Acyl-homoserine lactones from Erwinia psidii R. IBSBF 435T, a guava phytopathogen (Psidium guajava L.)

Article abstract of DOI:10.1021/jf050586e

The phytopathogen Erwinia psidii R. IBSBF 435^T causes rot in branches, flowers, and fruits of guava (Psidium guajava L.), being responsible for crop losses, and has no effective control. It was demonstrated that this strain produces two compounds [S-(-)-N-hexanoyl and N-heptanoyl-homoserine lactone], both belonging to the class of quorum-sensing signaling substances. A protocol using gas chromatography - flame ionization detection with chiral stationary phase is described for the absolute configuration determination of a natural acyl-homoserine lactone. Biological assays with specific reporter and synthesis of identified substances are also described. This is the first report on the N-heptanoyl-homoserine lactone occurrence in the Erwinia genus.

Full text of DOI:10.1021/jf050586e

6262 J. Agric. Food Chem. 2005, 53, 6262−6265
T
Acyl-homoserine Lactones from Erwinia psidii R. IBSBF 435 , a
Guava Phytopathogen (Psidium guajava L.)
†
‡
§
ARMANDO M. POMINI, GILSON P. MANFIO, WELINGTON L. ARA UÄ JO, AND
,
†
ANITA J. MARSAIOLI*
Chemistry Institute, State University of Campinas, CP 6154, CEP 13083-970 Campinas - SP, Brazil,
Natura Inova c¸ a˜ o e Tecnologia de Produtos Ltda. Rod. Anhag u¨ era s/n Km 30.5,
CEP 07750-000 Cajamar - SP, Brazil, and Departamento de Gen e´ tica, ESALQ, USP, CP 83,
CEP 13400-970 Piracicaba - SP, Brazil
T
The phytopathogen Erwinia psidii R. IBSBF 435 causes rot in branches, flowers, and fruits of guava
(Psidium guajava L.), being responsible for crop losses, and has no effective control. It was
demonstrated that this strain produces two compounds [S-(-)-N-hexanoyl and N-heptanoyl-
homoserine lactone], both belonging to the class of quorum-sensing signaling substances. A protocol
using gas chromatography-flame ionization detection with chiral stationary phase is described for
the absolute configuration determination of a natural acyl-homoserine lactone. Biological assays with
specific reporter and synthesis of identified substances are also described. This is the first report on
the N-heptanoyl-homoserine lactone occurrence in the Erwinia genus.
KEYWORDS: Acyl-homoserine lactones; Erwinia psidii R.; Psidium guajava L.; GC-MS; quorum-sensing
INTRODUCTION
premature production of virulence factors can alert host defense
systems before a high population density is reached (4).
In Gram-negative bacteria, the main class of signaling
substances is acyl-homoserine lactones (acyl-HSL). The first
study of phenotypic expression control by these substances was
reported for the marine luminescent bacterium Vibrio fischeri
B. In the free-living bacterium, light emission is not observed,
however, in high cellular concentrations, V. fischeri displays
bioluminescence with blue-green light. During growth, this
microorganism releases an autoinducer, N-(3-oxo-hexanoyl)-
HSL, which regulates the expression of genes responsible for
bioluminescence once its critical concentration is reached. This
control of phenotypic expression is essential for this micro-
organism in ecological relationships with the squid Euprymna
scolopes B. Genes controlling synthesis and detection of the
autoinducer are called luxI and luxR, respectively, and are well
characterized in this bacterium (5, 6).
Since its discovery, the quorum-sensing system has been
demonstrated in a great number of Gram-negative bacteria, many
of which employ acyl-HSL in intercellular communication. The
structural diversity of these metabolites relies on the acyl side
chain, while the homoserine lactone moiety is maintained. The
most common structural variations are the chain length, oxo
and hydroxy substituents at the 3 position, and unsaturations.
This variety can be illustrated by N-butanoyl-HSL (biofilm
synthesis control in Pseudomonas aeruginosa S.), N-(3-hydroxy-
butanoyl)-HSL (bioluminescence control in Vibrio harVeyi J.
and S.), N-(3-oxo-octanoyl)-HSL (plasmid conjugal transfer in
Agrobacterium tumefaciens S. and T.), and N-(3-hydroxy-7-
cis-tetradecenoyl)-HSL (expression of rhizosphere genes in
Rhizobium leguminosarum F.) (7).
