Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Ectoine: a compatible solute in radio-halophilic

Stenotrophomonas sp. WMA-LM19 strain to prevent
ultraviolet-induced protein damage
W. Sajjad1,2,3, S. Qadir1, M. Ahmad1, M. Rafiq1,4, F. Hasan1, R. Tehan2, K.L. McPhail2 and A.A. Shah1
1 Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
2 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA
3 Department of Multidisciplinary Studies, National University of Medical Sciences, Rawalpindi 46000, Pakistan
4 Department of Microbiology, Abdul Wali Khan University, Mardan, Pakistan

Keywords Abstract
anti-oxidant, ectoine, proteins, radio-halophilic,
Stenotrophomonas sp., ultraviolet radiation. Aim: Thiss study was conducted to investigate the possible role of a
compatible solute from radio-halophilic bacterium against desiccation and
Correspondence ultra-violet radiation-induced oxidative stress.
Aamer Ali Shah, Department of Microbiology, Methods and Results: Nine different radio-resistant bacteria were isolated from
Faculty of Biological Sciences, Quaid-i-Azam
desert soil, where strain WMA-LM19 was chosen for detailed studies on the basis
University, Islamabad, Pakistan.
of its high tolerance to ultraviolet radiation among all these isolates. Here, 16S
E-mail: [email protected]
rRNA gene sequencing indicated the bacterium was closely related to
2017/1697: received 28 August 2017, revised Stenotrophomonas sp. (KT008383). A bacterial milking strategy was applied for
3 February 2018 and accepted 15 April 2018 extraction of intracellular compatible solutes in 70% (v/v) ethanol, which were
purified by High Performance Liquid Chromatography (HPLC). The compound
doi:10.1111/jam.13903 was characterized as ectoine by 1H and 13C Nuclear Magnetic Resonance (NMR),
and Mass Spectrometry (MS). Ectoine inhibited oxidative damage to proteins and
lipids in comparison to the standard ascorbic acid. It also demonstrated more
efficient prevention (54
80%) against lysis to erythrocytes membrane by surface
active agents than lecithin. Furthermore, a high level of ectoine-mediated
protection of bovine serum albumin against ionizing radiation (1 500–
2 000Jm2) was observed, as indicated by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
Conclusion: The results indicated that ectoine from Stenotrophomonas sp. WMA-
LM19 can be used as a potential mitigator and radio-protective agent to overcome
radiation- and salinity-mediated oxidative damages in extreme environment.
Significance and Impact of the Study: Due to its anti-oxidant properties,
ectoine from a radio-halophilic bacterium might be used in sunscreen
formulation for protection against UV-induced oxidative stress.

extremophiles in a given habitat is strictly dependent

Introduction
upon their ability to maintain an internal osmotic pres-
The capability of micro-organisms to regulate osmotic sure. In order to maintain an osmotic equilibrium, these
pressure is a critical consideration when evaluating their extremophiles (bacteria, archea and eukrya) can equili-
ability to successfully compete and grow in a given habi- brate to saline environments by accumulating compatible
tat. This unique feature of extremophiles facilitate exten- solutes (Csonka and Hanson 1991). The diverse chemical
sive applications in biotechnology (Gabani and Singh structures of compatible solutes range from sugars and
2013). Most living cells have to adapt themselves with polyols to amino acid derivatives, and are not only con-
respect to fluctuations in the osmotic strength of their sidered as osmoregulatory solutes, but also valuable to
environment within certain limits. The proliferation of bacterial cells in not only protecting proteins but also

Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 457
Ectoine from Stenotrophomonas sp. WMA-LM19 W. Sajjad et al.

