esterase as an enzymatic signature of geodermatophilaceae adaptability to sahara desert stones and...
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ORIGINAL ARTICLE
Esterase as an enzymatic signature of Geodermatophilaceaeadaptability to Sahara desert stones and monumentsI. Essoussi1, F. Ghodhbane-Gtari1, H. Amairi1, H. Sghaier2, A. Jaouani1, L. Brusetti3, D. Daffonchio3,A. Boudabous1 and M. Gtari1
1 Laboratoire Microorganismes & Biomolecules Actives, Departement de Biologie, Faculte des Sciences de Tunis, Campus Universitaire, Tunis,
Tunisia
2 Unite de Microbiologie et de Biologie Moleculaire, Centre National des Sciences et Technologies Nucleaires (CNSTN), Sidi Thabet, Tunisia
3 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita degli Studi, Milano, Italy.
Introduction
Stone surfaces are complex habitats in which environ-
mental conditions resulting from solar radiation, temper-
ature, aridness and lack of nutrients fluctuate widely
(Gorbushina 2007). These surfaces, however, have been
shown to harbour a large variety of bacterial genera that
demonstrate a high tolerance to such stressful factors.
Geodermatophilaceae are among the main settlers of these
stringent environments (Urzı et al. 2001, 2004) with other
sympatric prevalent bacteria of various phylogenetic
affiliations. Members of these genera are predominantly
recovered from environments characterized by dry
conditions including those extreme like Antarctic or hot
desert soils (Mevs et al. 2000), rocks and monument
surfaces (Eppard et al. 1996; Urzı and Realini 1998;
Urzı et al. 2001; Salazar et al. 2006). The presence of
Geodermatophilaceae has been frequently associated to
Keywords
16S rRNA gene, Blastococcus, Esterase PAGE,
Geodermatophilus, Modestobacter, Sahara
desert.
Correspondence
Maher Gtari, Laboratoire Microorganismes &
Biomolecules Actives, Departement de
Biologie, Faculte des Sciences de Tunis,
Campus Universitaire, 2092 Tunis, Tunisia.
E-mail: [email protected]
2009 ⁄ 0818: received 8 May 2009, revised
and accepted 18 September 2009
doi:10.1111/j.1365-2672.2009.04580.x
Abstract
Aim: To assess esterase profiling of members of Geodermatophilaceae isolated
from desert stones and monuments in Tunisia and Egypt.
Methods and Results: Members of Geodermatophilaceae family isolated from
desert stones and monuments in Tunisia and Egypt were characterized by
partial 16S rRNA sequences. Twenty-five strains were clustered in three dissimilar
groups of the genera Geodermatophilus (12 strains), Blastococcus (5 strains) and
Modestobacter (3 strains). Isolates were also screened and typed based on major
groups of esterase hydrolytic activity. Their esterase patterns were determined
and compared to those of ten reference strains belonging to Geodermatophilaceae
family. Strains exhibited a diverse and complex pattern of electrophoretic esterase
bands, and 31 haplotypes were obtained for the 35 investigated strains. Esterases
produced by members of Geodermatophilaceae family have an optimal activity
around 40�C and at pH 8. Esterases from Geodermatophilus strains display a high
resistance to thermal inactivation and alkaline pH and retaining 30 and 20% of
activity after heating for 20 min at 120�C and at pH 12, respectively, and were
completely inactivated after 30 min at 120�C. Enzyme activity has been strongly
activated in the presence of Ca2+and Mg2+ ions and moderately by Zn2+ and was
markedly inhibited by Cu2+ and Co2+ ions.
Conclusions: Geodermatophilaceae isolates share a rich and particular pool of
esterase activities that could be directly linked to harsh conditions characteriz-
ing their ecological habitat including high level of aridity, temperature, ionic
strength and low nutrient availability.
Significance and Impact of the Study: Esterase could be considered as enzy-
matic signature that outlines adaptability of Geodermatophilaceae in arid area.
Journal of Applied Microbiology ISSN 1364-5072
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732 1723No claim to Tunisian Government works
aesthetic alteration of stones like orange, black and grey
stains and patinas as well as with mechanical damages,
like biopitting and powdering proving their active
involvement in decay process (Urzı et al. 2001).
Esterases (EC 3Æ1Æ1.x) represent a diverse group of
hydrolases catalyzing the cleavage and formation of ester
bonds. They are widely distributed in animals, plants and
micro-organisms. As an alternative typing approach,
esterase electrophoretic polymorphism has proved its reli-
ability to differentiate several bacterial species on the basis
of their genetic relationships. (Goullet and Picard 1995).