The guava tree (Psidium guajaVa L.) is a perennial plant
belonging to the Myrtaceae family, popularly known in Brazil
as “goiabeira”. It is cultivated in ca. 11 500 hectares, being an
important commercial culture mainly for small farmers. Their
fruits are appreciated in natura and as juices, candies, and jellies
(1).
One of the major drawbacks to guava production is caused
by the phytopathogen Erwinia psidii R. This disease is
characterized by wilt of new branches (which become black or
brown), lesions and cell death in leaves, and necrosis and
mummification of flowers and fruits. The bacterium is intro-
duced into the orchards by contaminated shoots and spreads
during the pruning process (2).
It has become clear that bacteria do not survive as solitary
microorganisms but exploit elaborate intercellular communica-
tion systems to adapt themselves to the fluctuations of envi-
ronmental conditions by a process known as quorum-sensing.
This process is based on the production of low-molecular weight
diffusible substances, whose extracellular concentrations are
related to the microorganism population density. Upon reaching
the critical concentration, the signaling substances can be
detected by the cells, inducing the population to initiate a
concerted action (3). This control of phenotypic expression may
be of extreme importance in pathogenic infestations, when the
*
To whom correspondence should be addressed. Tel: +55 19 37883098;
e-mail: anita@iqm.unicamp.br.
†
Chemistry Institute, State University of Campinas.
‡
Natura Inova c¸ a˜ o e Tecnologia de Produtos Ltda.
§
Departamento de Gen e´ tica, ESALQ, USP.
1
0.1021/jf050586e CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/01/2005

Acyl-homoserine Lactones from Erwinia psidii
J. Agric. Food Chem., Vol. 53, No. 16, 2005 6263
Many phytopathogens exploit acyl-homoserine lactone based
communication systems. Pectobacterium carotoVorum H.
Erwinia carotoVora J.), which causes the soft rot disease, is
one of the best-studied examples. It has been demonstrated that
the carbapenem antibiotic and plant-cell-wall-degrading enzyme
syntheses are under the control of the same autoinducer, the
N-(3-oxo-hexanoyl)-HSL (8). Furthermore, the communication
system can be disrupted by antagonists, such as halogenated
furanones from the red algae Delisea pulchra M., thus decreas-
ing antibiotic and exoenzyme production (9).
0.5% NaCl, and 0.5% yeast extract (Oxoid). Solid medium was prepared
with 2% agar (Oxoid). X-Gal (5-bromine-4-chloro-3-indolyl-â-D-
galactopyranoside) was purchased from Sigma (Aldrich Chemical Co.,
Milwaukee, WI).
(
Bioassay with Reporter Agrobacterium tumefaciens NTL4(pZLR4).
Inoculums of strains P. aeruginosa CCT 1987, A. tumefaciens NTL4-
T
(
pZLR4), and E. psidii IBSBF 435 were prepared in test tubes with
LB liquid medium (2 mL). These were maintained at 28 °C for 24 h.
To four test tubes containing LB liquid medium (2 mL) the following
solutions were added: tube 1 (blank control): 20 µL of inoculum of
E. psidii and 20 µL of X-Gal (stock solution at 50 mg/mL in DMF);
tube 2 (blank control): 20 µL of inoculum of A. tumefaciens
NTL4(pZLR4) and 20 µL of X-Gal; tube 3 (positive control): 20 µL
of inoculum of A. tumefaciens NTL4(pZLR4), 20 µL of X-Gal, and
This example shows the importance of the quorum-sensing
system during development of some phytopathogenic bacteria
in hosts. Suppression of this mechanism is considered a new
research field to control bacteriosis (10). Therefore, it is
important to understand how these biological mechanisms occur,
starting from the chemical nature of substances involved. The
current communication deals with the acyl-homoserine lactones
produced by Erwinia psidii, a particularly destructive phyto-
pathogen of Brazilian guava crops (P. guajaVa). The study of
possible quorum-sensing signaling substances from this bacte-
2
0 µL of inoculum of P. aeruginosa CCT 1987; tube 4 (test): 20 µL
of inoculum of A. tumefaciens NTL4(pZLR4), 20 µL of X-Gal, and
0 µL of inoculum of E. psidii. The four tubes (in duplicate) were
incubated at 28 °C and the colorations were visually evaluated after
4 h.