DNA from the damaging effects of drying, freezing, high intra-molecular hydrogen bonding (secondary structures)
temperatures and ultraviolet radiation (Ventosa and and the diminution in solubility of the peptide backbone
Nieto 1995; Kunte et al. 2014; Hahn et al. 2017). (Kunte et al. 2014). Halomonas elongata, an ectoine-pro-
The compatible solute ectoine (1, 4, 5, 6-tetrahydro-2- ducing strain, has widely been studied, and recycled to
methyl-4-pyrimidinecarboxylic acid) is a cyclic derivative develop the fermentation technology at large scale (Hahn
of aspartate, which has achieved extensive interest as a et al. 2015). Optimal production of ectoines was condi-
protective agent, and appears to be a universal organic tioned by the interplay of carbon and nitrogen metabo-
solute in bacteria. Ectoine was formerly discovered in a lism. C. salexigens was challenged to grow in medium
phototrophic and halophilic bacterium Ectothiorhodospira with a carbon/nitrogen (C/N) imbalance employing the
halochloris (Galinski et al. 1985). However, its biosynthe- fed-batch fermentation, and observed that the limited
sis has now been documented in a great variety of halo- supply of carbon and ammonium was best conditions for
tolerant and halophilic species, particularly in those cultivation of C. salexigens (Salar-Garc
ıa et al. 2017). The
having simple growth demands. In the bacterial domain, bacterial milking strategy and improvement of ectoine
biosynthesis of ectoine is far more widespread than excreting mutants (“leaky mutants”) are the two vital
betaine and glycine (Oren 2008), and it contributes in a technologies that allow ectoine’s annual production on a
wide array of applications including cosmetic additives, large scale. Despite being the most successful method,
biological processes and pharmaceutical industries (Kana- bacterial milking also has some drawbacks. This method
pathipillai et al. 2005; Lentzen and Schwarz 2006; Zhang is inexorable with decline in cell growth rate and diffi-
et al. 2006). The wide range of applications arise interest culty of downstream handling due to discontinuous pro-
to develop methods for efficient production of ectoine. duction pattern and high salt mediation (Schubert et al.
Ectoine producing halophilic bacteria isolated from salt 2007).
stress environments have also been studied (Pastor et al. To overcome these shortcomings, a radio halophilic
2010). In order to meet the continuously increasing mar- bacterium Stenotrophomonas sp. strain WMA-LM19, iso-
ket demand, many efforts have been made in the last two lated from desert soil, was able to nurture and excrete
decades to modify the process of ecotine production from ectoine in high UV, manganese and salt concentration,
bacterial strains. Enhanced yield of ectoine from H. elon- using monosodium glutamate as a carbon and nitrogen
gata was achieved by a bioprocessing technique called source in 0
5 mol l1 NaCl. Ectoine was purified and its
“bacterial milking” (Sauer and Galinski 1998), which possible protective role in radiation and salt tolerance, as
involves a cyclic upturn and a diminution of the salt con- an anti-oxidant to protect the cellular protein in stress
centration for ectoine synthesis and secretion, corre- was also evaluated. Protective effect of ectoine on bovine
spondingly. In addition, these solutes are not only limited serum albumin (BSA) and red blood cell (RBC) mem-
to use as stress protectants, but can serve as an important brane was also evaluated, using SDS-PAGE and mem-
source of carbon and nitrogen for intracellular energy brane damage assay.
production (Salar-Garc
ıa et al. 2017). Moreover, these
solutes can induce cell death or hypo-osmotic shock in
Materials and methods
other micro-organisms when released to the surrounding
medium. Some of the compatible solutes produced by
Bacterial strain and growth conditions
extremophiles are shown in Fig. 1.
Ectoine’s protective properties for biological proteins Soil samples were collected from the desert of District
can be enlightened through its strong uptake of the Lakki Marwat, Khyber Pukhtoonkhwa, Pakistan, asepti-
organic solute by interface with water and the consequent cally in sterile zipper plastic bags and kept at 4°C until
omission from protein surfaces, the strengthening of investigation for radio-resistant microbial communities.
Serially diluted soil samples were inoculated by spread
O plate method on Spizizen’s minimal medium (SMM)
O consisting of (NH4)2SO4 (2 g l1), Na3-citrate. 2H2O
H
H3C O
(1 g l1) K2HPO4 (14 g l1), KH2PO4 (6 g l1), MgSO4.
+ +
N H N OH
H3C
– H – 7H2O (0
2 g l1), MnCl2. 4H2O 0
01 g l1 and NaCl
NH2
H3C O O 30 g l1, with 0
5% (w/v) glucose as the carbon source
supplemented with phenylalanine (18 mg l1), and tryp-
Structure of Glycine zwitterion Structure of
tophan (20 mg l1). SMM plates were exposed to UV
Betaine glycine
radiation for 5 min in the UV chamber
Figure 1 Compatible solutes produced by microbes in extreme (119 9 69 9 52 cm) supplied with a 20 W, 280 nm UV
environment. light, prior to incubation. The UV fluence rate [energy

