ARTICLE IN PRESS

Microbiological Research 163 (2008) 200207

www.elsevier.de/micres

Production of an antimicrobial substance against Cryptococcus neoformans by Paenibacillus brasilensis Sa3 isolated from the rhizosphere of Kalanchoe brasiliensis
Tiago Oliveira Fortesa,b, Daniela Sales Alvianob, Gleiser Tupinambab, c d Tha Souto Padron , Angelo Roberto Antoniolli , s Celuta Sales Alvianob, Lucy Seldina,
a

Laboratorio de Genetica Microbiana, Centro de Ciencias da Saude, Instituto de Microbiologia Prof. Paulo de Goes, ` Universidade Federal do Rio de Janeiro, Bloco I, Ilha do Fundao, CEP 21941-590 Rio de Janeiro, RJ, Brasil b Laboratorio de Estruturas de Superf cie de Microrganismos, Centro de Ciencias da Saude, Instituto de Microbiologia ` Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Bloco I, Ilha do Fundao, CEP 21941-590 Rio de Janeiro, RJ, Brasil c Laboratorio de Biologia Celular e Ultraestrutura, Centro de Ciencias da Saude, Instituto de Microbiologia Prof. Paulo ` de Goes, Universidade Federal do Rio de Janeiro, Bloco I, Ilha do Fundao, CEP 21941-590 Rio de Janeiro, RJ, Brasil d Laboratorio de Farmacologia/Bioqu mica, CCBS, Universidade Federal do Sergipe, Sao Cristovao, SE, Brasil Accepted 10 May 2006

KEYWORDS Antimicrobial substance; Cryptococcus neoformans; Paenibacillus brasilensis; Rhizosphere of Kalanchoe brasiliensis

Summary
An antifungal substance produced by Paenibacillus brasilensis strain Sa3 was preliminary characterized and showed to be stable after treatment with different enzymes and organic solvents and at a wide range of pH, and presented a molecular weight between 3 and 10 kDa. In vitro antagonism of this strain towards Cryptococcus neoformans was investigated by optical and electronic microscopic analyses and a fungicidal effect on C. neoformans was observed. Ultrastructural analysis showed intense changes on the fungus when it was paired cultured with strain Sa3, mainly the detachment of the capsule from the cell wall and the presence of altered organelles in the cytoplasm. This novel antifungal substance produced by P. brasilensis Sa3 may represent a new insight in antifungal therapy mainly against emergent fungi. Also, prospective studies on rhizobacteria of plants as Kalanchoe brasiliensis may offer a potential source for the discovery of bioactive compounds with medical value. & 2006 Elsevier GmbH. All rights reserved.

Corresponding author. Tel.: +55 21 2562 6741; fax: +55 21 2560 8344.

E-mail addresses: lucyseldin@uol.com.br, lseldin@micro.ufrj.br (L. Seldin). 0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2006.05.003

ARTICLE IN PRESS
Antimicrobial substance of P. brasilensis 201 strain, denominated Sa3, was further characterized phenotypically. Also, preliminary characterization of the antifungal substance produced by strain Sa3 was determined. In vitro antagonism of this strain towards Cryptococcus neoformans was observed by optical and electronic microscopic analyses. This novel antifungal substance may represent a new insight in antifungal therapy mainly against emergent fungi that tend to persist in the future.