Culture of E. psidii and Detection of Acyl-HSL. E. psidii IBSBF
435 was grown in test tubes containing liquid NB medium (10 mL),
2
2
T
was incubated at 30 °C for 24 h, and then was transferred to NB medium
(1L) to be incubated at 30 °C under shaking at 100 rpm. After 24 h,
the culture medium was centrifuged at 5000 rpm for 20 min under
refrigeration (5 °C). The aqueous medium was extracted with ethyl
acetate (3 × 500 mL). The combined organic phases (1.5 L) were
washed with distilled water (1 × 500 mL), dried over anhydrous sodium
sulfate, and evaporated under reduced pressure at 50 °C. The whole
procedure was repeated eight times yielding a crude extract (0.728 g),
which was separated by silica column chromatography (15 g) eluted
with hexane, dichloromethane, and ethyl acetate, recovering 84 fractions
of 50 mL, monitored by TLC. Similar fractions were combined and
analyzed by GC-MS. Acyl-HSL were detected in fractions FRA32 (2.0
mg) and FRA33-35 (2.5 mg).
T
rium was carried out employing the IBSBF 435 type strain,
isolated from infected guava trees.
MATERIALS AND METHODS
General Experimental Procedures. NMR spectroscopic data were
acquired from a Varian Inova spectrometer, operating at 499.88 MHz
1
13
for H NMR and 125.71 MHz for C NMR. CDCl
3
was used as the
0.0 ppm).
solvent and TMS as the internal reference (δ and δ
H
C
Chemical shifts δ were recorded in ppm, and coupling constants J were
recorded in Hertz (Hz). Optical rotation was measured on a Perkin-
Elmer 341 polarimeter at 17 °C, and the results were converted to 20
+
°
C by the usual equations. Fourier transform infrared (FTIR) spectra
N-Hexanoyl-HSL. GC-MS (EI, 70 eV) m/z: 199 (M , 2), 156 (12),
were obtained with a Bomem MB Michelson spectrometer, using KBr
143 (100), 125 (21), 115 (6), 102 (12), 101 (15), 99 (22), 57 (45), 43
(47).
N-Heptanoyl-HSL. GC-MS (EI, 70 eV, SIM) m/z: 213 (M , 1), 156
(Merck, Darmstadt, Germany) as sample support. Silica gel for CC
+
(0.035-0.070 mm) was from Merck. TLC analyses were made on silica
gel 60 F254 plates (Merck) and were visualized under exposure to UV
light (254 nm) or chemically with solution of p-anisaldehyde (5%),
acetic acid (50 mL), and concentrated sulfuric acid (1 mL) and were
heated to 90-100 °C for 5 min.
(10), 143 (100), 125 (20), 113 (15), 102 (14), 101 (15), 85 (16), 57
(52), 43 (40).
Acyl-HSL Synthesis. General Procedure. To a round-bottom flask
-
4
(5 mL) containing distilled water (2.5 mL), triethylamine (1.05 × 10
GC-MS Analysis. GC-MS analyses were carried out on an HP 6890/
mol), R-amino-γ-butyrolactone hydrobromide [racemic or S-(-), Al-
-4
5
973 instrument, equipped with a 30 m × 0.25 mm × 0.25 µm i.d.
drich Chemical Co., Milwaukee, WI] (1.05 × 10 mol), and hexanoic
-4
HP5 fused silica capillary column. Mass spectra were recorded over
or heptanoic acids (1.57 × 10 mol) were added. To the stirred solution
the 40-450 amu range at 3.54 scans/s, with an ionization energy of
at room temperature, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
-
3
7
0 eV. Helium was the carrier gas at a flow rate of 1 mL/min. The
hydrochloride (1.57 × 10 mol) was added. The reaction was further
stirred at room temperature for 24 h. Extraction with ethyl acetate (3
× 10 mL) and the usual workup yielded pure acyl-HSL.
injector temperature was maintained at 250 °C. The initial oven
temperature was 100 °C and was programmed to increase to 290 °C at
1
0 °C/min and then was held for 10 min. One-microliter samples were
(()-N-Hexanoyl-homoserine Lactone. 54% yield. GC-MS (EI, 70
1
13
injected, without splitting.
