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Gluconacetobacter diazotrophicus, a sugar cane endosymbiont, produces a bacteriocin against Xanthomonas albilineans, a sugar cane pathogen
Research in Microbiology 153 (2002) 345–351 www.elsevier.com/locate/resmic
Gluconacetobacter diazotrophicus, a sugar cane endosymbiont, produces a bacteriocin against Xanthomonas albilineans, a sugar cane pathogen Dolores Piñón a , Mario Casas a , María Blanch b , Blanca Fontaniella b , Yolanda Blanco b , Carlos Vicente b,∗ , María-Teresa Solas c , María-Estrella Legaz b a National Institute of Sugar Cane Research, 17203 Van Troy Av., Boyeros, Havana, Cuba b Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain c Department of Plant Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain
Received 18 January 2002; accepted 17 May 2002
Abstract Gluconacetobacter diazotrophicus in liquid culture secretes proteins into the medium. Both medium containing Gluconacetobacter protein and a solution of this protein after acetone precipitation appeared to inhibit the growth of Xanthomonas albilineans in solid culture. This apparent inhibition of bacterial growth has, in fact, been revealed to be lysis of bacterial cells, as demonstrated by transmission electron microscopy. Fractionation of the Gluconacetobacter protein mixture in size-exclusion chromatography reveals a main fraction with lysozyme-like activity which produces lysis of both living bacteria and isolated cell walls. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Gluconacetobacter diazotrophicus; Xanthomonas albilineans; Bacteriocin; Lysozyme
1. Introduction Gluconacetobacter diazotrophicus is a N2 -fixing, aerobic Gram-negative bacterium found as an endophyte in roots, stems and leaves of sugar cane [5], as well as in Pennisetum purpureum, Ipomoea batatas [29] and Coffea arabica [14]. Bacteria were present in the intercellular apoplast of sugar cane stems [7] and they have also been detected in the xylem vessels at the base of the stem [13]. Moreover, Bellone et al. [4] reported the occurrence of intracellular bacteria in sugar cane root tips. The mechanism by which bacteria enter sugar cane seedlings has not yet been established conclusively. G. diazotrophicus was not found in Brazilian soils where sugar cane was cultured [3] whereas Li and MacRae [22] reported that the bacterium was present on the Australian sugar cane rhizosphere. James et al. [13] suggested that G. diazotrophicus colonized the root surface only after bacterial consumption of sucrose in the habitat. Then bacteria would enter the root apoplast via lateral root * Correspondence and reprints.
E-mail address: [email protected] (C. Vicente).
junctions and the loose cells of the root cap. Ashbolt and Inkerman [2] showed the occurrence of G. diazotrophicus in the mouth stylus of the pink mealybug, Saccharococcus sacchari, an insect that feeds on the meristematic tissue between the leaf sheath and the stem, the leaf sheath pocket. Other Gluconacetobacter spp. can be isolated from mealybugs as well as other bacteria also present in the leaf sheath pocket, such as Leuconostoc mesenteroides and Erwinia amylovora, although the latter microorganisms do not enter the host cells. Thus, the insect may be involved in transferring bacteria from plant to plant [12]. Some anionic glycoproteins [20,21] produced by sugar cane stalks [27] bind the specific sites on the bacterial cells walls of G. diazotrophicus being desorbed by sucrose. Their glycidic moiety is composed of β-1,2 linked polyfructose and galactitol in the ether bond [19]. The same glycoproteins did not show binding activity against the epiphytic bacterium L. mesenteroides [18]. Thus, sugar cane plants have developed a chemical, discriminatory ability to choose the compatible endophyte from several possible ones. In contrast, many other sugar cane endophytes behave as pathogenic agents. Leaf scald, a bacterial-vascular dis-
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 3 6 - 0