Introduction
Kalanchoe brasiliensis (popularly known as saiao, thick leaf or lucky leaf) is one of the many species of the family Crassulaceae often found along the southeast coast of Brazil. The juice of K. brasiliensis leaves contains many polysaccharides, avonoids, ascorbic acid and different trace elements, and it is used in folk medicine for the treatment of the oral mucosa inammation, bronchitis and nasal congestion. Its anti-inammatory effects have been already proved on carrageenininduced rat paw edema (Mourao et al., 1999). Because of the wide use of this plant, it is maintained in a Living Pharmacy in Sergipe, Brazil; however, data concerning the microbial population living associated to K. brasiliensis are very scarce. It has been previously demonstrated that bacterial adaptation to specic, heterogeneous and uctuating environments, such as the plant rhizosphere, is dependent on the diversity of the bacterial population (Roszak and Colwell, 1987), and that the genus Paenibacillus is widely distributed in different plant rhizospheres (Lindberg and Granhall, 1984; Seldin et al., 1984; Holl et al., 1988; von der Weid et al., 2000, 2002; among others). Different species of Paenibacillus have been described as having an eventual role in the interaction between antagonistic micro-organisms and soil-born plant pathogens (Rosado and Seldin, 1993; Dijksterhuis et al., 1999; Seldin et al., 1999, Budi et al., 2000; von der Weid et al., 2003, 2005). Moreover, strains of Paenibacillus polymyxa usually found associated to different plant rhizospheres were shown to produce antimicrobial substances (AMSs) against human pathogenic bacteria and fungi (Vogler and Studer, 1966; Nakajima et al., 1972; Kurusu et al., 1987; Kajimura and Kaneda, 1997; Seldin et al., 1999). Fungal infections in humans range from supercial to deeply invasive and disseminated (such as candidiasis and cryptococcosis) have increased dramatically in recent years (Rodrigues et al., 1999). The treatment of fungal infections has lagged behind bacterial chemotherapy and fewer antifungal than antibacterial substances are available. Therefore, a search for new antifungal drugs is extremely necessary and to know more about the gram-positive spore forming Paenibacillus community found associated with plants in tropical soils, roots of K. brasiliensis were sampled and investigated. Exploring the diversity of these populations could represent a potential source for discovery of novel strains and bioactive compounds. In this study, different strains were isolated and their antifungal spectra were determined and one

Materials and methods

Sampling site
The study area was located in Aracaju, Sergipe State, Brazil. Plants were collected from the Living Pharmacy. The soil of this area is characterized as a clayish soil with an organic matter content of 4.3%, P (127 ppm), K (365 ppm), Ca+Mg (9.5 emg) and Na (35 ppm). The values of soil pH varied from 5.6 to 6.4.

Isolation of Paenibacillus strains from the rhizosphere of K. brasiliensis

Spore-forming bacteria were isolated from the rhizosphere soil of K. brasiliensis by the method described by Seldin et al. (1998). Plants were harvested and the roots shaken to remove the loosely attached soil. Adhering soil (1 g) was mixed with 9 ml of distilled water, shaken for 10 min and pasteurized (80 1C, 10 min). Two-fold serial dilutions of the rhizosphere sample was plated onto TBN agar (thiaminebiotinnitrogen; Seldin and Penido, 1986) and incubated for 3 days at 32 1C. Bacterial cultures of the isolates were stored at room temperature on GB (glucose broth) agar with 1% (wt/vol) CaCO3 (Seldin et al., 1983).

AMS assay
The overlay method described by Rosado and Seldin (1993) was used to detect antifungal activity. It was performed using GB agar plates on which the different isolates were inoculated as a 5 ml spot from an overnight culture. After incubation at 28 1C for 48 h, the cells were killed by exposure to chloroform vapor for 15 min. The plates were then ooded with suspensions containing different pathogenic fungi. AMS production was indicated by clear zones of inhibition around the bacterial colonies after incubation for 48 h at 28 1C. The fungal species used as indicators (Table 1) were

ARTICLE IN PRESS
202
Table 1. Antagonistic activity of Paenibacillus brasilensis Sa3 against human pathogenic fungal strains assessed by the overlay method Human pathogenic fungal species Cryptococcus neoformans var. neoformans sorotype A Candida albicans sorotype B ATCC36802 C. parapsilosis C. dubliniensis C. guillermondii C. tropicalis Fonsecaea pedrosoi Sporothrix schenckii Trichophyton rubrum Histoplasma capsulatum Aspergillus fumigatus ATCC 16913 A. versicolor ATCC 16853 Fusarium moniliforme LGM-2 F. solani LGM-1
a

T.O. Fortes et al. also used for conrmation of strain identication as described by the manufacturer.

