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Keywords:

  • Streptomyces lunalinharesii;
  • chitin;
  • LEA (lectin from Lycopersicum esculentum);
  • IR spectroscopy;
  • NMR spectroscopy;
  • transmission electron microscopy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chitin from Streptomyces lunalinharesii spores, detected on its outermost surface layer, was isolated and characterized by chemical and spectroscopic methods, transmission electron microscopy and flow cytometry analysis. Gold–chitinase- and gold–lectin (Lycopersicum esculentum agglutinin, LEA)-conjugated labels were used in microscopy experiments, whereas a fluorescence–lectin (LEA) conjugate was used in flow cytometry analysis. Chitin isolation consisted of several steps of hot alkali and nitrous acid treatment, and the final material was obtained in the colloidal form. The infrared and the 13C CP/MAS NMR spectra of Streptomyces sp. colloidal chitin and colloidal chitin obtained from commercial crab shell chitin were very similar. Incubation of the spores with gold-labeled lectin, or gold-labeled recombinant chitinase, showed the presence of gold particles around the spore surface, indicating the specific binding of the lectin or the recombinant chitinase with the chitin present on the outermost surface. Flow cytometry analysis, using the fluorescence–lectin conjugate, confirmed these results. According to scanning electron microscopy, S. lunalinharesii presented spore surface ornamentation belonging to the spiny group. This is the first detailed characterization of chitin on the spore's outermost layer from a Streptomyces species.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chitin is a linear polysaccharide composed of (1[RIGHTWARDS ARROW]4)-linked 2-acetamido-2-deoxy-β-d-glucopyranosyl units occurring in the biosphere (Gooday, 1990). Although the most abundant reservoirs of chitin in nature are the cell wall of fungi and exoskeleton of arthropods, this polysaccharide is also present in protists and higher animals (Mulisch, 1993; Zhang et al., 2005). Studies relating its presence in eubacteria are rare because there is no information about this subject in the literature in the last few years (Cabib, 1987; Gooday, 1990; Mulisch, 1993; Dahiya et al., 2006; Gohel et al., 2006).

The genus Streptomyces comprises a group of bacteria (actinomycetes) with elongated cells or filaments that usually show some degree of true branching. Streptomyces spore ornamentation has been considered a stable and therefore a useful taxonomic characteristic (Shirling & Gottlieb, 1966). According to spore morphology, there is an extracellular, relatively delicate fibrous sheath, loosely attached to free mature spores that covers the spores of most actinomycete genera during their formation (Locci & Sharples, 1984). Smucker & Pfister (1978) have already shown the presence of chitin in Streptomyces coelicolor rodlet mosaic. This structure is placed just outside the spore wall, in an internal position in relation to the external sheath, and was suggested to be composed of interwoven parallel fiber pairs, these fibrils being carbohydrate polymers of N-acetylated glucosamine. More recently, Claessen et al. (2004) have demonstrated that the rodlet layer covering the smooth surface of some Streptomyces species is mainly composed of two essential proteins called rodlins and chaplins. In conclusion, chitin localization in Streptomyces spores remains unclear.

Streptomyces lunalinharesii strain RCQ 1071, a highly chitinolytic actinomycete recently described as a new species (Souza et al., 2008), was isolated from a Brazilian cerrado soil, and is promising for use in biological control against phytopathogenic fungi (Gomes et al., 2001). The aim of the present work was the isolation and characterization of chitin in S. lunalinharesii spores, using infrared and nuclear magnetic resonance (NMR) spectroscopic analysis, and the detection and localization of chitin on the outermost sheath spore, by electron microscopy and flow cytometry analysis. A lectin (LEA –Lycopersicum esculentum agglutinin) specific for N-acetyl-d-glucosamine-chitotriose and a purified recombinant chitinase (rec-chitinase) from Vibrio parahemolyticus were used for specific recognition of chitin on the spore surface.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Microorganism

Streptomyces lunalinharesii strain RCQ 1071 was previously isolated from a Brazilian Cerrado soil, located on the Central Plateau, Brasilia, Distrito Federal, Brazil (Gomes et al., 2001). It has been maintained in 20% glycerol at −20 °C. After growth in yeast extract–malt extract–agar plates (Shirling & Gottlieb, 1966) for 14 days at 28 °C, spore suspension was obtained according to Hopwood et al. (1985).