Thanks to their extent biochemical diversity and their
great electrophoretic variability (Gillepsie 1991), esterase
has widely been used to highlight genetic diversity of
microbial communities.
Many Geodermatophilaceae show a wide substrate toler-
ance, which led to the assumption that they have evolved
to enable access to various carbon sources or to be
involved in catabolic pathways (Bornscheuer 2002),
improving their surviving abilities in dry environments
and favouring the final colonization and alteration of
stone and monuments. The aim of the present study is to
assess the esterase profiling of member of Geodermato-
philaceae genera isolated from desert stones and monu-
ments in Tunisia and Egypt in comparison with their
relative type strains and to outline the usefulness of the
esterases as enzymes signature of Geodermatophilaceae in
arid area.
Materials and methods
Strains isolation and growth conditions
Isolation and cultivation of Geodermatophilaceae were
performed as described by Urzı et al. (2001). Depending
on sampled surface extend (monument, building or rock
from desert), 0Æ5–1 g of rock samples or powders from
scraped surfaces were taken. Samples were individually
ground to a powder in a sterile mortar and suspended
(1 : 10 w ⁄ v) in physiological saline solution (0Æ9% NaCl)
supplemented with 0Æ01% (w ⁄ v) tween 80. The suspen-
sion was stirred for 60 min, and 1 ml of each suspension
and its decimal dilutions were plated on Luedemann
medium (Luedemann 1968) supplemented with cyclo-
heximid 50 mg l)1 to avoid the growth of fungi. Incuba-
tion was carried out at 30�C. Bacterial colonies were
observed and described morphologically after 7, 15 and
30 days, those resembling the morphological features of
Geodermatophilaceae were picked out and sub cultured
twice to three times on Luedemann medium. Geoderma-
tophilus obscurus ssp. obscurus (DSM 43160T) (Luede-
mann 1968), G. obscurus ssp. utahensis (DSM 43163)
(Luedemann 1968), G. obscurus ssp. dictyosporus (DSM
43161) (Eppard et al. 1996), Blastococcus aggregatus DS17
and DS10 (Urzı et al. 2001), B. saxobsidens (DSM44509T)
(Urzı et al. 2004) and Modestobacter multiseptatus
BC501, BC498, BC499 and DS13 and Geodermatophila-
ceae DD13 (Urzı et al. 2001) were used as reference
strains.
DNA extraction, ARDRA and 16S rRNA gene sequencing
Bacterial isolates were grown at 30�C for 3 days on
Luedemann medium. Three to five well-isolated colonies
were washed three times in sterile physiological saline
solution. Total genomic DNA was extracted by a CTAB-
SDS lysis protocol (Ausubel et al. 1994). PCR amplifica-
tion of 16S rRNA gene was performed using the following
primers: S-D-Bact0008-a-S-20 and S-D-Bact-1495-a-A-20
as described by Gtari et al. (2004). ARDRA profiles were
determined using the following restriction enzymes:
HpaII, RsaI and CfoI (Promega, Madison, WI, USA). The
16S rRNA gene PCR products were purified from PCR
mixtures using the QIAquick Wizard PCR purification
Kit (Promega), according to manufacturer instructions.
The sequences were determined by cycle sequencing using
the Taq Dye Deoxy Terminator Cycle Sequencing Kit
(Applied Biosystems, HTDS, Tunisia) and underwent
fragment separation in an ABI Prism 3130 DNA sequenc-
ing as previously described (Gtari et al. 2004). Similarity
matrix of 16S rRNA gene sequences with closest neigh-
bours and identification was achieved using RDP utilities
(Ribosomal Database Project II: http://rdp.cme.msu.edu/
html).
Extraction of cellular proteins and electrophoretic
esterase profiling
Extraction of cellular protein was performed as described
by Ouzari et al. (2006). Cells were grown at 30�C for
3–5 days on Luedemann medium, washed twice with
distilled water and suspended in 400 ll of Tris buffer
[100 mmol)1 KCl, 5% w ⁄ v glycerol, 20 mmol)1 Tris, (pH
7Æ5)]. Cells were lysed by sonication for 3 min under
cooling (cycle 0Æ25 s, amplitude 50 W). The resulting
cellular extracts were centrifuged at 20 000 g for 30 min.