Origina 1 1 2 2 2 2 3 3 3 3 4 4 5 5

Inhibition by Sa3b +++ ++ ++ + ++ ++ ++ + ++ ++ ++ +

Preparation of genomic DNA and amplied ribosomal DNA (rDNA) restriction analysis (ARDRA)
Total DNA was extracted from strain Sa3 using the method described by Pitcher et al. (1989). For amplication of Paenibacillus 16S rDNA fragments, the primer set consisting of the specic forward primer PAEN515F and the universal reverse primer 1377 and the amplication conditions were those described by Shida et al. (1997). Based on the results presented in Coelho et al. (2003), a sample (10 ml) of the 16S rRNA amplied product was then digested with the endonuclease HinfI (Invitrogen), for 16 h, according to the specications of the manufacturer. Agarose (2%) gel electrophoresis of restricted DNA was performed at 80 V for 4 h at room temperature.

(1) strains from Universidade Federal de Sao Paulo, SP; (2) Faculdade de Odontologia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ; (3) Hospital Clementino Fraga Filho, UFRJ, RJ; (4) Laboratorio de Micologia Medica, FIOCRUZ, RJ; and (5) EMBRAPA/CNPMS, Sete Lagoas, Brazil. b Diameters of inhibition zones were scored as follows: () no inhibition, (+) weak inhibition of the indicator strain around the spot (clear zones of inhibition less than 1 cm), (+ +) inhibition of the indicator strain around the spot (clear zones between 1 and 2 cm) and (+ + +) strong inhibition of the indicator strain around the spot (clear zones bigger than 2 cm).

Preparation of culture supernatant of strain Sa3

Preparation of culture supernatant was done in GB medium. After incubation of the culture of the producer strain for 48 h at 32 1C, it was then centrifuged (12,000g, 20 min) and the supernatant (crude preparation) was ltered-sterilized (Millipore 0.45 mm), lyophilized and resuspended (to a ten-fold concentration) in GB. Activity was determined by the highest dilution giving a halo against the indicator strain. In this case and for partial characterization of the AMS produced by strain Sa3, a strain of Micrococcus sp. was used as the indicator strain (as done by von der Weid et al., 2003, 2005). Arbitrary units (AU) per ml were dened as the result of: [reciprocal of the greatest dilution of the supernatant that showed a zone of inhibition]/ [volume of supernatant applied on the spot] 1000.

grown in Sabouraud broth at 28 1C from 2 to 7 days and stored at 4 1C in the same medium.

Identication of AMS producer strain Sa3

For morphological and physiological characterization, strain Sa3 was generally cultivated in GB and incubation was carried out still at 32 1C. Most biochemical tests were performed by using the methods and media described by Gordon et al., (1973). Catalase activity was determined by bubble production in a 3% (v/v) hydrogen peroxide solution. Growth under anaerobic conditions was determined after incubation for 7 days in anaerobic Gaspak jars (BBL) containing an atmosphere of 80% N2, 10% CO2 and 10% H2. Cellular morphology, form and position of spores were observed by using a microscope Axioplan 2 (Zeiss). The kit API 50CH (Appareils et Procedes d0 Identication bioMerieux sa, Lyon, France) was

Estimation of molecular weight of the AMS

An estimation of the molecular weight of the AMS was performed by placing dialysis membranes of two different pore sizes (molecular weight cut-off mwco of 3500 and 12,000) on the surface of GB agar. Cells (5 ml) were then inoculated on the membranes and incubated at 32 1C for 24 h. The membranes were then removed and the cell-free medium was ooded with the indicator strain (Micrococcus sp.). After an 18 h incubation period, the plates were examined for inhibition of growth