Chitin isolation

Obtainment of the alkali-insoluble fraction

The alkali-insoluble fraction from spores of S. lunalinharesii was obtained as described previously (Hearn & Sietsma, 1994). Briefly, lyophilized spores were treated twice with 1 M KOH for 20 min at 60 °C, under N2, and the insoluble material was collected by centrifugation (at 3000 g for 10 min), washed five times with water and lyophilized. The residue was suspended in 2 M sodium nitrite, 2 M HCl and water (3.0 : 1.0 : 2.0 v/v) and subjected to vigorous stirring for 90 min at room temperature. The insoluble fraction was collected by centrifugation, washed with water and further treated with 1 M KOH at 60 °C for 20 min. The alkali-insoluble residue was washed five times with water, once with 64% (v/v) ethanol and lyophilized.

Obtainment of colloidal chitin

The alkali-insoluble residue obtained from S. lunalinharesii spores was initially bleached with 5.25% commercial sodium hypochlorite and stirred overnight at room temperature. Then it was washed five times with water, followed by centrifugation (3000 g for 10 min) to remove the bleach, and freeze dried. Then, it was dissolved in concentrated HCl by stirring for 30 min at room temperature. The soluble fraction was precipitated as a colloidal suspension by the slow addition of water at 4 °C. The precipitate was collected by centrifugation, washed five times with water and lyophilized (Hsu & Lockwood, 1975). Chitin from a commercial crab shell (Sigma Chemical Co., St Louis, MO) was subjected to the same procedure, in order to obtain standard colloidal chitin.

Chitin characterization

Infrared studies in solid state

Measurements were carried out on a Nicolet Magna 550 FT-IR spectrophotometer using a KBr pellet method. KBr discs were prepared according to Stevenson & Gohn (1974), from dry mixtures of about 1 mg of the sample and 100 mg of KBr. Both colloidal chitin, from S. lunalinharesii and from commercial crab shell (Sigma), were subjected to this procedure.

Solid-state 13C cross-polarization magic-angle-spinning (CP/MAS)–NMR spectroscopy

13C CP/MAS-NMR spectra were recorded at 100 MHz on a Varian Inova 300 spectrometer operated at room temperature at a rotating frequency of 4000 c.p.s.

Chitin detection

Transmission electron microscopy (TEM)
Pre-embedding colloidal gold labeling

Spores were washed with phosphate-buffered saline (PBS), pH 7.2, and incubated in PBS-bovine serum albumin (BSA) 1%, for 60 min at room temperature in the presence of gold-labeled LEA lectin, specific for N-acetyl-d-glucosamine-chitotriose (E-Y Laboratories, San Mateo, CA), diluted 1 : 20, or in the presence of gold-labeled rec-chitinase. Spores were rinsed in PBS and then fixed in a solution containing 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. After fixation, the spores were washed three times with cacodylate buffer, postfixed in osmium tetroxide and dehydrated in graded acetone. The infiltration was performed in PolyBed resin and the polymerization was performed at 60 °C for a duration of 3 days. Ultrathin sections were collected on 400-mesh copper grids, stained with uranyl acetate and lead citrate and observed in a transmission electron microscope (Zeiss EM-900). Controls consisted of the bacterial spores treated with purified rec-chitinase (0.1 U for 16 h at 42 °C in PBS, pH 6.0) from V. parahemolyticus (V-Labs Inc., Convington, LA).

Preparation of chitinase–gold complex

Purified rec-chitinase was used. Gold particles, 8–10 nm in diameter, were prepared according to Frens (1973). The minimal amount of chitinase required to stabilize a given volume of colloidal gold was visually determined by the salt flocculation test at pH 7.0 (Geoghegan & Ackerman, 1977). Concentration and purification of the complex was achieved by ultracentrifugation for 45 min at 4 °C, using a fixed-angle rotor with an acceleration of 60 000 g. Following centrifugation, the supernatant was carefully aspirated, and the pellet containing the protein–gold complexes was resuspended in PBS containing 0.02% (w/v) PEG 20 000 and 0.2% (w/v) NaN3 and stored at 4 °C.