Total protein content of the supernatant was evaluated
using the Bradford method (Bradford 1976). Enzyme
extracts were analysed in 12% native PAGE at constant
voltage of 150 V during 14 h in Tris–glycin running
buffer (pH 8Æ3) at 4�C (Sambrook et al. 1989). Upon
migration, the gel was equilibrated at 4�C in 0Æ15 mol l)l
potassium phosphate buffer (pH 6Æ5) for 20 min. The
staining procedure was performed as described by De
Carvalho et al. (1986) by incubating the gel for 30 min
with gentle agitation in potassium phosphate buffer
Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments I. Essoussi et al.
1724 No claim to Tunisian Government works
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732
containing a-naphthyl acetate (a-NA), b-naphthyl acetate
(b-NA), a-naphthyl propionate (a -NP) or b-naphthyl
propionate (b -NP) (1% w ⁄ v in acetone) as substrates.
Esterase activity signals were visualized as deep purple or
black bands in the gel after incubation, in darkness, with
the staining solution (1% w ⁄ v fast Garnet GBC salt in KP
buffer). The evaluation of electrophoretic esterase profile
was repeated twice for each strain on different cellular
extracts.
Statistical analysis of enzyme mobility data was carried
out using the mvsp 3.131 software (MultiVariate Statisti-
cal Package,Kovach, 2004). Bands from all gels were man-
ually detected using as marker esterase bands of a strain
having a specific pattern (DSM43160 = G20) and loaded
in all the gels. Esterase bands were scored by 0 for
absence and 1 for presence of a band with the same elec-
trophoretic mobility. Similarity levels among Geodermato-
philaceae strains were determined by the band-sharing
coefficient calculated by the formula of Jaccard (Sneath
and Sokal 1973). Clustering into groups was performed
by the unweighted pair group method with arithmetic
average (UPGMA).
Enzyme assay
Esterase activity was measured with 1 mm 4-nitrophenyl
acetate as a substrate previously dissolved in acetone in
50 mm phosphate buffer (pH 7). A stock solution of
4-nitrophenyl acetate is prepared by dissolving substrates
in acetone. Measurements were continuously monitored
at 410 nm using SmartSpect� Plus spectrophotometer
(BIO-RAD). The reaction was started by adding enzyme
to the reaction mix. One unit of esterase activity was
defined as the amount of enzyme that catalyses the release
of 1lmol of nitrophenol from 4-nitrophenyl acetate
per min. Measurements were corrected for background
hydrolysis in the absence of enzyme. Measurements were
carried out at least three times, and the molar extinction
coefficient of nitrophenol was determined for every
condition prior to each measurement. Activity was deter-
mined from the initial rate of the hydrolysis reaction.
pH, temperature and cationic strength effects
The effect of pH on esterase activity was studied in a
pH range of 2Æ0–12 using 4-nitrophenyl acetate as a
substrate. The buffers used were 50 mmol l)1 citrate-
phosphate (pH 2Æ2–8Æ0), 50 mmol l)1 borate-NaOH (pH
8–10Æ2) and Na2HPO4-NaOH (pH 10Æ9–12), reaction
mixture was incubated at 30�C for 3 h, and measurements
were monitored at 410 nm. The effect of temperature
on esterase activity was investigated in the range of
20–120�C. Activity was measured after incubating enzyme
solution with 4-nitrophenyl acetate for10 min at each
temperature. The pH of the buffers was adjusted at each
temperature.
Effect of different metal ions (Mg2+, Ca2+, Zn2+, Cu2+,
Mn2+, Co2+) on enzyme activity was determined after
incubating of 2 U of the enzyme with various concentra-
tions (10, 30 and 50 mmol l)1) of different metal salts for
30 min at 30�C. The activity of esterase without addition
of metal ions was defined as 100%. Enzyme activity was
assayed at 410 nm.
Thermostability was measured by incubating the
enzyme extracts at 120�C for various intervals of time,
enzyme activity was assayed towards 4-nitrophenyl acetate
at 410 nm.
Nucleotide sequence accession numbers
The NCBI accession numbers for the 16S rRNA gene
sequences of Geodermatophilaceae BMG isolates deter-
mined in this study are from FJ966172 to FJ966187,
respectively.
Results
Identification of actinobacterial isolates
Fifty-one samples of stones and rocky substrate from
monuments located in Tunisia and Egypt were examined
for the presence of Geodermatophilaceae. Geodermatophil-
aceae-like isolates and the characteristics of their origins
of isolation were listed in Table 1. Twenty-five strains
showing the typical morphology of Geodermatophilaceae
were isolated from 10 of the 51 stone samples analysed.