ARTICLE IN PRESS
Antimicrobial substance of P. brasilensis of the indicator strain. The Sa3 culture supernatant, previously ltered with a 0.45 mm membrane (Millipore), was also submitted to Amicon Diao (Millipore) membranes with molecular weight cut-offs of 10, 3 and 1 kDa. Three distinct molecular weight fractions ranging in size from lower than 10 kDa and higher than 3 kDa (fraction A), lower than 3 kDa and higher than 1 kDa (fraction B) and lower than 1 kDa (fraction C) were obtained, lyophilized, resuspended in distilled water (tenfold-concentrated) and tested for antimicrobial activity as described above. 203 preparations were heated during different periods at 42, 65 and 100 1C, and autoclaved (121 1C, 15 min). For pH stability, 10 ml of crude preparations were mixed to the same volume of citric acidNa2HPO4 buffer to achieve different pH values lower than 6.0 and with TrisHCl buffer for pHs higher than 6.5. Antimicrobial activities were determined before and after all treatments, using Micrococcus sp. as the indicator strain.

In vitro antagonism of P. brasilensis Sa3 towards C. neoformans in liquid medium

The pathogenic fungus, C. neoformans, was precultured in 20 ml Sabouraud medium for 48 h at room temperature and then inoculated (106 cell/ ml) with the same number of Sa3 cells grown overnight in GB medium or with the same volume of the molecular weight fraction A (400 AU/ml) obtained through Amicon ltration. At different time intervals, samples from the paired cultures and from Sa3 supernatant plus fungal were taken to count the total number of fungal cells using a Neubauer chamber. In addition, for microscopic analysis of interactions between the bacterium and the fungus and to determine the percentage of viable cells, the samples were prepared by mixing the same volume of the culture treated with the supernatant active fraction and 0.08% trypan blue dye (Sigma, Sao Paulo, Brazil). Immediately after, the slides were observed with a Zeiss Axioplan 2 Epiuorescence microscope (Oberkochen, Germany). The number of viable fungal cells was also evaluated by inoculating each time interval sample in Sabouraud agar plates.

Sensitivity of the AMS to proteolytic enzymes, organic solvents, chemicals, heat and pH
Crude preparations (15 ml of ten-fold-concentrated supernatant about 600 AU/ml obtained after growth of strain Sa3 in GB) were submitted to the different treatments with 5 ml of each of proteolytic enzymes, organic solvents and chemicals (Table 2) and incubated for 2 h at 37 1C (for enzymes) or room temperature (for organic solvents and chemicals). For heat treatment, the
Table 2. Properties of the antimicrobial substance produced by P. brasilensis Sa3 Response to different enzymes (1 mg/ml)a Pronase E Proteinase K DNase I RNase Trypsin Solvents (50%)a Methanol Ethanol Acetone Toluene Temperature treatmenta 42 1C for 20, 45 and 60 min 65 1C for 20, 45 and 60 min 100 1C for 20, 45 and 60 min Autoclaved (121 1C for 15 min) pHc 3.59.5 in 0.5 steps Diffusion through dialysis membranes mwco 12,00014,000 3500 b + + Yes No

Electron microscopy (EM)

For routine EM, two different samples were processed: a culture containing only the fungus C. neoformans (106 cell/ml) and an additional sample containing the fungus (106 cell/ml) and the bacterial cells (106 cell/ml). Both were harvested by centrifugation at 4000g during 15 min, rinsed in PBS and then xed in 4% formaldehyde, 2.5% glutaraldehyde, in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. Cells were rinsed twice, in 0.1 M cacodylate buffer and post-xed in 1% OsO4 plus 0.8% potassium ferrocyanide and 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2 for 1 h at room temperature. After that, samples were rinsed in PBS, dehydrated in acetone and embedded in Polybed 812. Ultra-thin sections obtained with a Reichert Ultracut S ultramicrotome were stained with uranyl acetate and lead citrate

a Tests performed on the supernatant ten-fold-concentrated containing the antimicrobial substance. b () Inhibition zones similar to those observed in control without treatment, (+) no inhibition zone observed. c pH of supernatants was adjusted from 3 to 9.5 varying 0.5 before testing their activity.