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ease of sugar cane, has Xanthomonas albilineans as a casual organism. The initial characteristic symptom is a white streak (“pencil-line”) 1-2-mm wide on the leaf which follows the direction of the main veins. The streaks may later become more enlarged and the affected leaf becomes wilted and necrotic. The white pencil line may also be visible on the leaf sheaths [23]. Symptoms of this phase are seen after ratooning or in young shoots growing from infected plant cane. Later, these symptoms may disappear, although plants remain infected. Alternatively, plants may be infected, but grow without showing any symptoms. Mature stalks may suddenly wilt and die, sometimes without the prior appearance of other symptoms. The bacterium is transmitted by infected cuttings and by implements used to cut stalks. There is evidence for soil and water transmission as well. The pathogen is confined mainly to the leaf and stalk vascular bundles which are often partially or completely occluded with a gum-like substance [26]. Many bacteria produce exocellular substances known as bacteriocins, active against other microorganisms [16]. In contrast with other antibacterial agents produced by bacteria, the bacteriocins are characterized by their primary lethal action, their inactivation by trypsin, their resistance to pH 2 (in the crude state) and their insensitivity to DNase I after treatment with 7 M urea [10]. Bacteriocins are proteins responsible for the lytic activity of the producing strains against other contaminating bacteria [30]. In this paper, we attempt to study the production of bacteriocin-like substances by G. diazotrophicus, active against the sugar cane pathogen X. albilineans.
2. Materials and methods 2.1. Bacterial strains and growth conditions X. albilineans was isolated from a selection clone (Cs 9343) of scalded sugar cane and cultured in the field in the experimental sugar cane station (Havana). The bacteria was cultured on Willbrink medium [8], at 37 ◦ C. G. diazotrophicus, strain 166 (CM-INICA) isolated from sugar cane, was maintained in the N-poor solid medium [5], which contains 10% sucrose and has a pH of 5.5. Culture was maintained for 5 days at 30 ◦ C and the formation of acid in parallel to the culture growth was tested by adding bromothymol blue to the medium [7,35]. This indicator is green at pH 5.5 and changes to yellow at pH 5.0. The size of the inoculum and the growth rate were measured by nephelometry following the absorbance changes at 710 nm. 2.2. Binding and lysis assays When growth reached the stationary phase (about 96 h), bacteria were removed from the media by centrifugation at 10 000 g for 20 min at 2 ◦ C, media were dialyzed overnight against 10 mM phosphate buffer, pH 6.24, and protein in
dialysates was measured according to Lowry et al. [24]. Then the dialysate was used for binding and lysis assays. Protein secreted into the medium in which G. diazotrophicus was grown for 96 h was labelled by adding 3.0 mg of fluorescein isothiocyanate (FITC) per mg protein, and the mixture was incubated for 12 h at 37 ◦ C. After this, the incubation mixture was dialyzed against 5.0 l of distilled water at 4 ◦ C. The bath of dialysis was changed 5 times until free FITC was completely removed. Then, 8.9 mg dry weight of X. albilineans were resuspended in 3.0 ml of dialysate containing 35 mg protein and this cell suspension was maintained for 6 h at 37 ◦ C. Absorbance at 710 nm was recorded each hour. Finally, the suspension was centrifuged at 12 000 g for 15 min at 2 ◦ C and the fluorescence spectrum of the supernatant was measured by exciting the sample with light of a 460-nm wavelength. After this, the pellet was resuspended in 3.0 ml 50 mM mannose, washed for 1 h at 37 ◦ C, newly centrifuged in the same conditions and the fluorescence emission of the supernatant was also measured. Mannose was selected for a desorption experiment as being most likely responsible for affinity binding of glycoproteins to the bacterial cell wall [18]. Alternatively, protein in 20 ml of culture media of G. diazotrophicus was precipitated with 80% (v/v) acetone, collected by centrifugation at 14 000 g for 20 min at 2 ◦ C and dried in a vacuum. Dry residue was redissolved in 20 ml of distilled water (containing a final volume of 17.5 mg protein ml−1 ) and used to assay its