Chiral GC-FID Analyses. Chiral gas chromatography analyses were
carried out with an HP 6890 instrument with flame ionization detection
eV) data were identical to the natural product. IR, H, and C NMR
data were consistent with those previously reported (11, 12).
S-(-)-Hexanoyl-homoserine Lactone. 54% yield. IR, GC-MS, 13C,
1
(
FID), equipped with a 25.0 m × 250.0 µm × 25.0 µm chiral capillary
H NMR, DEPT-135, and DEPT-90 data were identical to those
obtained for the racemic compound. [R]20
-22.86° (c. 0.35 MeOH).
column Chrompack CP chirasil-dex coating 7502 (Chrompack Inter-
national BV, 4330 EA Middelburg, The Netherlands). The initial oven
temperature was 50 °C and was programmed to increase at 2 °C/min
to 180 °C and then was held for 5 min. Hydrogen was the carrier gas
at a flow rate of 1 mL/min. The injector and detector (FID) temperatures
were kept at 220 °C and 240 °C, respectively. One-microliter samples
were injected, with a 1:100 split ratio.
D
(()-N-Heptanoyl-homoserine Lactone. 64% yield. GC-MS (IE, 70
eV) data were identical to the natural product. IR (KBr) 3315 (m),
-
1
2955 (m), 1776 (s), 1646 (s), 1545 (m), 1173 (m), 1013 (m), 646 cm
1
(w). H NMR (499.88 MHz, TMS, CDCl
3
) δ (ppm) 0.88 (t, 3, J )
H
7.3 Hz, H-7′), 1.29 (m, 6, H-4′, H-5′, H-6′), 1.64 (quintet, 2, J ) 7.6
Hz, H-3′), 2.14 (m, 1, H-4), 2.25 (t, 1, J ) 8.9 Hz, H-2′), 2.84 (m, 1,
H-4), 4.28 (ddd, 1, J ) 5.8, 11.3, 9.5 Hz, H-5), 4.47 (t, 1, J ) 8.9 Hz,
H-5), 4.56 (ddd, H, J ) 5.8, 11.6, 8.6 Hz, H-3), 6.15 (d, 1, J ) 3.7 Hz,
Bacterial Strains and Cultivation Media. The strain Erwinia psidii
T
IBSBF 435 ()ATCC 49406 type strain) was kindly provided by Dr.
NH). 13C NMR (125.71 MHz, TMS, CDCl
J u´ lio Rodrigues Neto, curator of the collection of cultures of Instituto
Biol o´ gico de S a˜ o Paulo, Campinas, Brazil. It was isolated from Psidium
guajaVa L. 1982, Brazil and maintained in solid nutrient broth (NB)
medium. Indicator strain Agrobacterium tumefaciens NTL4(pZLR4)
was maintained in Luria-Bertani (LB) medium supplemented with
gentamicin (50 µg/mL). Pseudomonas aeruginosa CCT 1987 was
maintained in solid NB medium. NB medium (20 g/L) was from Oxoid
3 C
) δ (ppm) 14.0 (C-7′);
22.4 (C-6′); 25.3 (C-3′); 28.8 (C-5′); 30.5 (C-4); 31.4 (C-4′); 36.1 (C-
2′); 49.2 (C-3); 66.1 (C-5); 173.7 (C-1′); 175.5 (C-2).
Biological Assays with Extract, Fractions, and Synthetic Prod-
ucts. Biological activities of the synthetic products, ethyl acetate extract,
and fractions from E. psidii culture medium with biosensor A.
tumefaciens NTL4(pZLR4) were evaluated as described above. The
tests were performed using solutions (20 µL) of each synthetic product
(Hampshire, England). LB medium is composed of 1% peptone (Oxoid),

6264 J. Agric. Food Chem., Vol. 53, No. 16, 2005
Pomini et al.