ARTICLE IN PRESS
204 before being analyzed and photographed in a ZEISS 900 or JEOL 1200 transmission electron microscopes. T.O. Fortes et al. with HinfI. The same pattern observed by Coelho et al. (2003) for the species P. brasilensis was obtained in this study for strain Sa3 (not shown). Therefore, this strain was considered to belong to the species P. brasilensis. Recently, von der Weid et al. (2005) have demonstrated the potential of strain PB177 of P. brasilensis to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. For the preliminary characterization of the AMS produced by strain Sa3, the effect of temperature, pH, organic solvents, chemicals and enzymes was evaluated. Table 2 summarizes the results obtained after different treatments of the concentrated supernatant. The antimicrobial activity was insensitive to different proteolytic enzyme treatments, RNase and DNase and to treatment with organic solvents. The substance was heat stable after incubation at 65 1C for 45 min, but did not maintain its activity neither after being treated at 100 1C (20 or 45 min) or autoclaved at 121 1C for 15 min. Finally, the substance produced by strain Sa3 was active in a wide range of pH values (3.59.5). Concerning the estimation of the molecular weight of the substance (Table 2), it was retained by dialysis membrane with molecular weight cut-off of 3500 Da and was able to pass molecular weight cutoff of 12,000 Da, indicating that this inhibiting substance has a molecular weight between 3500 and 12,000 Da. This estimation of the molecular weight of the substance was conrmed when the concentrated supernatant of Sa3 was partioned in three fractions with molecular weight ranging from 10 to 1 kDa. The inhibitory activity against Micrococcus sp. could be detected only in fraction A (between 10 and 3 kDa), indicating that the AMS has a molecular weight lower than 10,000 but higher than 3000 Da. This result implies that very low molecular weight peptide antibiotics were not responsible for the inhibitory effect observed in Sa3. Many of these characteristics presented by the AMS produced by Sa3 are shared by the substances produced by P. polymyxa SCE2 (Rosado and Seldin, 1993) and P. peoriae NRRL-BD62 (von der Weid et al., 2003). The close relationship between P. brasilensis and P. polymyxa and P. peoriae, both species able to produce antifungal substances (Nakajima et al., 1972; Kurusu et al., 1987; Kajimura and Kaneda, 1997; von der Weid et al., 2003) has already been demonstrated genetically and phenotypically (Heyndrickx et al., 1996; Coelho et al., 2003). To determine whether the AMS produced by strain Sa3 shows a fungicidal or a fungistatic action, paired cultures of P. brasilensis Sa3 and C. neoformans, as well as the antimicrobial

Results and discussion

Among the different spore-forming bacterial strains isolated from the rhizosphere of K. brasiliensis, six strains were conrmed to belong to the genus Paenibacillus by the PCR amplication of an 800 bp fragment using the 16S rRNA-based primers PAEN515F and 1377 described by Shida et al. (1997). All of them were able to inhibit the bacterial indicator strain Micrococcus sp. in plate assays (data not shown). When these strains were tested against 14 human pathogenic fungi (presented in Table 1), all of them showed the capability to inhibit 12 (more than 85%) of these fungal strains. On the other hand, none of the strains were able to inhibit the growth of Trichophyton rubrum and Aspergillus fumigatus. One of these strains, named Sa3, was chosen for further phenotypic characterization and for preliminary studies of the AMS produced because of the strong inhibition of C. neoformans (clear zones bigger than 2 cm, Table 1). This fungal species is the agent of cryptococcosis and cryptococcal meningitis, which are prevalent infections in immunosuppressed patients. The importance of C. neoformans as an opportunistic pathogenic fungus has increased over the last decades, owing to the intensive chemotherapy of cancer patients, the use of immunosuppressive drugs in organ transplant recipients and the spread of AIDS (Rodrigues et al., 1999). Strain Sa3 was characterized based on its colonial morphology, physiology and nutritional requirements. Colonies grown on TBN agar were convex, circular, with entire margins, mucoid and bright yellow. Growth was observed in pH 5.7, the strain was facultatively anaerobic, catalase, VogesProskauer (VP) test and reduction of nitrate to nitrite positive. Strain Sa3 was able to hydrolyze both starch and casein. The spores showed to be oval to ellipsoidal and predominantly central to subterminal and distend the sporangium. Gelatin was liqueed by strain Sa3 and no crystalline dextrins were formed in rolled oat medium. Acid and gas were produced from glucose but not from glycerol, D-xylose and L-arabinose. Strain Sa3 was also characterized by using API 50CH and the result obtained was the same as observed for P. brasilensis (von der Weid et al., 2002). To conrm its identication as P. brasilensis, the PCR product (800 bp fragment) obtained using the 16S rRNAbased primers PAEN515F and 1377 DNA was digested