inhibitory action on X. albilineans as well as untreated medium, containing 14.08 mg protein ml−1 . A volume of 50 µl of crude medium, medium after culture of G. diazotrophicus or redissolved acetone precipitate, as well as the same volume of both diluted samples, were deposited in a central hole in a petri dish containing solid medium and inocula of X. albilineans were sown in streaks from this hole. The halo of inhibition was measured after 48 h at 38 ◦ C. 2.3. Ultrastructural study To study ultrastructural modifications produced by secreted protein from G. diazotrophicus on X. albilineans, about 9.0 mg of the latter bacterium were resuspended in 1.0 ml of Gluconacetobacter medium containing bacterial protein and maintained for 6 h at 37 ◦ C. Following this, bacteria were collected by centrifugation and the pellet was fixed and dehydrated. Embedding was done in Spurr’s, low viscosity epoxy resin [34], heated at 70 ◦ C for 18 h and polymerization was prolonged for 24 h more. Ultra-thin sections (600 Å), obtained with an OmU2 Reitcher Ultratome, were post-stained with lead citrate [32] and examined on a Philips 300 electron microscope. 2.4. Lysozyme assay and inactivation After dialysis, medium on which G. diazotrophicus grew was filtered through a Sephadex G-100 column (74 cm ×
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3.0 cm) equilibrated with 10 mM phosphate buffer, pH 6.24. Fractions of 5.0 ml were assayed for their protein content [24] and for their lytic activity, incubating with each fraction about 9.0 mg in dry weight of X. albilineans for 6 h at 37 ◦ C and measuring the decrease in absorbance at 710 nm. Lytic activity of selected fractions from Sephadex G-100 was assayed by measuring the decrease in the absorbance at 450 nm of a suspension of cell walls from Micrococcus luteus in the same buffer [9]. Alternatively, the enzyme was inactivated by incubation at 60 ◦ C for 1 h in the absence of substrate, or by incubation of 10 µg of bacterial protein with 10 µg trypsin (grade III, Sigma Chem. Co.) for 1 h at 25 ◦ C in 10 mM Tris–HCl buffer, pH 7.6 containing 10 µM magnesium sulfate [1], in a final volume of 1.0 ml.
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in Laemmli buffer containing 5 µM 2-mercaptoethanol [17]. Samples were then clarified of debris by centrifugation in a laptop centrifuge at 20 000 g for 15 min at 4 ◦ C, boiled for 5 min at 100 ◦ C, loaded onto a 15% polyacrilamide gel and subjected to PAGE/SDS employing 50 mM Tris/0.1 M glycine/0.1% SDS as running buffer. When samples ran to the end of the gel slab, electrophoresis was stopped, the gel extracted from the running slab and stained for 30 min with Coomassie Brilliant Blue reagent (Sigma Chemical Co., St. Louis, MO). Destaining was performed by immersion of the gel in 20% methanol/10% acetic acid over night. The gel was then vacuum-dried and scanned. Molecular weight markers were SeeBlue 2 plus from Novex Innogenetics.
2.5. PAGE/SDS Samples were prepared and resolved in PAGE/SDS according to standard protocols. Briefly, samples of unfractionated culture supernatant and the biologically active fraction (5 ml) of the same culture medium eluted from a Sephadex G100 column (1 m × 3 cm) employing 75 mM phosphate buffer, pH 6.9 as eluent, were lyophilized and resuspended
Fig. 1. Inhibition halo of X. albilineans growth on solid medium produced by secreted substances from G. diazotrophicus to the culture media (2) or by protein (") extracted from these media, at different dilutions. The inset shows dimensions of the halo produced by undiluted fresh medium, undiluted medium after culture and undiluted protein solution.
Fig. 2. A. Fluorescence emission spectra of FITC-conjugated protein secreted to the medium from G. diazotrophicus (continuous line), the supernatant of the same solution after incubation of X. albilineans for 6 h in the solution of FITC-protein (dashed line), and the supernatant after incubation of FITC-treated X. albilineans in 3.0 ml 50 mM mannose. B. Measurement of the turbidity at 710 nm of a cell suspension of X. albilineans in 3.0 ml of a solution of secreted protein from G. diazotrophicus containing 35 mg of total protein.