Table 1. Results of Chiral GC-FID Analyses and Absolute Configuration Determination of Natural S-(
−)-N-Hexanoyl-HSL
(
±
)-N-he xanoyl-HSL
S-(-)-N-hexanoyl-HSL
S
FRA32 from E. psidii IBSBF435T
S
FRA32
R
+ (±)-N-h exanoyl -HSL
enantiomers
R
S
S
retention times (min)
relative abundances (%)
56.72
50.00
56.89
50.00
56.93
96.34
56.97
>99.00
56.79
42.16
56.96
57.83
∼
∼
[(()-N-hexanoyl-HSL, S-(-)-N-hexanoyl-HSL, and (()-N-heptanoyl-
HSL], ethyl acetate extract from E. psidii cultivation medium, and
fraction FRA32 in ethanol (2 mg/mL). The blank was performed with
ethanol (20 µL).
N-Hexanoyl-HSL Absolute Configuration. The optimum analytical
conditions for the enantiomeric discrimination were established using
synthetic (()-N-hexanoyl-HSL (two peaks in an approximately 1:1
ratio), according to the conditions described above. The identification
of the R- and S-N-hexanoyl-HSL retention times was obtained by
analysis of the synthetic S-(-)-N-hexanoyl-HSL. The absolute config-
uration of the natural product was determined by comparing the
retention times and relative abundances of the R- and S-(-)-N-hexanoyl-
HSL in the synthetic racemic standard, in FRA32, and by co-injection
of both samples (Table 1).
Figure 1. Acyl-homoserine lactones identified in extracts from Erwinia
psidii IBSBF 435 culture medium and origin of the diagnostic base peak
T
fragments in MS spectra.
RESULTS AND DISCUSSION
Initially, the production of acyl-homoserine lactones in
E. psidii was evaluated using Agrobacterium tumefaciens
NTL4(pZLR4) as reporter, by a methodology adapted from the
literature (13, 14). This bioassay has been extensively employed
in screenings of acyl-HSL producers, mainly because of its
simplicity, quickness, and high sensitivity. This mutant cannot
produce its own acyl-HSL by deletion of the traI gene. The
detection system was inserted by the pZLR4 plasmid, which
contains the traR gene and the fusion traG::lacZ. Exogenous
acyl-HSL bind to the TraR receptor, and this complex regulates
the expression of lacZ gene, which codifies the synthesis of
â-galactosidase enzymes. Finally, the reagent X-Gal is metabo-
lized by the â-galactosidase enzymes, yielding a blue color.
Bioassays with E. psidii and P. aeruginosa (used as positive
control) (15) exhibited blue coloration, furnishing evidence of
the presence of acyl-HSL in both strains.
As previously reported (15, 16), acyl-HSL are usually
synthesized in low concentrations, requiring therefore large
amounts of culture medium for spectroscopic investigations.
Consequently, E. psidii extracts from 8 L of culture medium
were purified by column chromatography, and fractions were
analyzed by GC-MS. N-Hexanoyl-HSL was detected in 7% of
relative abundance in fraction FRA32 (a mixture of 2.0 mg,)
and in 1% in FRA33-35 (a mixture of 2.5 mg), and trace
amounts of N-heptanoyl-HSL were identified in fraction FRA33-
these substances are produced in low amounts and their analysis
requires appropriate methodologies; in the present case, the
chiral configuration of N-hexanoyl-HSL was determined using
gas chromatographic analyses with chiral stationary phase and
flame ionization detection. The optimum analytical conditions
for the enantiomeric separation were obtained using a synthetic
racemic mixture (R stereoisomer at 56.72 min and S stereo-
isomer at 56.89 min). Elution of synthetic stereoisomer S-
(-)-N-hexanoyl-HSL (92% ee) under the same conditions
furnished the retention time (56.93 min) for the S stereoisomer.
Analysis of fraction FRA32, obtained from E. psidii culture
medium, displayed a signal with retention time (56.97 min) very
close to that of the synthetic S stereoisomer. Co-injection of
the natural product and of the synthetic racemic mixture resulted
in perfect signal overlap, with increase in the relative abundance
of the S stereoisomer (56.96 min) in comparison with that of
the R stereoisomer (56.79 min). Thus, the natural product was
the S-(-)-N-hexanoyl-HSL (Table 1). The N-heptanoyl-HSL
is a minor constituent (trace amounts), and the same procedure
could not be applied. The methodology proved to be very
efficient in situations where the amount of substance is small
or when they are present in complex mixtures. Furthermore,
chiral GC-FID is applied to determine the absolute configuration
of a natural acyl-homoserine lactone for the first time, providing
an additional tool in quorum-sensing researches.