A unique fragment of approximative 1500 bp was ampli-
fied for all the tested strains with the set of primers used
in this study. Amplified ribosomal DNA restriction analy-
sis (ARDRA), performed on the 25 strains selected
among the 57 isolates and on the 9 Geodermatophilaceae
reference strains, was performed using three restriction
enzymes HpaII, RsaI and CfoI to obtain a significant
sampling of the sequences (Andreoni et al. 2000). Twenty
different haplotypes with different incidence among the
analysed strains have been detected; six of them were
represented each by 2, 3, 4 and 7 isolates (Table 1).
Partial sequencing (nearly 1200 nt) of 16S rRNA genes
processed on 16 strains belonging to each of the identi-
fied haplotypes whereas those from reference strains were
retrieved from public database. All sequences confirm
the membership of the strains to the family Geodermato-
philaceae; 12 strains have been found to be affiliated to
Geodermatophilus genus, 5 strains are clustered with
Blastococcus genus and 3 with Modestobacter genus
(Table 1).
I. Essoussi et al. Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732 1725No claim to Tunisian Government works
Table 1 Sampling locations, genotypic characterization and Phylogenetic affiliation of Geodermatophilaceae strains
Strains Sample
Restriction
enzymes patternsARDRA
types
Esterase
types
Closest identified
relative (accession
number)*MspI RsaI CfoI
Geodermatophilus
obscurus ssp.
dictyosporus
(DSM 43 161)�
Soil, Westward Pass,
California, USA
M2 R2 C3 11 E6
G. obscurus ssp.
obscurus
(DSM 43160T)
Soil, Amargosa Desert,
Nevada, USA
M1 R1 C3 12 E23
G. obscurus ssp.
utahensis (DSM 43 163)
Soil, Zion National Park,
Utah, USA
M1 R1 C3 12 E3
Blastococcus
saxobsidens
DSM 44 509
Globigerine limestone,
Church Sta Marija Ta’Cwerra,
Siggiewi, Malta
M1 R1 C3 21 ND
Geodermatophilaceae
DD13
Calcarenite ancient wall,
Cagliari, Italy
M2 R2 C3 10 E2
Modestobacter
multiseptatus BC501
Carrara marble, Italy M6 R10 C9 14 E6
Blastococcus
aggregatus DS17
Calcarenite ancient wall,
Cagliari, Italy
M2 R2 C4 14 E4
Modestobacter
multiseptatus DS13
Calcarenite ancient wall,
Cagliari, Italy
M2 R2 C4 14 E14
Blastococcus
aggregatu DS10
Calcarenite ancient wall,
Cagliari, Italy
M2 R2 C4 13 E8
Modestobacter
multiseptatus BC499
Carrara marble, Italy M2 R3 C4 14 E7
BMG571 Marble Bulla Regia
monument, Tunisia
M1 R2 C8 1 E26 Geodermatophilus
obscurus (X92359)
BMG575 Marble Bulla Regia
monument,Tunisia
M1 R2 C2 2 E23 Geodermatophilus
obscurus (L40620)
BMG572 Marble Bulla Regia
monument,Tunisia
M1 R2 C2 2 E26 Geodermatophilus
obscurus (X92359)
BMG5712 Limestone Desert, Tunisia M1 R1 C2 3 E11 ND
BMG576 Limestone Tomb
monument, Egypt
M1 R1 C2 3 E21 Geodermatophilus
obscurus (L40620)
BMG5714 Limestone Desert, Tunisia M1 R1 C2 3 E12 Geodermatophilus
obscurus (X92355)
BMG574 Marble Bulla Regia
monument, Tunisia
M1 R1 C2 3 E19 ND
BMG573 Marble Bulla Regia
monument, Tunisia
M1 R1 C2 3 E25 Geodermatophilus
obscurus (X92359)
BMG5711 Marble Bulla Regia
monument, Tunisia
M1 R1 C2 3 E26 Geodermatophilus
obscurus (X92359)
BMG5710 Limestone Bulla
Regia monument, Tunisia
M1 R1 C2 3 E1 ND
BMG5715 Limestone Desert,Tunisia M1 R2 C1 4 ND ND
BMG579 Marble Bulla Regia, Tunisia M1 R1 C1 5 E27 ND
BMG578 Limestone Tomb
monument, Egypt
M1 R1 C1 5 E20 Geodermatophilus
obscurus (X92359)
BMG5729 Marble Bulla Regia
monument, Tunisia
M2 R8 C2 6 E24 Geodermatophilus
obscurus (L40620)
BMG5753 Limestone Bulla
Regia monument, Tunisia
M2 R4 C5 7 E15 ND
Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments I. Essoussi et al.