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3. Results As shown in Fig. 1, the inhibitory action of Gluconacetobacter culture media on X. albilineans growth on solid medium was revealed as a function of the volume of media used in the experiment. Inhibition seemed to be due to the protein secreted from Gluconacetobacter during culture, since an almost identical quantitative effect was achieved by using acetone-precipitated protein instead of the crude medium. Fluorescence emission spectra of FITC-labelled protein from Gluconacetobacter medium before and after incubation with X. albilineans cells did not significantly vary (Fig. 2A). This could imply that fluorescent protein from Gluconacetobacter neither bound to the cell wall nor entered Xanthomonas cells, and thus they were completely recovered after incubation. However, Gluconacetobacter medium contained 14.08 mg protein ml−1 before incubation with Xanthomonas whereas 26.26 mg protein ml−1 were recovered after 6 h of contact with pathogenic bacteria. This could only be explained by assuming that the inhibitory effect of Gluconacetobacter protein on Xanthomonas growth was in
fact a lytic action such that non-labelled protein removed from lysed bacteria were recovered together with those initially contained in Gluconacetobacter medium. This lytic action could explain the continuous decrease in absorbance at 710 nm recorded during the period of Xanthomonas incubation in these media (Fig. 2B). The wall of this Gram-negative microorganism seems to be extremely complex. Three dense layers can be seen in the wall structure overlying the protoplast membrane by TEM (transmission electron microscopy). In some preparations, the innermost of the three dense layers is considerably thicker than the other two layers (Fig. 3A). In some cases, a three-layered structure probably composing the protoplast membrane can be seen immediately inside the innermost layer of the cell wall. When cells of X. albilineans were incubated for 6 h in a solution of protein recovered from media in which Gluconacetobacter was grown, some of those cells showed the disappearance of the wall structure at a polar region, membrane breakage and the cytoplasm was spread outside the cell. The rest of the intact wall preserved its three-layered structure. Moreover, the innermost, thick layer of the cell wall disappeared in ghosts and only
Fig. 3. Transmission electron micrographs of X. albilineans cells immediately removed from the medium on which they were grown (A), or incubated for 6 h in a solution of protein secreted from G. diazotrophicus (B), as specified in Fig. 2B. Black arrow (B) indicates the point at which the cell wall is broken, whereas white arrow indicates a transversal cut of a ghost in which only the outer double track of the cell wall can be seen; cw: cell wall; ic: intact cell; lc: lysed cell.
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Fig. 5. Lytic activity of fractions eluted from Sephadex G-100 showing the highest lytic activity against X. albilineans cells. Values are the means of three replicates. Vertical bars give standard error where larger than the symbols.
Fig. 4. Elution profile of protein from G. diazotrophicus from a column of Sephadex G-100, measuring protein (2) and lytic activity against X. albilineans (") of each fraction, as described in Methods. Inset: Samples of unfractionated culture medium (lane 3) and fraction 67 from a Sephadex G100 column (lane 2) were loaded and resolved in a 15% polyacrylamide gel by standard PAGE/SDS. At least seven bands of molecular mass over 30 kDa in addition to a major band of ∼ 12.5 kDa can be observed in the unfractionated culture medium, whereas only the latter band appeared after fractionation through a Sephadex G-100 column, (lane 2). Lane 1 corresponds to SeeBlue2 plus molecular weight markers. Molecular mass of each band is indicated.
