2±6.9 pixels;
P<0.0001, t-test). Image analyses also revealed that cell walls
were thicker in suberin-treated bacteria (46.6±8.8 nm) than
in control bacteria (36.6±6.7 nm; P<0.0001, t-test).
Bacteria grown in the presence of suberin showed higher
resistance to mechanical lysis
Protein content of supernatants obtained after mechanical
lysis of bacterial suspensions was significantly higher for
bacteria grown for 7 d in CM than in SM, in all Streptomyces
strains tested (Table 2).
An additional experiment was consequently conducted
to assess resistance to mechanical lysis over a 7-d period. S.
scabiei was grown in CM and SM and resistance to
mechanical lysis was measured every day, as described above.
The amount of protein released by mechanical treatment was
Typical morphology of isolated colonies of Streptomyces
scabiei EF-35, S. acidiscabies ATTC 49003, S. avermitilis ATTC
31267, S. coelicolor M145 and S. melanosporofaciens EF-76 after 5 d
of growth on solid minimal starch (1%) medium, complemented or not
with 0.1% suberin.
Streptomyces Differentiation by Suberin39
similar for both experimental conditions after incubation
periods of 1 and 2 d (Fig. 3); however, from day 3 to day 7,
resistance to mechanical lysis was significantly higher (i.e.,
protein concentration was lower) in bacteria grown in the
presence of suberin (Fig. 3).
Membrane fluidity and fatty acid composition
Anisotropy measurements performed with the DPH probe,
1 d after inoculation in minimal medium, revealed that
membrane fluidity of S. scabiei was significantly higher (i.e.,
anisotropy was lower) in bacteria grown in the presence of
suberin than in control bacteria. This pattern was observed
at all temperatures tested (Fig. 4A). The greater membrane
fluidity of bacteria grown in the presence of suberin was
maintained over the 4 d time course performed at 25°C (Fig.
The analysis of fatty acid composition revealed that the
membranes of S. scabiei contained a majority of two
branched-chain (iso-16:0 and anteiso-15:0), an unsaturated
(16:1  cis, i.e., palmitoleic acid) and a straight-chain (16:0,
i.e., palmitic acid) fatty acids (Table 3). Differences between
bacteria grown in the absence or presence of suberin were
observed after 1 d of growth in minimal medium. Suberin
induced a higher proportion of total branched-chain fatty
acids. The abundance of two of these, iso-16:0 and anteiso-
17:0, increased significantly in the presence of suberin while
the abundance of iso-15:0 and anteiso-15:0 remained
unchanged; however, suberin induced a significantly lower
proportion of unsaturated acids. No variation in the propor-
tions of straight-chain fatty acids was observed (Table 3).
Suberin is a polymer recalcitrant to microbial degradation
in nature. Unambiguous evidence for the presence of suberin
in soil organic matter has been revealed by different groups
Bacterial growth and production of typical secondary metabolites by five Streptomyces species grown for 4 d in MM in the absence or
presence of suberin
Dry mycelial weight (mg±SD)
Metabolite production (μg mg DW−1±SD)a
S. scabiei28±490±3***n.d. 3.61±0.14***
S. acidiscabies 60±388±2***0.05±0.00 1.44±0.42**
S. avermitilis 27±4134±3***n.d. n.d.
S. coelicolor38±164±4***0.35±0.20 1.22±0.45*
S. melanosporofaciens 41±469±6**n.d. 1.32±0.79***
Values are the means of three replicates.
aMetabolites assayed were thaxtomin A for S. scabiei and S. acidiscabies, abamectin for S. avermitilis, γ-Actinorhodin for S. coelicolor and
geldanamycin for S. melanosporofaciens.
bValues from suberin medium are significantly different from control at *: P<0.05, **: P<0.01 and ***: P<0.001 (t-test).
n.d.: not detected; detection limits were 0.05 μg, 0.05 μg and 0.1 μg of total thaxtomin A, abamectin and geldanamycin, respectively.
Proteins (mg mL−1±SD) released by mechanical lysis performed on five Streptomyces species grown for 7 d in the absence or presence
S. scabieiS. acidiscabies S. avermitilisS. coelicolorS. melanosporofaciens
Control0.26±0.05 0.75±0.060.71±0.06 1.05±0.10 2.23±0.11
P value (t-test)0.00050.0013 0.00010.0223
Values are the means of four replicates.
Electron microscopy images of Streptomyces scabiei EF-35
after 7 d of growth in minimal medium (A) or in suberin-supplemented
medium (B), at a 35,590× magnification. Arrows show thicker cell wall
in bacteria grown in the presence of suberin.