ARTICLE IN PRESS
Antimicrobial substance of P. brasilensis molecular weight active fraction and the fungus were sampled. The results obtained clearly demonstrated that the presence of bacteria or its supernatant led to a quick death of fungal cells. Figs. 1A and B show the result of paired cultures containing P. brasilensis Sa3 culture supernatant (Amicon fraction A between 10 and 3 kDa) and C. neoformans on the cell growth. After 2 h incubation period, cells of C. neoformans stopped growing (Fig. 1A) while, after 14 h, 100% of the fungal cells were already dead (Fig. 1B). Microscopic analysis revealed that the bacterial or supernatantfungal interactions led to loss of cellular impermeability to trypan blue. Dead conidia were visualized as blue conidia due to the incorporation of trypan blue 205 dye, which could be observed 24 h after the start of the interactions (Fig. 1C). The ultrastructural analysis of C. neoformans showed the same classical images well known where the fungus irrespective of the angle of section appears to be round and surrounded by a limiting membrane (although this is not always well dened), an electron dense cell wall and a thick polysaccharide capsule (Figs. 2AC). The cytoplasm of all cells in that sample was electron dense presenting several organelles and vacuoles (Figs. 2A and B). When C. neoformans was incubated in the presence of P. brasilensis an intense change on the fungus ultrastructure could be observed. First, we observed many irregular cells, which is not a characteristic of the C. neoformans culture in the absence of the bacteria. Then, fungus presenting normal or irregular shapes was surrounded by a cell wall not so electron dense, and presenting irregular limits when compared with the fungus growth in the absence of bacteria. The capsule was not altered in width or electron density, but in some irregular cells it was detached from the cell wall (Fig. 2D). Furthermore, cells in close proximity of bacteria almost devoid of capsule could be observed (Fig. 2F). The most dramatic effect of the bacterial presence on the fungus ultrastructure was observed in the cytoplasm. It presented an empty aspect with many membrane proles and altered organelles (Figs. 2D and F). C. neoformans is unique among fungal pathogens for its major virulence factor, a complex polysaccharide capsule. It has been shown that highly virulent isolates produce more capsule than weakly virulent isolates (Blackstock and Casadevall, 1997). Although we observed an alteration of capsule attachment to the cell wall or the almost absence of capsule when strain Sa3 was co-cultured with C. neoformans, to what extent the AMS is active against this fungus in vivo is yet to be determined. In conclusion, the present study demonstrated an antifungal activity of P. brasilensis strain Sa3 on different pathogenic fungi in vitro, including C. neoformans. This is particularly relevant, considering that cryptococcosis is still incurable in immunocompromised patients (Cenci et al., 2004). Additional biochemical and genetic characterization of this AMS produced by strain Sa3 should be encouraged to facilitate the development, design and synthesis of more efcient, broad-spectrum therapeutic AMSs. Finally, prospective studies on rhizobacteria of not well studied plants may offer a potential source for the discovery of bioactive compounds with medical value.