the outer double track can be seen in a similar way to that observed after lysozyme treatment (Fig. 3B). This evidence suggested that the thick layer is composed at
least partially of mucopeptides. Quantitative analysis of six different replicates, similar to that shown in Fig. 3B, showed that about 75% of total bacteria were lysed during incubation whereas the cell wall of the rest (25%) remained unaltered. Gluconacetobacter medium was then filtered through a column of Sephadex G-100 equilibrated with 10 mM phosphate buffer, pH 6.24. Protein was eluted by using the same buffer by collecting fractions of 5.0-ml volume. Protein was recovered from 270 to 345 ml of eluate with a maximum at 290 ml of elution volume. However, lytic activity against X. albilineans was restricted to a narrow peak at 335 ml (fraction 67) although fractions 66 and 68 showed low values of lytic activity (Fig. 4). Analysis by PAGE/SDS of this active fraction revealed only one band corresponding to a protein with a molecular mass of about 12.5 kDa (inset in Fig. 4). The same analysis of the medium containing secreted proteins from G. diazotrophicus revealed three major bands, in addition to that of 12.5 kDa, of 150, 70, and 55 kDa, respectively, and several minor bands with molecular masses ranging from 50 to 33 kDa. In addition, fractions 66 to 68 were active against a suspension of cell walls isolated from M. luteus (Fig. 5). The most active fraction, as expected, was that which showed the highest lytic activity against the living microorganism. Lytic activity was completely lost after 1 h at 60 ◦ C or by incubation with trypsin for 1 h at 25 ◦ C (data not shown).
4. Discussion G. diazotrophicus colonize the rhizosphere and the surface of roots. However, the maximal efficiency at improving the growth of sugar cane plants is achieved by bacteria colonizing the interior of roots and shoots [25,33], although G. diazotrophicus cells are also found in the apoplast, i.e., intercellular spaces and xylem vessels of sugar cane stalks [11]. Since this endophyte occupies spaces through which pathogens such as X. albilineans develop an infection path-
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way, [31], the hypothesis exists as to possible competition between these two bacteria, in a similar way to that found for the antagonistic competition between G. diazotrophicus and the fungus Colletotrichum falcatum, the causal organism of red-rot disease of sugar cane [28]. In fact, G. diazotrophicus in liquid culture produce a factor, probably a secreted protein (Fig. 1), able to impede the growth of X. albilineans on solid medium. This factor rapidly diffuses in agar medium, a characteristic of lactobacillus-synthesized bacteriocin [16]. Many other bacteria produce bacteriocins involved in the biological control of some plant diseases. For example, excellent biological control of crown gall is obtained by soaking germinated seeds or dipping nursery seedlings in a suspension of a particular strain of Agrobacterium radiobacter, antagonistic to most strains of A. tumefaciens. The antagonist controls crown gall initiation by establishing itself on the surface of the plant tissues where it produces the bacteriocin agrocin 84, an inhibitor of the growth of most virulent A. tumefaciens strains [15]. There is no binding of G. diazotrophicus bacteriocin to the cell wall of Xanthomonas cells (Fig. 2) but lysis of these cells is achieved by destroying the innermost thick layer of the cell wall and producing the loss of cytoplasmic content to the medium (Fig. 3B). Lytic protein eluted from the size-exclusion chromatography column in a volume fraction in which cytochrome c (Mr . 14 kDa) was recovered from the standard mixture (Fig. 4). However, its electrophoretic mobility in SDS/PAGE gives a molecular mass of about 12.5 kDa, lower than other lysozymes the molecular mass of which is 14.6 kDa [6]. The lytic activity is in reality supported by a proteinic structure since this fraction is compeltely inactivated at 60 ◦ C for 1 h or after tryptic digestion. By considering that the Gluconacetobacter protein was able to lyse isolated cells walls of X. albilineans in in vitro conditions (Fig. 5), this lytic protein could thus be defined as a lysozyme-like bacteriocin, similar to pyocines described for several species of Pseudomonas [30].
Acknowledgements We wish to thank Dr. M. Vicente-Manzanares for help and criticism and Miss Raquel Alonso for her excellent technical assistance. This work was supported by a grant from the Dirección General de Investigación Científica y Tecnológica (Ministerio de Educación y Cultura, Spain) BFI2000-0610 and a grant from the Subdirección General de Cooperación Internacional (Ministerio de Educación y Cultura, Spain) PR77/00-9027.