458 Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology
W. Sajjad et al. Ectoine from Stenotrophomonas sp. WMA-LM19

per area per time (J/m2)] of the test sample was calcu-
Extraction and determination of ectoine by NMR
lated, using the following equation (Sajjad et al. 2017).
spectroscopy
He ¼ Ee  t Extraction of intracellular compatible solutes was carried
where He is the energy that reaches a surface area due to out by a slight modification of the previously described
irradiance (Ee) maintained for time duration (t). The method (Wang et al. 2006). Strain WMA-LM9 was grown
total UV dose was calculated by exposure time to the UV in 200 ml of SMM having a high salt concentration [8%
fluence rate. The bacterial isolates from irradiated plates (w/v) NaCl] to induce accumulation of ectoine. When
were subcultured for further confirmation and to deter- optical density (OD600) reached up to 1 (1 OD per
mine their survival curve. ml = 0
30 g DCW per l), cells were collected by centrifuga-
tion (1000 9 g) for 10 min at 4°C and washed with
50 mmol l1 potassium phosphate buffer (pH 7
2) contain-
Bacterial survival curves at UVB and oxidative stress
ing an isotonic NaCl concentration. The cells were trans-
The survival curve of strain WMA-LM19 was plotted ferred to deionized water containing low salt concentration
against various doses of UVB radiation by the method as [hypo-osmotic shock from 8 to 3% (w/v) NaCl] that rapidly
previously described (Mattimore and Battista 1996). Cell released accumulated solutes from the cells to achieve osmo-
of strain WMA-LM19 were serially diluted (1:1000), tic equilibrium. These solutes were extracted by re-suspend-
using phosphate buffer saline (PBS) and used to spread ing the pellets in 80% (v/v) ethanol for 10 h then
on tryptone glucose yeast (TGY) agar plates. The plates centrifuged. After the centrifugation, the supernatant was
were exposed to various doses at 280 nm UV radiation collected, dried and dissolved in 0
8 ml methanol-d4
and then incubated for 3 days at 30°C. The survival rate (CD3OD) for proton (1H) and carbon (13C) NMR.
was determined by dividing number of the colonies on NMR data were acquired at ambient temperature on
irradiated plates to the colonies appeared on un-irra- Bruker Avance 500 MHz NMR spectrometer in CD3OD
diated plates. Oxidative stress was determined by diluting referenced to residual protonated solvent (dH 3
35 ppm,
an overnight grown culture of the strain WMA-LM19 dC 49
3 ppm) with tetramethylsilane (TMS) as an internal
with sterile normal saline up to an optical density standard (temperature of 20–22°C).
(OD600) 0
5, then treated with different molar concentra-
tions (0–10 mmol l1) of hydrogen peroxide (H2O2) for
Reversed-phase high-performance liquid chromatography
30 min, prior to inoculation on TGY plates. The plates
of ectoine
were incubated at 30°C for 3 days, and the survival rate
was determined by comparing a number of colonies The extract was further purified, using Whatman filter
obtained from the treated and untreated samples. All paper, applying a second cross flow filtration in a mixture
experiments were carried out in triplicate. of water and ethanol for HPLC analysis. The cellular
extract was filtered using a small pore size membrane fil-
ter (0
22-um) and subjected to isocratic HPLC Shimadzu
Identification of radio-resistant bacterium
Dual LC-20AD solvent delivery system with a Shimadzu
Strain WMA-LM19 was identificed both morphologically SPD-M20A UV/vis photodiode array detector C18 Phe-
as well as biochemically by previously described method nomenex Synergi Hydro-RP 250 9 4
6 mm, 4l. The
(Murray et al. 1981). For molecular identification, DNA extract was eluted with a mixture of methanol/water
was extracted using DNA extraction kit (QIAGEN), and (70:30) as the mobile phase for 10 min at a flow rate of
16S rRNA gene sequence was amplified using universal 0
8 ml min1, using UV detection at 210 nm.
primers [27F0 (50 -AGAGTTTGATCCTGGCTCAG-30 ) and
1492R’ (50 -CTACGGCTACCTTGTTACGA-30 )]. The
Liquid chromatography–mass spectrometry analysis of
amplified product was sequenced at Macrogen Service
ectoine
Center (Geunchun-gu, Seoul, South Korea) and the near-
est relative sequences in the NCBI database were com- The purified compatible solute was subjected to LC-MS
puted, using Blast tool and Molecular Evolutionary on an ABScieX 3200 Q-TRAP mass spectrometer
Genetic Analysis (MEGA) ver. 6 (Tamura and Nei 1993). equipped with a TurbolonSpray ESI source, and con-
A neighbour-joining tree was constructed to identify the nected to a Shimadzu HPLC system with dual LC-20
isolated strain and examine the diversity among UV-resis- pumps, an SPD-M20A UV/Vis photodiode array (PDA)
tant extremophiles. The sequence obtained was submitted detector. The extract was eluted in a mixture of water
to the NCBI GenBank for acquisition of the accession and methanol (5:95) with 0
1% (v/v) formic acid with
number. mass detection from m/z 80-1500 in the positive

Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 459
Ectoine from Stenotrophomonas sp. WMA-LM19 W. Sajjad et al.

ionization mode. The capillary voltage and atomizing gas level (Yang et al. 2006). Cells were pretreated separately
pressure were 4
0 kV and 35 psi, respectively, flow rate of with ectoine (1% w/v) and lecithin (1% w/v), and then
drying gas (nitrogen as collision gas) 12 ml min1 and kept under stress for 1 h at 37°C in the presence of sur-
temperature of solvent removal was set at 350°C. face-damaging substances like Benzalkonium chloride
(BZK), Cocamidopropyl betaine (CAPB), Sodium dode-
Evaluation of anti-oxidants and radio-protective cyl sulphate (SDS), Sodium lauryl ether sulphate (SLES)
properties of ectoine and Alkylpoly glucoside (APG). About 200 ll of the reac-
tion mixture was taken and centrifuged at 3 000 9 g for
Lipid peroxidation inhibition assay 5 min. The supernatant was collected and absorbance was
Thiobarbituric acid reactive substances assay was carried determined at 540 nm. The per cent haemolysis was cal-
out for lipid peroxidation,using lipid rich medium (mice culated by the following formula:
liver homogenate) (Ohkawa et al. 1979). Liver tissue
(1 g) was homogenized in Tris HCl buffer (pH 7
4) and %haemolysis ¼ ðcontrol - test/controlÞ  100
centrifuged at 3 000 9 g for 5 min. About 1 ml of the
homogenized mixture was mixed with different ectoine Analysis of radio-protection to BSA by ectoine
concentrations (2–10 lg). 10 lmoll1 of FeSO4 was also The structural damage to BSA in response to different
added and raised the total volume up to 3 ml using doses of irradiation (1 500–2 000 Jm2) and protection
deionized water. In each 3 ml of the mixture, 0
2 ml of in the presence of ectoine was confirmed by SDS-PAGE
SDS (8
1%), 0
5 ml of acetic acid (pH 3
4) and an equal analysis. Protein was irradiated with different UV doses
volume of thiobarbituric acid (0
6% v/v) was added and in the presence and absence of ectoine, then added
incubated for 1 h at 100°C. Absorbance of the coloured (20 lg) to PAGE loading buffer [0
0625 mmol l1Tris/
mixture was measured at 532 nm and was compared with HCl, pH 6
8, 5% (v/v) glycerol, 2% (v/v) 3-mercap-
the standard curve of malondialdehyde (MDA), using toethanol and 0
01% (w/v) bromophenol blue]. Sample
blank without FeSO4. was loaded on 10% (w/v) polyacrylamide gel and elec-
trophoresis was carried out at constant voltage (90 V).
Protein oxidation inhibition assay The gel was stained in 0
1% (w/v) Coomassie Brilliant
The inhibitory effect of ectoine on protein oxidation Blue R-250 by gentle shaking at room temperature for
in vitro was quantified, using BSA as the standard protein 1 h and detained in methanol/acetic acid/water mixture
(Tian et al. 2009). Different concentrations of ectoine (2– (4:2:4), bands were observed over a clear background.
10 lg ml1) were incubated with 200 ll of BSA
(1 mg ml1). The mixture was then treated with 100 ll
of oxidant FeSO4 (1 mmol l1), 100 ll H2O2 Statistical analysis
(80 mmol l1) and incubated for 1 h at 37°C. The reac- All the experiments were run in triplicates where n = 3.
tion was stopped by adding 15 unit (U) of catalase. The error bars in each figure representing  Standad Devi-
Finally, 100 ll of 10 mmol l1 2:4-dinitrophenylhydra- ation (SD). Student’s pairwise t-test was used to assess the
zine (DNPH) was added to the mixture and incubated significance between results, and P < 0
05 was considered
for 1 h. The unbound proteins to DNPH were precipi- as significant. Bacterial % survivability to UV, H2O2 and
tated by adding 10% trichloroacietic acid (TCA). The scavenging % activities offered by ectoine were studied by
supernatant was dissolved in 200 ll of guanidine regression analysis with their respective concentrations and
hydrochloride (6 mol l1) and protein-oxidation was to that of control (Ascorbic acid 10 lg ml1).
quantified spectrophotometrically at 370 nm using H2O
to replace FeSO4 and H2O2 as blank. Per cent inhibition
offered by ectoine was calculated using the following Results
formula: Among nine isolated radio-resistant bacterial strains,
Inhibition of protein oxidationð%Þ maximum survival (50%) exhibited by isolate WMA-
¼ ðControl - sample/controlÞ  100 LM19 under given UV dose (280 nm) at 1
3 9 103 Jm2,
therefore, selected for further investigation.