A Number of cells

1.0E+11 1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+06 0 2 4 8 10 Time (h) 12 14 24

B Viable cells (%)

120 100 80 60 40 20 0 0 2 4 6 8 10 Time (h) 12 14 24

Control (after 24h)

Treated (after 24h)

Figure 1. Inuence of paired cultures containing P. brasilensis Sa3 culture supernatant (Amicon fraction A between 10 and 3 kDa) and C. neoformans on the cell growth (A) and viability (B) of C. neoformans: , untreated cells; m, cells treated with antimicrobial substance. Microscopic analysis (C) of untreated and treated fungal cells, after 24 h with the addition of 0.08% trypan blue. Dark staining indicates dead conidia. Bar 10 mm.

ARTICLE IN PRESS
206 T.O. Fortes et al.

Figure 2. Transmission electron microscopy of paired cultures of P. brasilensis Sa3 and C. neoformans. (AC) C. neoformans in the absence of P. brasilensis presents a regular shape. The capsule is thick and regular (C). The cell wall is electron dense and presents regular limits (double white arrows between gures (B and C); (D and E) When C. neoformans is incubated in the presence of P. brasilensis (Pb) the morphological aspect of the fungus changes signicantly. Cell becomes irregular (asterisk in D), the cell wall presents a more irregular aspect (black arrows in D and F and double white arrows between D and E), the cytoplasm shows several membrane fragments and empty organelles (arrowheads in D) and some cells are almost devoid of capsule (white star in F). Bars in A, B, D and F 0.5 mm; Bars in C and E 0.1 mm.

Acknowledgments
We thank the Brazilian National Research Council (CNPq) and Fundaco de Amparo ` Pesquisa do Rio a -a de Janeiro (FAPERJ) for nancial support.

References
Blackstock, R., Casadevall, A., 1997. Presentation of cryptococcal capsular polysaccharide (GXM) on activated antigen-presenting cells inhibit the T-supressor response and enhanced delayed-type inhibits hypersensitivity and survival. Immunology 92, 334339.

Budi, S.W., van Tuinen, D., Arnould, C., Dumas-Gaudot, E., Gianinazzi-Pearson, V., Gianinazzi, S., 2000. Hydrolytic enzyme activity of Paenibacillus sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-born pathogenic fungi. Appl. Soil Ecol. 15, 191199. Cenci, E., Bistoni, F., Mencacci, A., Perito, S., Magliani, W., Conti, S., Polonelli, L., Vecchiarelli, A., 2004. A synthetic peptide as a novel anticryptococcal agent. Cell. Microbiol. 6, 953961. Coelho, M.R.R., von der Weid, I., Zahner, V., Seldin, L., 2003. Characterization of nitrogen-xing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus

ARTICLE IN PRESS
Antimicrobial substance of P. brasilensis
enzyme electrophoresis. FEMS Microbiol. Lett. 222, 243250. Dijksterhuis, J., Sanders, M., Gorris, L.G.M., Smid, E.J., 1999. Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum. J. Appl. Microbiol. 86, 1321. Gordon, R.E., Haynes, W.C., Pang, H.N., 1973. The genus Bacillus. Agriculture Handbook no 427. US Department of Agriculture, Washington, DC. Heyndrickx, M., Vandemeulebroecke, K., Scheldeman, P., Kersters, K., De Vos, P., Logan, N.A., Aziz, A.M., Ali, N., Berkeley, R.C.W., 1996. A polyphasic reassessment of the genus Paenibacillus, reclassication of Bacillus lautus (Nakamura 1984) as Paenibacillus lautus comb. nov. and Bacillus peoriae (Montefusco et al., 1993) as Paenibacillus peoriae comb. nov., and emended description of P. lautus and P. peoriae. Int. J. Syst. Bacteriol. 46, 9881003. Holl, F.B., Chanway, C.P., Turkington, R., Radley, R.A., 1988. Response of crested wheatgrass (Agropyron cristatum L.), perenial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) to inoculation with Bacillus polymyxa. Soil Biol. Biochem. 20, 1924. Kajimura, Y., Kaneda, M., 1997. Fusaricidins B, C, and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8. Isolation, structure elucidation and biological activity. J. Antibiot. 50, 220228. Kurusu, K., Ohba, K., Arai, T., Fukushima, K., 1987. New peptide antibiotics LI-FO3, FO4, FO5, FO7, and FO8, produced by Bacillus polymyxa I. Isolation and characterization. J. Antibiot. 40, 15061514. Lindberg, T., Granhall, U., 1984. Isolation and characterization of dinitrogen-xing bacteria from the rhizosphere of temperate cereals and forage grasses. Appl. Environ. Microbiol. 48, 683689. Mourao, R.H.V., Santos, F.O., Franzotti, E.M., Moreno, M.P.N., Antoniolli, A.R., 1999. Antiinammatory activity and acute toxicity (LD50) of the juice of Kalanchoe brasiliensis (Comb.) leaves picked before and during blooming. Phytother. Res. 13, 352354. Nakajima, N., Chihara, S., Koyama, Y., 1972. A new antibiotic, gatavalin. I. Isolation and characterization. J. Antibiot. 25, 243247. Pitcher, D.G., Saunders, N.A., Owen, R.J., 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151156. Rodrigues, M.L., Alviano, C.S., Travassos, L.R., 1999. Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms. Microb. Infect. 1, 293301.