1726 No claim to Tunisian Government works
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732
Esterase profiling of the isolates
Esterases have been characterized on native PAGE for
the 25 isolates of Geodermatophilus, Blastococcus and
Modestobacter genera with nine reference strains related to
Geodermatophilaceae family (Table 1). Most of the strains
exhibit a complex esterase pattern (Fig. 1). A large varia-
tion in number, colour, intensity and electrophoretic
Table 1 (Continued )
Strains Sample
Restriction enzymes
patternsARDRA
types
Esterase
types
Closest identified
relative (accession
number)*MspI RsaI CfoI
BMG5754 Limestone Tome
monument, Egypt
M2 R6 C5 8 ND Blastococcus jejuensis
(DQ200983)
BMG5756 Marble Bulla Regia
monument,Tunisia
M5 R7 C6 9 E17 ND
BMG5755 Limestone Bulla
Regia monument, Tunisia
M2 R5 C4 15 E16 ND
BMG5726 Marble Bulla
Regia monument, Tunisia
M2 R3 C4 16 E18 Modestobacter
versicolor (AJ871304)
BMG5731 Sandstone Bulla
Regia monument, Tunisia
M2 R3 C4 16 E28 ND
BMG5758 Sandstone Bulla
Regia monument, Tunisia
M2 R3 C4 16 E13 ND
BMG5724 Limestone Khufu
pyramid, Egypt
M4 R3 C4 17 E9 Blastococcus jejuensis
(DQ200983)
BMG5721 Limestone khafre
pyramid, Egypt
M1 R3 C4 18 E10 Blastococcus jejuensis
(DQ200983)
BMG5749 Limestone Worker Tomb
monument, Egypt
M3 R9 C4 19 E29 Blastococcus saxobsidens
(AJ316570)
BMG5737 Sandstone Desert, Tunisia M3 R3 C4 20 E30 Blastococcus jejuensis
(DQ200983)
ND, not determined; ARDRA, Amplified ribosomal DNA restriction analysis.
*The phylogenetic affiliation of the isolates was achieved using RDP utilities (Ribosomal Database Project II: http://rdp.cme.msu.edu/html).
�Reference strains are in bulk.
BM
G5.
7.8
BM
G5.
7.9
BM
G5.
7.10
BM
G5.
7.11
BM
G5.
7.12
BM
G5.
7.1
BM
G5.
7.14
BM
G5.
7.2
BM
G5.
7.4
BM
G5.
7.3
BM
G5.
7.5
BM
G5.
7.6
BM
G5.
7.55
BM
G5.
7.58
BM
G5.
7.56
BM
G5.
7.53
BM
G5.
7.31
BM
G5.
7.26
BM
G5.
7.37
BM
G5.
7.15
BM
G5.
7.24
BM
G5.
7.21
BM
G5.
7.29
BM
G5.
7.49
DS
M43
160
DS
M43
160
DS
M43
163
DS
M43
161
DS
M43
160
DD
13
DS
13
DS
M43
160
DS
10D
S17
BC
498
BC
499
BC
501
DS
M43
160
Figure 1 Esterase patterns of Geodermatophilaceae reference strains and isolates. Esterase activities were revealed by incubating gel slices with
fast garnet GBC salt and a mix of a-naphthyl acetate, b-naphthyl acetate and b-naphthyl propionate.
I. Essoussi et al. Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732 1727No claim to Tunisian Government works
mobility is noted among strains of the same genus and
those of the different studied genera. Strains showed two,
three or multiple esterase bands (2 ‡ n £ 16) that hydroly-
sed substrates with different affinity; red colour bands are
active against b-naphthyl propionate. Those with dark
brown colour are specific to both a and b-naphthyl acetate
(result not shown). A total of 30 polymorphic esterase
bands active against a and b-naphthyl esters of short chain
fatty acids were distinguished assigning the Geodermato-
philaceae-like strains and the reference ones to a 31 distinct
esterase electrophoretic types. Among them only one
(E27) is found to include three strains of Blastococcus
with a complete homology in their esterase patterns. The
remaining 30 esterase types represent individual strains or
group of strains with a low level of similarity (£0Æ4). The
general analysis revealed a large amount of heterogeneity
among the Geodermatophilaceae genera that were very
dissimilar and within strains of the same genus (Fig. 2).