Membrane protection assay

Resistance to UV radiation, oxidative stress and
Human RBCs (erythrocytes) were isolated by centrifuga-
mitomycin C
tion of citrated blood at 2 000 9 g for 15 min. Cells
were washed with phosphate buffer saline, then resus- Strain WMA-LM9 exhibited 50% viability at 1 300 Jm2
pended in the same buffer up to the desired hematocrit of UV radiation (280 nm), in comparison to the control,

460 Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology
W. Sajjad et al. Ectoine from Stenotrophomonas sp. WMA-LM19

E. coli (10536), which was unable to resist such high genus Stenotrophomonas. The phylogenetic tree con-
energy dosage (Fig. 2a). During oxidative stress analysis, structed on the basis of sequences obtained from NCBI,
it was observed that survival of strain WMA-LM19 grad- clustered the strain WMA-LM19 around the group Proe-
ually decreased with an increase in concentration of teobacteria (Fig. 3). The nucleotide sequence reported
H2O2 and maintained up to 50% viability at 6 mmol l1 here can be obtained from the NCBI nucleotide sequence
of H2O2 for 60 min (Fig. 2B). Results are expressed as database under accession number KT008383.
means  SD and were compared using the Student’s
unpaired t-test. Moreover, the percentage values had an
Purification and identification of compatible solute from
exponential distribution. Error bars represent standard
strain WMA-LM19
deviation for triplicate experiments. P < 0
05 is consid-
ered significant. LC-MS analysis of the extract
The ethanol extract of compatible solute from Stenotro-
phomonas sp. strain WMA-LM19 exhibited a single peak
Identification of strain WMA-LM19
on HPLC at 3
0 min retention time (Fig. 4). Mass data
Strain WMA-LM19 was aerobic, Gram-negative rod were collected in positive ionization mode and the mass
shaped and form white to light pink, raised, mucoid scan width was set to m/z 100–1 700. A major peak for
colonies on TGY agar medium. 16S rRNA gene sequence m/z 143
68, matched the formula C6H11N2O2 (Fig. 4),
of strain WMA-LM19 exhibited 93% similarity with the corresponding to protonated ectoine.

1
H and 13C NMR
Representative 1H NMR spectrum of the extract from the
bacterial cells (Fig. 5A) showed the presence of diastereo-
topic protons in the compound, which together with the
N-H singlet at dH 2
2 ppm, was consistent with the struc-
ture of ectoine. The 1H NMR data was found to be iden-
tical to that already reported in the literature for ectoine,
as was the 13C NMR data (Fig. 5B). The presence of sig-
nals at dC 161
67 (C-CH3) and 175
99 (C=O) in the 13C
spectrum confirmed the presence of a carboxyl group,
while signals at 18
7, 23
1, 38
7, and 55
0 ppm showed
the 2-CH and 3-CH aliphatic carbons in ectoine
(Fig. 5C). Thus, on the basis of the NMR and LC-MS
data, we conclude that highly pure ectoine accumulated
in the cells. The structure was also predicted, using
ChemDraw software.