Extracellular protein contents (±SD) released by mechanical
lysis of Streptomyces scabiei EF-35 grown in minimal control medium
(open circles) and in suberin-minimal medium (solid circles) over a 7-d
LERAT et al. 40
(24, 28, 31). Only rare studies have investigated the
biochemical mechanisms associated with suberin degradation
(16). Nevertheless, some authors have suggested that actino-
mycetes might be involved in the degradation process (10,
32). All Streptomyces strains used in this study showed better
growth in the presence of the polymer. This enhanced growth
is probably not the effect of the utilization of suberin
constituents as a carbon source (suberin concentration was
low), but would rather result from an increase in membrane
fluidity that facilitates the transport of nutrients and waste
The fatty acid monomers associated with the suberin
structure act as membrane perturbants of phospholipid
vesicles (8). Here, suberin affected both the membrane
composition and fluidity of living S. scabiei cells. The
anisotropy test unequivocally showed that in the S. scabiei
EF-35 membrane, fluidity was overall higher in bacteria
grown in the presence of suberin. Iso and anteiso branched-
chain fatty acids in the bacterial membrane generally
contribute to its fluidity (6, 34) and the proportions of
branched-chain fatty acids increased in bacterial cells grown
in the presence of the polymer. On the other hand, unsaturated
fatty acids also have the ability to increase membrane fluidity
(6, 7), but their proportions decreased in suberin-treated
membranes, showing that adjustment of membrane fluidity
is a complex mechanism. This decrease in the proportions of
unsaturated fatty acids may, however, represent a protection
mechanism against phenolic compounds present in the
suberin polymer. Denich et al. (6) stated that saturated fatty
acids help in preventing the access of phenol molecules to
the membrane interior.
The presence of suberin in the growth media of the five
Streptomyces species tested conferred relative protection
against mechanical stress, suggesting that this plant polymer
triggers changes not only in the bacterial membrane, but also
in the cell wall. This was demonstrated in S. scabiei since
cell walls were thicker in bacteria grown in the presence of
suberin. The cell wall also appeared to contain more electron-
dense material. The increase in cell-wall thickness may
explain the higher resistance of S. scabiei to mechanical lysis.
Once again, the presence of phenols in suberin may be
responsible for the higher thickness of cell walls associated
with bacteria grown in the presence of suberin. It was
suggested that a high number of peptidoglycan layers may
effectively be caused by the exposure of bacterial cells to
environmental stresses such as phenols (21).
In the present study, potato suberin altered the develop-
ment of five bacterial species of the genus Streptomyces
(S. scabiei, S. acidiscabies, S. avermitilis, S. coelicolor and
S. melanosporofaciens). The general morphological patterns
of isolated colonies of the five Streptomyces species used
here point toward the capacity of suberin to promote cellular
differentiation. On solid minimal medium, colonies of the
five species tested here presented a hairy morphotype in the
presence of suberin (although this phenotype was only
slightly visible in S. avermitilis), while in the absence of the
biopolymer, colonies were bald, or to some extent hairy. The
onset of morphological differentiation attributable to suberin
concurs with the production of secondary metabolites whose
presence, after 4 d of growth in liquid minimal medium,
boosted the synthesis of characteristic phytotoxins and
antibiotics by S. scabiei, S. acidiscabies, S. coelicolor and
Anisotropy (±SD) of the DPH probe incorporated into the
membrane of Streptomyces scabiei EF-35 grown in minimal medium
(open circles) and in suberin-supplemented medium (solid circles) after
1 d of incubation, as a function of temperature (A); and measured at
25°C over a 4-d period (B).
Membrane fatty acid composition of Streptomyces scabiei
EF-35 grown for 1 d in minimal medium in the absence or
presence of suberin
Fatty acids Control (%±SD)Suberin (%±SD)
anteiso-13:0 1.7±0.3 1.7±0.3
anteiso-15:0 11.3±0.3 12.2±0.5
iso-17:0 2.9±0.2 2.7±0.1
16:1 (9) cis*16.4±3.1 13.0±0.1
17:0 cyclopropane (9, 10)3.7±1.14.2±0.4
Values are the means of six replicates (three replicates of two repeats).
ANOVA; *: P<0.05, **: P<0.01.
Streptomyces Differentiation by Suberin 41
S. melanosporofaciens. Although no abamectin was produced
by S. avermitilis under the tested conditions, the production
of other unidentified metabolites was apparently stimulated
by the presence of suberin. Abamectin is only one secondary
metabolite potentially produced by S. avermitilis and other
antibiotics may also be synthesized by this bacterium (20).
It has been speculated that complex polymers could
influence the development and production of bioactive
molecules by soil-dwelling streptomycetes (33). For instance,
N-acetylglucosamine and cellobiose, the main degradation
products of chitin and cellulose, lock S. coelicolor (33) and
S. scabiei (19) in the vegetative state. The effect of cellulose
and cellooligosaccharides on morphological development has
also been described in S. griseus (23). Interestingly, it has
been demonstrated that suberin counteracts the effect of
cellobiose and could promote morphological differentiation,
even in the presence of the disaccharide (19). In S. coelicolor,
environmental stresses trigger cell morphological differenti-
ation associated with secondary metabolism (36) and it was
shown that suberin is perceived by streptomycetes as a stress
The data presented in this paper not only confirm previous
observations suggesting that suberin triggers the onset of
secondary metabolism (17, 19) but also bring to light new
characteristics of the changes induced by the presence of
suberin. When exposed to this plant polymer, streptomycetes
undergo profound morphological modifications. While mech-
anisms linked to suberin biodegradation in streptomycetes
are still largely unknown, determining which suberin constit-
uents trigger differentiation is challenging. Characterization
of the secretome of Streptomyces species grown in the
presence of suberin is in progress.
This work was supported by the National Sciences and Engineer-
ing Research Council of Canada (NSERC) and the Fonds Québécois
de Recherche en Nature et Technologie. AL gratefully acknowledges
the receipt of a scholarship from NSERC.
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