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Gluconacetobacter diazotrophicus, a sugar cane endosymbiont, produces a bacteriocin against Xanthomonas albilineans, a sugar cane pathogen Dolores Piñón a , Mario Casas a , María Blanch b , Blanca Fontaniella b , Yolanda Blanco b , Carlos Vicente b,∗ , María-Teresa Solas c , María-Estrella Legaz b a National Institute of Sugar Cane Research, 17203 Van Troy Av., Boyeros, Havana, Cuba b Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain c Department of Plant Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain
Received 18 January 2002; accepted 17 May 2002
Abstract Gluconacetobacter diazotrophicus in liquid culture secretes proteins into the medium. Both medium containing Gluconacetobacter protein and a solution of this protein after acetone precipitation appeared to inhibit the growth of Xanthomonas albilineans in solid culture. This apparent inhibition of bacterial growth has, in fact, been revealed to be lysis of bacterial cells, as demonstrated by transmission electron microscopy. Fractionation of the Gluconacetobacter protein mixture in size-exclusion chromatography reveals a main fraction with lysozyme-like activity which produces lysis of both living bacteria and isolated cell walls. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Gluconacetobacter diazotrophicus; Xanthomonas albilineans; Bacteriocin; Lysozyme
1. Introduction Gluconacetobacter diazotrophicus is a N2 -fixing, aerobic Gram-negative bacterium found as an endophyte in roots, stems and leaves of sugar cane [5], as well as in Pennisetum purpureum, Ipomoea batatas [29] and Coffea arabica [14]. Bacteria were present in the intercellular apoplast of sugar cane stems [7] and they have also been detected in the xylem vessels at the base of the stem [13]. Moreover, Bellone et al. [4] reported the occurrence of intracellular bacteria in sugar cane root tips. The mechanism by which bacteria enter sugar cane seedlings has not yet been established conclusively. G. diazotrophicus was not found in Brazilian soils where sugar cane was cultured [3] whereas Li and MacRae [22] reported that the bacterium was present on the Australian sugar cane rhizosphere. James et al. [13] suggested that G. diazotrophicus colonized the root surface only after bacterial consumption of sucrose in the habitat. Then bacteria would enter the root apoplast via lateral root * Correspondence and reprints.
E-mail address: [email protected] (C. Vicente).
junctions and the loose cells of the root cap. Ashbolt and Inkerman [2] showed the occurrence of G. diazotrophicus in the mouth stylus of the pink mealybug, Saccharococcus sacchari, an insect that feeds on the meristematic tissue between the leaf sheath and the stem, the leaf sheath pocket. Other Gluconacetobacter spp. can be isolated from mealybugs as well as other bacteria also present in the leaf sheath pocket, such as Leuconostoc mesenteroides and Erwinia amylovora, although the latter microorganisms do not enter the host cells. Thus, the insect may be involved in transferring bacteria from plant to plant [12]. Some anionic glycoproteins [20,21] produced by sugar cane stalks [27] bind the specific sites on the bacterial cells walls of G. diazotrophicus being desorbed by sucrose. Their glycidic moiety is composed of β-1,2 linked polyfructose and galactitol in the ether bond [19]. The same glycoproteins did not show binding activity against the epiphytic bacterium L. mesenteroides [18]. Thus, sugar cane plants have developed a chemical, discriminatory ability to choose the compatible endophyte from several possible ones. In contrast, many other sugar cane endophytes behave as pathogenic agents. Leaf scald, a bacterial-vascular dis-
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 3 6 - 0
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ease of sugar cane, has Xanthomonas albilineans as a casual organism. The initial characteristic symptom is a white streak (“pencil-line”) 1-2-mm wide on the leaf which follows the direction of the main veins. The streaks may later become more enlarged and the affected leaf becomes wilted and necrotic. The white pencil line may also be visible on the leaf sheaths [23]. Symptoms of this phase are seen after ratooning or in young shoots growing from infected plant cane. Later, these symptoms may disappear, although plants remain infected. Alternatively, plants may be infected, but grow without showing any symptoms. Mature stalks may suddenly wilt and die, sometimes without the prior appearance of other symptoms. The bacterium is transmitted by infected cuttings and by implements used to cut stalks. There is evidence for soil and water transmission as well. The pathogen is confined mainly to the leaf and stalk vascular bundles which are often partially or completely occluded with a gum-like substance [26]. Many bacteria produce exocellular substances known as bacteriocins, active against other microorganisms [16]. In contrast with other antibacterial agents produced by bacteria, the bacteriocins are characterized by their primary lethal action, their inactivation by trypsin, their resistance to pH 2 (in the crude state) and their insensitivity to DNase I after treatment with 7 M urea [10]. Bacteriocins are proteins responsible for the lytic activity of the producing strains against other contaminating bacteria [30]. In this paper, we attempt to study the production of bacteriocin-like substances by G. diazotrophicus, active against the sugar cane pathogen X. albilineans.