Blastococcus aggregatus DS17 G. obscurus ssp. utahensis DSM43163
Geodermatophilaceae DD13
Geodermatophilus sp. BMG5710
Blastococcus aggregatus DS10
Modestobacter multiseptatus BC499
Modestobacter multiseptatus BC501
G. obscurus ssp. dictyosporus DSM43161
Modestobacter multiseptatus BC498
Modestobacter multiseptatus DS13
Modestobacter sp. BMG5758
Geodermatophilus sp.BMG5714
Geodermatophilus sp.BMG5712
Blastococcus sp.BMG5721
Blastococcus sp.BMG5724
Geodermatophilus sp. BMG578
Geodermatophilus sp. BMG574
Modestobacter sp. BMG5726
Geodermatophilaceae BMG5756
Geodermatophilaceae BMG5755
Geodermatophilaceae BMG5753
Geodermatophilus sp. BMG571
Geodermatophilus sp. BMG573
Geodermatophilus sp. BMG5729
Geodermatophilus sp. BMG575
G. obscurus ssp. obscurus DSM43160
Geodermatophilus sp. BMG576
Blastococcus sp. BMG5737
Blastococcus sp. BMG5749
Modestobacter sp. BMG5731
Geodermatophilus sp. BMG579
Geodermatophilus sp. BMG5711
Geodermatophilus sp. BMG572
0 0·2 0·4 0·6 0·8 1
Figure 2 Phylogenetic relationship between Geodermatophilaceae isolates based on esterase patterns. The dendrogram was generated by
UPGMA ⁄ Jaccard coefficient cluster analysis of esterase band.
Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments I. Essoussi et al.
1728 No claim to Tunisian Government works
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732
Characterization of crude esterase activity
Esterase activity was determined on Geodermatophilaceae
reference strains and two isolates belonging to Geoderma-
tophilus genus (BMG575 and BMG576) using 4-nitrophe-
nyl acetate as substrate. Temperature and pH effects on
crude esterase activity were given in Figs 3 and 4, respec-
tively. The highest esterase activities were observed for
members of Geodermatophilus genus when compared to
those of Blastococcus and Modestobacter that showed a
relatively faint activity. An optimal esterase activity was
obtained around 40�C and at pH 8 for all strains. Ester-
ase from Blastococcus is active between 20 and 50�C and
in a wide pH range from 5 to 12. This activity is kept at
pH ranging from 4 to 12 and rapidly decreased above
60�C for Modestobacter strains. Esterase activities from
Geodermatophilus strains displayed a high resistance to
thermal inactivation and alkaline pH. Enzyme activities
were maintained for temperature higher than 100�C and
retained 30 and 20% of the maximum activity by heating
for 20 min at 120�C and at pH 12, respectively. Enzymes
were completely inactivated after 30 min at 120�C. The
effect of metal ions on Geodermatophilaceae esterase
activity was tested using various metal ions: Ca2+, Mg2+,
Zn2+, Cu2+, Mn2+ and Co2+ at concentrations of 10, 30
and 50 mmol l)1. An inhibitory effect was observed with
concentrations of 30 and 50 mmol l)1, while at 10 mmol
l)1 produced esterase was enhanced strongly in the pres-
ence of Ca2+ and Mg2+ ions, moderately by Zn2+ ions
and markedly inhibited by Cu2+ and Co2+ ions (Table 2).
5
6
7(a)
(b)
(c)
0
1
2
3
4
0 20 40 60 80 100 120 140
Spe
cific
act
ivity
(u
µg–1
)
Temperature (°C)
Spe
cific
act
ivity
(u
µg–1
)
0 20 40 60 80 100 120 140
Temperature (°C)
6
7
2
3
4
5
0
1
Spe
cific
act
ivity
(u
µg–1
)
0 20 40 60 80 100 120 140
Temperature (°C)
5
6
7
8
0
1
2
3
4
Figure 3 Temperature effect on activity of crude esterases of (a)
Blastococcus: , DS10; , DS17; , DSM44509; , DD13.
(b) Modestobacter: , BC501; , BC499; , BC498; , DS13.
