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: maher.gtari@fst.rnu.tn

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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