Flow cytometry analysis

Washed spores of S. lunalinharesii were fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, for 30 min at room temperature. Thereafter, spores were washed in PBS and incubated in the presence of rec-chitinase from V. parahemolyticus (0.1 U at 42 °C) in PBS, pH 6.0, for 24 h. Treated and untreated rec-chitinase spores were then washed three times in PBS-BSA and incubated with a lectin-fluorescein conjugate of LEA–fluorescein isothiocianate (FITC) (1 μg mL−1 for 15 min) at room temperature. Finally, cells were washed three times with PBS and examined in a fluorescence-activated cell sorter (FACS) Epics Elite flow cytometer (Coulter Eletronics, Hialeah, FL) equipped with a 15-mW argon laser emitting at 488 nm. Untreated spores were used as control.

Analysis of the spore surface ornamentation

This was determined by scanning electron microscopy (SEM), based on Garcia, (1995). Streptomyces lunalinharesii was grown as lawns on cellophane films placed on solid glucose–asparagine–yeast extract medium (Shirling & Gottlieb, 1966). Spores were carefully removed from the cellophane with PBS, fixed in osmium tetroxide vapor and then dehydrated. The samples were coated with gold, and the surface morphology of the spores and their imprints was examined by a JEOL JSM 6100 scanning electron microscope at 15 kV.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Infrared studies in solid state

The infrared spectrum of the chitinous materials from S. lunalinharesii spores (Fig. 1a) shared several bands with the spectrum of the standard colloidal crab shell chitin (Fig. 1b). Two amide bands ascribed to the CONH group vibration modes appeared at 1659 cm−1 (H-bonded C=O stretching of amide I) and 1557 cm−1 (H-bonded NH bending of amide II), respectively. Also, a vibrational absorption band at 1378 cm−1, assigned to the rocking of the methyl group, could be observed in both spectra. The absorption peak at 1540 cm−1 assigned to the stretching vibration of protein was absent (Matján et al., 2007).

image

Figure 1.  Infrared spectra of chitinous material (a) isolated from spores of Streptomyces lunalinharesii and (b) standard colloidal chitin from crab shell. Typical bands at 1655 (amide I) and 1550 (amide II) are indicated by arrows.

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Solid-state 13C CP/MAS-NMR spectroscopy analysis

NMR analysis of the S. lunalinharesii colloidal chitin gave peak patterns similar to those of commercial colloidal chitin from crab shell (Fig. 2a and b). Each spectrum presented six well-defined signals between 50 and 110 p.p.m. arising from resonances of C1–C6 carbons of the N-acetylglucosamine monomeric unit, indicating high structural homogeneity (Table 1). The methyl and the carbonyl of the acetyl group of chitin gave rise to signals at 23.24 and 174 p.p.m., respectively.

image

Figure 2. 13C CP/MAS NMR spectra of (a) chitinous material isolated from Streptomyces lunalinharesii spores and (b) standard colloidal chitin from crab shell. Typical signals at 76.051 and 73.947, corresponding to C5 and C3, respectively.

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Table 1. 13C CP/MAS-NMR spectral data of the Streptomyces lunalinharesii and crab chitin (Sigma)
13C signal assignmentChitin samples
Streptomyces lunalinharesiiCrab (Sigma)
C=0174.618174.67
C-1104.415104.307
C-483.73983.671
C-576.05175.984
C-373.94773.920
C-661.44461.417
C-255.65855.591
CH323.24821.180

Chitin detection

After incubation of the spores with gold-labeled lectin (LEA), the presence of gold particles around the spore surface (Fig. 3b–d) was detected by TEM, indicating the specific binding of the lectin with the chitin present on the outermost spore surface. No labeling of the microorganism was observed in the control, where spores were previously treated with rec-chitinase (Fig. 3e and f). In a second experiment, spores were incubated with gold-labeled rec-chitinase, and the presence of gold particles on the outermost spore surface could also be seen (Fig. 4), indicating the chitin–chitinase linkage. No labeling was observed in the control, using spores previously treated with rec-chitinase (data not shown).

image

Figure 3.  Ultrastructural analysis of Streptomyces lunalinharesii spores. SEM shows the spiny spore surface (arrows) (a, scale bar=0.2 μm). Cytochemical analysis at the TEM of S. lunalinharesii incubated in the presence of gold-labeled Lycopersicum esculentum lectin shows an intense labeling of the spores (arrows) (b, scale bar=0.2 μm; c, scale bar=0.2 μm; d, scale bar=0.5 μm). When spores were previously treated with rec-chitinase from Vibrio parahemolyticus for 24 h, no labeling was observed (e, scale bar=0.3 μm and f, scale bar=0.2 μm).