2. Materials and methods 2.1. Bacterial strains and growth conditions X. albilineans was isolated from a selection clone (Cs 9343) of scalded sugar cane and cultured in the field in the experimental sugar cane station (Havana). The bacteria was cultured on Willbrink medium [8], at 37 ◦ C. G. diazotrophicus, strain 166 (CM-INICA) isolated from sugar cane, was maintained in the N-poor solid medium [5], which contains 10% sucrose and has a pH of 5.5. Culture was maintained for 5 days at 30 ◦ C and the formation of acid in parallel to the culture growth was tested by adding bromothymol blue to the medium [7,35]. This indicator is green at pH 5.5 and changes to yellow at pH 5.0. The size of the inoculum and the growth rate were measured by nephelometry following the absorbance changes at 710 nm. 2.2. Binding and lysis assays When growth reached the stationary phase (about 96 h), bacteria were removed from the media by centrifugation at 10 000 g for 20 min at 2 ◦ C, media were dialyzed overnight against 10 mM phosphate buffer, pH 6.24, and protein in
dialysates was measured according to Lowry et al. [24]. Then the dialysate was used for binding and lysis assays. Protein secreted into the medium in which G. diazotrophicus was grown for 96 h was labelled by adding 3.0 mg of fluorescein isothiocyanate (FITC) per mg protein, and the mixture was incubated for 12 h at 37 ◦ C. After this, the incubation mixture was dialyzed against 5.0 l of distilled water at 4 ◦ C. The bath of dialysis was changed 5 times until free FITC was completely removed. Then, 8.9 mg dry weight of X. albilineans were resuspended in 3.0 ml of dialysate containing 35 mg protein and this cell suspension was maintained for 6 h at 37 ◦ C. Absorbance at 710 nm was recorded each hour. Finally, the suspension was centrifuged at 12 000 g for 15 min at 2 ◦ C and the fluorescence spectrum of the supernatant was measured by exciting the sample with light of a 460-nm wavelength. After this, the pellet was resuspended in 3.0 ml 50 mM mannose, washed for 1 h at 37 ◦ C, newly centrifuged in the same conditions and the fluorescence emission of the supernatant was also measured. Mannose was selected for a desorption experiment as being most likely responsible for affinity binding of glycoproteins to the bacterial cell wall [18]. Alternatively, protein in 20 ml of culture media of G. diazotrophicus was precipitated with 80% (v/v) acetone, collected by centrifugation at 14 000 g for 20 min at 2 ◦ C and dried in a vacuum. Dry residue was redissolved in 20 ml of distilled water (containing a final volume of 17.5 mg protein ml−1 ) and used to assay its inhibitory action on X. albilineans as well as untreated medium, containing 14.08 mg protein ml−1 . A volume of 50 µl of crude medium, medium after culture of G. diazotrophicus or redissolved acetone precipitate, as well as the same volume of both diluted samples, were deposited in a central hole in a petri dish containing solid medium and inocula of X. albilineans were sown in streaks from this hole. The halo of inhibition was measured after 48 h at 38 ◦ C. 2.3. Ultrastructural study To study ultrastructural modifications produced by secreted protein from G. diazotrophicus on X. albilineans, about 9.0 mg of the latter bacterium were resuspended in 1.0 ml of Gluconacetobacter medium containing bacterial protein and maintained for 6 h at 37 ◦ C. Following this, bacteria were collected by centrifugation and the pellet was fixed and dehydrated. Embedding was done in Spurr’s, low viscosity epoxy resin [34], heated at 70 ◦ C for 18 h and polymerization was prolonged for 24 h more. Ultra-thin sections (600 Å), obtained with an OmU2 Reitcher Ultratome, were post-stained with lead citrate [32] and examined on a Philips 300 electron microscope. 2.4. Lysozyme assay and inactivation After dialysis, medium on which G. diazotrophicus grew was filtered through a Sephadex G-100 column (74 cm ×