207
Rosado, A.S., Seldin, L., 1993. Production of a potentially novel anti-microbial substance by Bacillus polymyxa. World J. Microbiol. Biotechnol. 9, 521528. Roszak, D.B., Colwell, R.R., 1987. Survival strategies of bacteria in the natural environment. Microbiol. Rev. 51, 365379. Seldin, L., Penido, E.G.C., 1986. Identication of Bacillus azotoxans using API tests. Antonie van Leeuwenhoek 52, 403409. Seldin, L., van Elsas, J.D., Penido, E.G.C., 1983. Bacillus nitrogen xers from Brazilian soils. Plant Soil 70, 243255. Seldin, L., van Elsas, J.D., Penido, E.G.C., 1984. Bacillus azotoxans sp. nov., a nitrogen-xing species from Brazilian soils and grass roots. Int. J. Syst. Bacteriol. 34, 451456. Seldin, L., Rosado, A.S., Cruz, D.W., Nobrega, A., van Elsas, J.D., Paiva, E., 1998. Comparison of Paenibacillus azotoxans strains isolated from rhizoplane, rhizosphere and non-rhizosphere soil from maize planted in two different Brazilian soils. Appl. Environ. Microbiol. 64, 38603868. Seldin, L., Azevedo, F.S., Alviano, D.S., Alviano, C.S., Bastos, M.C.F., 1999. Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms. Lett. Appl. Microbiol. 28, 423427. Shida, O., Takagi, H., Kadowaki, K., Nakamura, L.K., Komagata, K., 1997. Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int. J. Syst. Bacteriol. 47, 289298. Vogler, K., Studer, R.O., 1966. The chemistry of the polymyxin antibiotics. Experientia 6, 345416. von der Weid, I., Nobrega, A., Paiva, E., van Elsas, J.D., Seldin, L., 2000. Diversity of Paenibacillus polymyxa strains isolated from the rhizosphere of maize planted in cerrado soil. Res. Microbiol. 151, 369381. von der Weid, I., Duarte, G.F., van Elsas, J.D., Seldin, L., 2002. Paenibacillus brasilensis sp. nov., a novel nitrogen-xing species isolated from the maize rhizosphere in Brazil. Int. J. Syst. Evol. Microbiol. 52, 21472153. von der Weid, I., Alviano, D.S., Santos, A.L.S., Soares, R.M.A., Alviano, C.S., Seldin, L., 2003. Antimicrobial activity of Paenibacillus peoriae against a broad spectrum of phytopathogenic bacteria and fungi. J. Appl. Microbiol. 95, 11431151. von der Weid, I., Artursson, V., Seldin, L., Jansson, J.K., 2005. Antifungal and root surface colonization properties of GFP-tagged Paenibacillus brasilensis PB177. World J. Microbiol. Biotechnol. 21, 15911597.