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image

Figure 4.  Cytochemical analysis at the TEM of Streptomyces lunalinharesii spores after incubation with gold-labeled rec-chitinase shows the presence of gold particles on the surface of the spores.

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In flow cytometry analysis, S. lunalinharesii spores incubated with FITC–LEA showed an intense fluorescence (Fig. 5b), as compared with control (Fig. 5a). The fluorescence intensity of the spores was significantly decreased after treatment with rec-chitinase for 24 h (Fig. 5c), where most of the spores incubated with FICT–LEA had lost the labeling capacity. These results indicated, once more, the presence of chitin on the spore surface.

image

Figure 5.  FACS showing the fluorescence of FITC–LEA binding to Streptomyces lunalinharesii spores. (a) Control, unlabeled spores, including auto-fluorescence and fluorescence background (region I); (b) spores incubated with FITC–LEA lectin, showing the fluorescence intensity dislocated to the right as compared with control (region J); (c) spores treated with chitinase for 24 h and incubated with FITC–LEA lectin, showing dislocation of fluorescence to region I, as observed in control (a).

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Spore ornamentation

The spore surface of actinomycetes is currently used as a taxonomic marker, being spiny, hairy, warty or smooth. According to Fig. 3a, S. lunalinharesii presents spore surface ornamentation belonging to the spiny group (Tresner et al., 1961).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the present work, we isolated and characterized chitin from the outermost surface layer of S. lunalinharesii spores, which belong to the spiny group ornamentation. Studies concerning the detection of chitin on the spore surface of actinomycetes are not common. According to the literature, the presence of chitin was detected previously only in the inner layer of spores of S. coelicolor (Smucker & Pfister, 1978) and Streptomyces bambergiensis (Smucker & Morin, 1985). The combination of sensitive and powerful techniques such as NMR spectroscopy, TEM and flow cytometry analysis has allowed us to show the presence of chitin on spore outermost surface of the chitinolytic actinomycete S. lunalinharesii and to characterize its structure.

Chitin characterization on S. lunalinharesii spores was achieved by infrared and solid-state 13C CP/MAS-NMR spectroscopy. Colloidal chitin from spores and colloidal chitin from crab shell exhibited very similar 13C NMR and infrared spectra. These spectra were also very similar to those described by Matján et al. (2007) for a bumblebee chitin, and also from the Aspergillus niger chitin (Teng et al., 2001).

Chitin was first detected in Streptomyces by Smucker & Pfister (1978). According to their model, based on their study with the smooth spores of S. coelicolor, fibrils of N-acetylated glucosamine-containing polymers would be part of the rodlet mosaic, placed just outside the two spore wall layers, but in an inner position in relation to the sheath observed on the outermost surface. In our study, according to electron microscopy and flow cytometry analysis, we can suggest that S. lunalinharesii chitin is located on the outermost layer of its spore, in the relatively delicate sheath. In S. bambergiensis (Smucker & Morin, 1985), a hairy spore surface actinomycete, as in S. coelicolor (Smucker & Pfister, 1978), chitin was detected in an inner layer, because the colloidal chitin–gold chitinase complex only labeled the spore surface in the sites corresponding to the broken hairs of the spore.

In conclusion, the presence of chitinous components was detected on the spore surface of S. lunalinharesii and their structure elucidation was confirmed. Moreover, according to the electron microscopy results, we can suggest that S. lunalinharesii, different from the other two Streptomyces species described in the literature (Smucker & Pfister, 1978; Smucker & Morin, 1985), presents the chitinous material located on the outermost layer of the spore, in the delicate, loosely attached sheath. This is the first description of the presence of chitin on the outer sheath of a Streptomyces species spore.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Brazilian agencies CNPq, FAPERJ, CAPES and FINEP-BID. We thank Dr Eliana Barreto-Bergter for critically reading the manuscript, Dr Pedro Persechini for the use of the flow cytofluorometer and Dr Wanderley de Souza for the use of the TEM. We also thank Dr Andrew Macrae for revising the English language.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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