Lipid peroxidation and protein carbonylation inhibition

Ectoine exhibited a concentration dependent inhibitory
effect on lipid peroxidation and protein carbonylation/
oxidation. The inhibitory effect of ectoine to lipid (mice
liver homogenate) and protein (BSA) oxidative damage
was 66
01 and 72
09%, respectively, at 10 lg ml1 of
ectoine that was comparatively higher than in case of
ascorbic acid [55
45% inhibition (P < 0
05)]. The study
demonstrated a significant effect on protein and lipid
Figure 2 Survivability of strain WMA-LM19 from desert soil at vary- oxidative damages in the presence and absence of ectoine
ing UV-B exposure. (a) UV radiation-resistant potential of WMA-LM9 (Fig. 6).
(■) in comparison to UV-sensitive E. coli (▲) (b) Resistance to differ-
ent concentrations of hydrogen peroxide (mM). % viability value is
calculated as N1/N0 9 100 where Ni is the value after exposure to Membrane protection
irradiation at different time in seconds (●) and H2O2 while N0 is the
value at time 0, for each condition tested. The results are significantly A marked protection was provided by ectoine against var-
different between groups, because P < 0
05. Values are mean  SD. ious surface membrane-damaging active substances used

Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 461
Ectoine from Stenotrophomonas sp. WMA-LM19 W. Sajjad et al.

Stenotrophomonas sp. |GQ901883·1|

18

17 Stenotrophomonas maltophilia |LK871646·1|

15 WMA-LM19 |KT008383|

Figure 3 Neighbour joining phylogenetic tree

based on 16S rRNA gene sequence analysis,
Stenotrophomonas sp. |gb|KJ541832·1| showing the position of isolate WMA-LM19
to other strains of Stenotrophomonas
obtained from NCBI. Accession numbers of
Stenotrophomonas sp. |KJ806210·1| the sequences used in this study are shown in
parentheses after the strain designation.
Numbers at nodes are percentage bootstrap
Stenotrophomonas sp. FSBRY18 |KJ184977·1| values based on 1,000 replications.

Figure 4 HPLC chromatogram/positive ESI-

MS spectrum of carotenoid extract exhibits
signals at m/z 143
20 [M+H]+. [Colour figure
can be viewed at wileyonlinelibrary.com]

in the membrane protection assay. Ectoine demonstrated efficacy of ectoine towards BSA against supralethal ioniz-
more efficient preventive activity (54
80%) to erythro- ing radiation-induced damage (Fig. 8).
cytes’ membrane in the presence of benzalkoniumchloride
in comparison to 1% (w/v) lecithin (28
915%) that
Discussion
served as a positive control (Fig. 7).
In this study, we have shown the close relationship
between radiation resistance and ectoine production in a
Protein protective efficacy of ectoine against radiation-
bacterium isolated from desert soil. Synthesis of ectoine
induced damage
and hydroxyectoine consumes glutamate as the donor of
The irradiation of an aqueous solution of BSA with dif- amino group for the synthesis of precursor aspartate
ferent doses of UV exhibited the radiation dose depen- (Salar-Garc
ıa et al. 2017). Strain WMA-LM19 (KT00
dent (1 500–2 000 Jm2) degradation of BSA that 8383) was found to produce ectoine intracellularly under
appeared as intense smeared bands on SDS-PAGE as UV radiation and salt stress via bacterial milking strategy.
compared to that of untreated controls. However, no This result was confirmed by the HPLC, LC–MS, and
smearing of BSA bands was observed upon irradiation NMR analyses. Furthermore, the inhibitory role of
(1 500–2 000 Jm2) in the presence of ectoine. The ectoine against protein and lipid oxidative damage was
results clearly demonstrated a significant radio-protective also determined. There are limited reports on production

462 Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology
W. Sajjad et al. Ectoine from Stenotrophomonas sp. WMA-LM19

(a) (b)

(c)

Figure 5 1H (a) and 13C (b) NMR spectra of purified compound LM19F2. The purified compound was identified as “Ectoine” chemical structure
(c) from NMR spectroscopy. [Colour figure can be viewed at wileyonlinelibrary.com]

100

80

60
% Inhibition

40

Figure 6 Protein oxidation inhibition (white 20

bars) and lipid peroxidation inhibition (black
bars) offered by ectoine using bovine serum
albumin (standard protein) and mice lipids as
test samples. Where n = 3 and the error bars 0
representing  SD. Ascorbic acid was used as 2 4 6 8 10 Control 10
standard control (10 lg) Concentration (µg)

of ectoine from wild-type strains of radio-resistant halo- rRNA gene sequencing indicated that strain WMA-LM19
philic bacteria. exhibited 93% similarity to genus Stenotrophomonas. Ste-
Among nine bacterial strains isolated from desert soil, notrophomonas sp. strain WMA-LM19 accumulated high
strain WMA-LM19 was found to grow under high UV concentration of ectoine intracellularly in the presence of
dosage and salt concentration (15%). The results of 16S manganese chloride, high salinity and UV radiation.

Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 463
Ectoine from Stenotrophomonas sp. WMA-LM19 W. Sajjad et al.

100

80
% Relative activity

60

40 Figure 7 Membrane damage preventing

assay by 1% ectoine (black bars) using
human RBCs and Lecithin (white bars) as a
20 positive control to different membrane
damaging substances. Where n = 3 and the
error bars representing  SD. BZK,
Benzalkonium chloride; CAPB,
0
Cocamidopropyl betaine,; SDS, Sodium
CAPB SDS BZK SLES APG
dodecyl sulphate; SLES, Sodium lauryl ether
Surface membrane-damaging active substances sulphate; APG, Alkylpoly glucoside.

quenches superoxides, thus preventing the cells from

L Pc T1 NC oxidative damage. However, the significant role of these
150 kDa compatible solutes in protection against dessication does
120 kDa not exclude the possible involvement of other cellular
95 kDa mechanisms like formation of water stress-proteins (Goyal
et al. 2005; Campos et al. 2013). Stenotrophomonas sp.
70 kDa strain WMA-LM19 survivability (up to 50%) against vari-
ous doses of UVB radiations as well as H2O2, suggested
66·6 kDa 66·6 kDa Smearing the presence of strong SOD (superoxide dismutase) anti-
oxidant and CAT (catalase) system, protecting cell from
50 kDa oxidative damage (Prazdnova et al. 2014). Similar findings
were observed previously that pigment producing strains
are more resistant to UV than nonproducing strains. Ste-
30 kDa notrophomonas maltophila, previously isolated from high
altitude of Aden lakes, showed resistance against UV radi-
ation due to a coloured pigment that absorbes radiation
(Flores et al. 2009). Previously, only one of the proteobac-
teria species has been reported from nuclear waste-con-
15 kDa taminated sediments that showed survival (0
00175%)
against 2
5 kGy of gamma radiation (Fredrickson et al.
2004). The ability of these UVR microbes to survive in
10 kDa several extreme conditions is suggested to be a result of
three combined mechanisms: prevention, tolerance and
repair (White et al. 1999).
Figure 8 Analysis of protein damage prevention offered by ectoine
Previously, the role of compatible solutes and dessica-
on SDS-PAGE. Observance of protein protection (20 lg of BSA)
tion stress upon survivability of Archaeoglobus fulgidus
offered by ectoine (10 lg ml1) at radiation doses 1 500–
2 000 Jm2. Lane PC: Positive control (only BSA); Lane NC: Negative and Hymenobacter marinus was correlated. The survival
control (BSA treated with hydrogen peroxide and UV radiation rate of both the species after dessication increased 30
2 000 Jm2). Lane T1: Test samples (BSA treated with hydrogen per- times under salt stress (Beblo-Vranesevic et al. 2017). A
oxide, UV radiation and ectoine in10 lg ml1). comparatively higher survivability of H. marinus might
be attributed to a number of significant factors that
Accumulation of Mn2+ and high ectoine might be two include repair enzyme, genome copy number and metals
interesting mechanisms of survival under high UV radia- (like Mn+2) (Webb and DiRuggiero 2012). On the other
tion and salinity, as Mn2+ block the Fenton reaction and hand, ectoine acts as an anti-oxidant and also prevents

464 Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology
W. Sajjad et al. Ectoine from Stenotrophomonas sp. WMA-LM19