(c) Geodermatophilus strains: , DSM43161; , DSM43163;
, DSM43160; , BMG575; , BMG576.
2·5 3
3·5 4
(a)
(b)
(c)
0 0·5
1 1·5
2
0 2 4 6 8 10 12 14pH
Spe
cific
act
ivity
(u
µg–1
)
0 2 4 6 8 10 12 14pH
1·52
2·53
00·5
1
Spe
cific
act
ivity
(u
µg–1
)
0 2 4 6 8 10 12 14pH
2 3 4 5 6 7
0 1
Spe
cific
act
ivity
(u
µg–1
)
Figure 4 pH effect on activity of crude esterases of (a) Blastococcus:
, DS10; , DS17; , DSM44509; , DD13, (b) Modestobact-
er: , BC501; , BC499; , BC498; , DS13. (c) Geodermato-
philus strains: , DSM43161; , DSM43163; , DSM43160;
, BMG575; , BMG576.
I. Essoussi et al. Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732 1729No claim to Tunisian Government works
Discussion
Twenty-five Geodermatophilaceae strains were isolated
from stone samples recovered from Tunisian desert and
monument surfaces in Tunisia and Egypt. Ten of the
51 analysed samples were found to harbour members of
Geodermatophilaceae family. These samples were charac-
terized by a high deterioration pattern described as black
and grey diffused patinas and by phenomena of detach-
ment, crumbling and powdering, typically because of
Geodermatophilaceae colonization (Urzı et al. 2001,
2004). Molecular analysis mainly based on 16S rRNA
gene sequencing grouped the isolated strains into three
groups in accordance with the subdivision of the family
into three genera, Geodermatophilus, Blastococcus and
Modestobacter.
Among them, isolates recovered from crumbly materials
have been affiliated to Geodermatophilus genus suggesting
that members of this genus are more frequently isolated
from internal parts of the rocks. The other isolates have
been clustered with the Blastococcus (five strains) and
Modestobacter (three strains) genera. Members of these
genera are known for their advanced adaptation to extreme
environments (Eppard et al. 1996; Mevs et al. 2000).
Whereof, they possess a well-defined pleomorphism
characterized not only by a transition from coccus to rod
shape (Ishiguro and Wolfe 1970, 1974; Mevs et al. 2000)
but also by the presence of a thick wall and elaboration of
dark coloured pigments that makes them particularly
adapted to survive under stressful conditions related to
high temperature, dryness and low nutrients availability.
Moreover, it has been reported that actinobacteria deprived
of an aerial mycelium such as those resembling to the
family Geodermatophilaceae demonstrate a relatively marked
genotypic and phenotypic diversity on exposed and arid
rock surfaces (Eppard et al. 1996; Urzı et al. 2001).
The esterase enzyme profiling showed a high degree of
genetic variation among strains. This was directly related
to large isoenzymatic dissimilarity among strains of the
same genus and those of different genera. Moreover,
this degree of variation of the enzymatic system proved
that the genetic structures of Geodermatophilaceae are
extremely diverse. Frankia, the close related genus to
Geodermatophilaceae within Frankineae sub-order (Gardes
et al. 1987; Normand 2006a,b), have been also reported
to have a large amount of variation in esterase as well as
highly ecological flexibility.
Cluster analysis performed on electrophoretic esterase
profiles on native polyacrylamide gel well highlighted the
genetic diversity within the Geodermatophilaceae members
with 31 different esterase electrophoretic types generated
for 35 investigated strains. However, no correlation
between esterase clustering has been noted with the taxo-
nomic schema of the family.
Esterase produced by member of Geodermatophilus
genus when compared to that of Blastococcus and
Modestobacter genera displayed a great tolerance to high
temperature that reach up to 100�C, enzyme kept 30% of
its maximal activity after heating for 120�C.
Such thermal stability is required for industrial applica-
tion. Enzymes from extremophiles and thermophiles in
particular are promising in this respect because these
enzymes have a high intrinsic thermal and chemical
stability (Levisson et al. 2007). Bacillus stearothermophilus
(Owusu and Cowan 1991; Shao and Wiegel 1995),
Sulfolobus shibatae (Huddleston et al. 1995), the hyper-
thermophilic archaea Archaeoglobus fulgidus (Manco et al.