3
5 (Figure 1). To improve the detection, some analyses were
In conclusion, it was demonstrated that Erwinia psidii IBSBF
435 produces acyl-HSL, which are well-recognized substances
T
carried out in the SIM mode. These substances were not detected
in the blank analyses with NB medium (data not shown).
The final structural confirmation of both acyl-HSL was
carried out by co-injection and fragmentation pattern comparison
of the natural products with the synthetic ones in GC-MS. These
synthetic standards were fully characterized by FTIR, GC-MS,
of the bacterial intercellular communication systems. N-hex-
anoyl-HSL is relatively common in quorum-sensing of Gram-
negative bacteria, for example, controlling the violacein pro-
duction (17) in Chromobacterium Violaceum B., and is reported
in trace concentrations in the phytopathogen Pectobacterium
chrysanthemi S. (18). However, N-heptanoyl-HSL, with an odd
carbon number acyl side chain, is an autoinducer of rare
occurrence, previously reported in Serratia marcescens B. (19)
and in Rhizobium leguminosarum (20). Therefore, this is the
first report of this substance in Erwinia genus. Odd-chain fatty
acids are biosynthesized by a route that exploits propionyl-CoA
and malonyl-CoA as starter and extender of the acyl chain,
respectively. Possibly, this biosynthetic route is explored by
these microorganisms in the production of N-heptanoyl-HSL
(21).
1
13
H, and C NMR spectroscopy in one- and two-dimensions
(1
H, H gCOSY; H, C J HSQC; H, C J gHMBC, n ) 2
1
1
13
1
1
13
n
1
3
and 3; C NMR DEPT-135 and DEPT-90; data not shown).
The synthetic substances, ethyl acetate extract, and fraction
FRA32 from E. psidii cultivation medium also exhibited positive
bioassays with A. tumefaciens NTL4(pZLR4) reporter, cor-
roborating the biological activity observed with the strain E.
T
psidii IBSBF 435 in vivo.
The absolute configuration of acyl-homoserine lactones plays
an important role in communication systems (11). However,

Acyl-homoserine Lactones from Erwinia psidii
J. Agric. Food Chem., Vol. 53, No. 16, 2005 6265
Further work should be done to understand the role of these
substances in the intercellular communication system in E. psidii.
As interferences in quorum-sensing mechanisms are promising
avenues for bacterial control, it is expected that these studies
could provide an alternative method to detain this guava
phytopathogen.
(11) Chhabra, S. R.; Stead, P.; Bainton, N. J.; Salmond, G. P. C.;
Stewart, G. S. A. B.; Williams, P.; Bycroft, B. W. Autoregulation
of carbapenem biosynthesis in Erwinia carotoVora by analogues
of N-(3-oxohexanoyl)-L-homoserine lactone. J. Antibiot. 1993,
4
6, 441-454.
(
12) Lao, W.; Kjelleberg, S.; Kumar, N.; deNys, R.; Read, R. W.;
Steinberg, P. 13C NMR study of N-acyl-S-homoserine lactone
derivatives. Magn. Reson. Chem. 1999, 37, 157-158.
ABBREVIATIONS USED
(
13) Ravn, L.; Christensen, A. B.; Molin, S.; Givskov, M.; Gram, L.
Methods for detecting acylated homoserine lactones produced
by Gram-negative bacteria and their application in studies of
AHL-production kinetics. J. Microbiol. Methods 2001, 44, 239-
HSL, homoserine lactone; X-Gal, 5-bromine-4-chloro-3-
indolyl-â-D-galactopyranoside.
2
51.
ACKNOWLEDGMENT
(
14) Cha, C.; Gao, P.; Chen, Y.-C.; Shaw, P. D.; Farrand, S. K.
Production of acyl-homoserine lactone quorum-sensing signals
by Gram-negative plant-associated bacteria. Mol. Plant-Microbe
Interact. 1998, 11, 1119-1129.
15) Pearson, J. P.; Gray, K. M.; Passador, L.; Tucker, K. D.;
Eberhard, A.; Iglewski, B. H.; Greenberg, E. P. Structure of the
autoinducer required for expression of Pseudomonas aeruginosa
virulence genes. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 197-
We wish to express our gratitude to Carol H. Collins (IQ/
UNICAMP) for revising the manuscript.
(
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