cells from desiccation and water loss. In this report, the amyloidogenic proteins (Arora et al. 2004; Furusho et al.
productivity of ectoine from Stenotrophomonas strain 2005; Botta et al. 2008). Ectoines have considerable
WMA-LM19 was 2
9 gl1d1, which is the highest level industrial and pharmacological interest due to function
of ectoine production by any strain other than Halomo- preservation and stabilizing effects (Pastor et al. 2010).
nas genera. The production of ectoine 3
3 g l1d1 from Ectoine and their derivatives have gained significant com-
H. elongate (an ectoine excreting strain) DSM 142 was mercial market value. Biotechnological techniques via
investigated using bacterial milking strategy (Sauer and “bacterial milking” enhanced the annual ectoine produc-
Galinski 1998). While, an anaerobic denitrifying halophi- tion and reached the scale of tons (Sauer and Galinski
lic bacterium Brevibacterium epidermis DSM 20659 was 1998; Schwibbert et al. 2011).
capable of producing ectoine up to 2 g l1d1 (Onraedt The native fluid systems (as opposed to dry stabiliza-
et al. 2005). tion of liposomes) with respect to its interaction with
HPLC analysis of the medium revealed no peak after other biomolecules, like lipids of plasma, still require a
centrifugation and filtration of the cell pellets, demon- lot of attention. Ectoine also stabilizes the membranes of
strating that ectoine is produced intracellularly preventing pretreated cells (erythrocytes from human RBCs) against
the excessive loss of water from cells in extreme environ- the damaging effect of stress factors and surfactants as
ment of high salinity and UV radiations. Ectoine from well that lead to dehydration (haemoglobin release). In
Stenotrophomonas sp. WMA-LM19 was investigated for the same way, skin cells also have a double lipid layer
its role in cellular protection against radiation-mediated configuration within targeted and linked proteins. Cell
oxidative damage. The reaction of various peroxides with survival largely depends upon function of this membrane
transition metals generate hydroxyl (OH) radical, which that control permeability, structure of molecules and ions
is considered as the most reactive oxygen. These OH in the cell by special ducts and pumps. A number of
radicals ultimately attack on cellular macromolecules such external parameters like temperature, pH, radicals and
as DNA, polyunsaturated fatty-acids and proteins, thus other chemical factors can disturb this equilibrium and
damaging the cell (Aruoma 1999). There seems to be a damage the membranes. However, it has been shown that
direct relationship between quenching ability of ectoine the cell membrane of pretreated cells are protected and
for prevention of the lipid peroxidation process. Ectoine stabilized by ectoine against damaging effect of surfac-
can react with the superoxides generated from radiation tants and suggesting its kosmotropic nature. Thus,
within cells to knockout electrons and thus provide the ectoine features long-term moisturizing efficacy as it is
protection to cellular lipids. The two fold stronger lipid more potent moisturizer than glycerol and other mem-
peroxidation inhibition abilities of ectoine may be the brane protective agent like lecithin (Graf et al. 2008).
result of keto group that can quench the super-oxides Ectoine attracted attention of industry due to its UV pro-
thus preventing the cell from photo-oxidation and exces- tective and stabilizing properties with a potential market
sive damages in extreme environments (Ventosa et al. value as an active component in cosmetics and health
1998; Buenger and Driller 2004). care products (B€ unger et al. 2001).
Anti-oxidant potential is considered as an important Based on this study, it can be concluded that ectoine
aspect of radio-protection, ectoine-mediated radio-pro- carried strong anti-oxidant properties, and thus can neu-
tection to BSA was evaluated by performing SDS-PAGE. tralize in vitro radiation-induced free radicals efficiently.
A clear, intense smearing of BSA was observed as a result Ectoine can be used as a potential mitigator and radio-
of oxidative damage to its secondary structure, due to protective agent to overcome the radiation and salinity
increase in radiation dosage in the absence of ectoine. mediated oxidative damages in extreme environments.
However, no smearing was observed in the presence of Further studies are required to evaluate the radio-protec-
ectoine, indicating its UV-protective activity. Ectoine tive efficacy of ectoine in vivo.
treatment of BSA contributes to inhibition of oxidative
damage in proteins and lipids in stress conditions, which Acknowledgements
might be attributed to the formation of a comparatively
more stable product (Cheng et al. 2014). The kos- This study was supported by Higher Education Commis-
motropic nature of ectoine decreasing the solubility of sion of Pakistan, through International Research Support
peptide back bone and strengthens the intramolecular Initiative Program (IRSIP).
hydrogen bonding or secondary structures thereby offer-
ing significant protection to proteins. Furthermore,
Conflict of Interest
ectoine and other compatible solutes stabilize the native
state, thus affecting the prevention against aggregation of The authors declare no conflict of interest.

Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 465
Ectoine from Stenotrophomonas sp. WMA-LM19 W. Sajjad et al.

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Journal of Applied Microbiology 125, 457--467 © 2018 The Society for Applied Microbiology 467