2000) and Thermotoga maritima (Levisson et al. 2007)
have been shown to contain thermostable esterases. Under
the effect of certain divalent cations, the produced ester-
ase have been activated strongly in presence of Ca2+ and
Mg2+ ions, moderately by Zn2+ ions and markedly inhib-
ited by Cu2+ and Co2+ ions. These divalent cation may
act as cofactor to these enzymes. The purified esterase
from Arthrobacter nicotinea is inhibited by Ca2+, Cu2+
and Zn2+ and is completely inactivated by Mg2+ (Smacchi
et al. 2000). It has also been reported that Ca2+ and Mg2+
ions had no effect on esterase activity purified from
Brevibacterium linens ATCC9174 (Rattray and Fox 1997);
however, they increased the activity of esterase from
Table 2 Effect of divalent cations on activity of crude esterases
Cation DS10 DS17 DSM44509 BC501 BC499 BC498 DS13 DSM43161 DSM43163 DSM43160 DD13 BMG575 BMG576
Control (100%) 1Æ45 1Æ3 1Æ38 1Æ53 1Æ48 1Æ34 1Æ37 3Æ53 3Æ15 3Æ6 1Æ27 2Æ97 3Æ01
Ca2+ 123 119 125 150 149 151 160 155 159 200 200 163 161
Mg2+ 129 131 131 135 136 131 129 134 134 132 139 139 141
Zn2+ 45 51 51 55 49 43 45 49 53 59 41 57 57
Cu2+ 10 9 9 7 8 8 10 9 8 9 11 8 8
Mn2+ 15 14 13 15 16 16 14 17 17 17 12 16 14
Co2+ 7 6 6 5 6 6 7 8 7 6 9 5 6
Activity was expressed in percentage. 100% correspond to the control without addition of cation and was expressed in U lg)1 protein.
Activity was determined at 10 mmol l)1 for each cation.
Esterase profiling of Geodermatophilaceae isolated from Sahara desert stones and monuments I. Essoussi et al.
1730 No claim to Tunisian Government works
ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732
Lactobacillus plantarum (Gobbetti et al. 1997). This could
be explained by the evolvement of such enzymatic system
to use divalent cations available in their environment as
cofactors.
Enhancement of Geodermatophilaceae esterase activity
in presence of Ca2+ and Mg2+ ions, tolerance to alkaline
pH as well as the thermostability observed among Geoder-
matophilus members could be directly linked to the nat-
ure of the ecological niche hosted by these actinobacteria
and also to the type of the colonized materials. Indeed, it
has been reported that alkaline pH as well as porosity
linked to the stone itself or to its deterioration status
could explain the prevalence of Geodermatophilaceae in
monuments and stone surfaces with marble and calcare-
ous substrates such as carbonate stones (Urzı et al. 2001).
Organic compounds such as aromatic hydrocarbons,
aliphatic incombustibles and other liberated molecules
from lyses of autotrophic bacteria were supposed to be
used as carbon source by actinobacteria occurring on
rock surface and monuments (Caneva et al. 1991). The
extreme conditions of such environment are traditionally
comparable to that in free space or on the Martian sur-
face. In their study on the physiological and metabolic
changes in bacteria that are present in space crafts or
space stations, Leys et al. (2004) have reported that intra-
cellular esterase activity and Ca2+ concentration in Ralso-
nia metallidurans and Rhodospiririllum rubrum were
significantly higher in space.
According to these data, it can be concluded that stone
and monument surfaces provide an ecological complex
habitat suitable for the proliferation of a large range of
actinobacteria that show a great similarity in ecotype but
seems larger different in genotype and phenotype. Other-
wise, the biodiversity of the rock-inhabiting flora when
compared to that of soils microflora is relatively high and
must be considered different from the ones found in the
same ecosystem (Eppard et al. 1996). Study of esterase
electrophoretic polymorphism proved its efficiency for the
differentiation and study of bacterial phylogenetic rela-
tionships and served as complementary taxonomic
approach to highlight population genetics diversity and
phylogeny. However, it was clarified that esterase is not
useful in phylogenetic analysis of Geodermatophilaceae.
The noted flexibility of esterase activities under high tem-
perature, alkaline pH and high cationic concentration
could be a fitting adaptation of these actinobacteria to
such extreme environment represented by calcareous and
limestone substrate under arid conditions.
Acknowledgement
This work was supported by the Tunisian Minister of
High Education, Scientific Research and Technology
(LabMBA grants). We would like to thank Dr David
P. Labeda, Curator of Actinobacterial Culture Collection
USDA ⁄ NRRL for providing Geodermatophilaceae reference
strains.
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ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1723–1732