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3.0 cm) equilibrated with 10 mM phosphate buffer, pH 6.24. Fractions of 5.0 ml were assayed for their protein content [24] and for their lytic activity, incubating with each fraction about 9.0 mg in dry weight of X. albilineans for 6 h at 37 ◦ C and measuring the decrease in absorbance at 710 nm. Lytic activity of selected fractions from Sephadex G-100 was assayed by measuring the decrease in the absorbance at 450 nm of a suspension of cell walls from Micrococcus luteus in the same buffer [9]. Alternatively, the enzyme was inactivated by incubation at 60 ◦ C for 1 h in the absence of substrate, or by incubation of 10 µg of bacterial protein with 10 µg trypsin (grade III, Sigma Chem. Co.) for 1 h at 25 ◦ C in 10 mM Tris–HCl buffer, pH 7.6 containing 10 µM magnesium sulfate [1], in a final volume of 1.0 ml.
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in Laemmli buffer containing 5 µM 2-mercaptoethanol [17]. Samples were then clarified of debris by centrifugation in a laptop centrifuge at 20 000 g for 15 min at 4 ◦ C, boiled for 5 min at 100 ◦ C, loaded onto a 15% polyacrilamide gel and subjected to PAGE/SDS employing 50 mM Tris/0.1 M glycine/0.1% SDS as running buffer. When samples ran to the end of the gel slab, electrophoresis was stopped, the gel extracted from the running slab and stained for 30 min with Coomassie Brilliant Blue reagent (Sigma Chemical Co., St. Louis, MO). Destaining was performed by immersion of the gel in 20% methanol/10% acetic acid over night. The gel was then vacuum-dried and scanned. Molecular weight markers were SeeBlue 2 plus from Novex Innogenetics.
2.5. PAGE/SDS Samples were prepared and resolved in PAGE/SDS according to standard protocols. Briefly, samples of unfractionated culture supernatant and the biologically active fraction (5 ml) of the same culture medium eluted from a Sephadex G100 column (1 m × 3 cm) employing 75 mM phosphate buffer, pH 6.9 as eluent, were lyophilized and resuspended
Fig. 1. Inhibition halo of X. albilineans growth on solid medium produced by secreted substances from G. diazotrophicus to the culture media (2) or by protein (") extracted from these media, at different dilutions. The inset shows dimensions of the halo produced by undiluted fresh medium, undiluted medium after culture and undiluted protein solution.
Fig. 2. A. Fluorescence emission spectra of FITC-conjugated protein secreted to the medium from G. diazotrophicus (continuous line), the supernatant of the same solution after incubation of X. albilineans for 6 h in the solution of FITC-protein (dashed line), and the supernatant after incubation of FITC-treated X. albilineans in 3.0 ml 50 mM mannose. B. Measurement of the turbidity at 710 nm of a cell suspension of X. albilineans in 3.0 ml of a solution of secreted protein from G. diazotrophicus containing 35 mg of total protein.
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3. Results As shown in Fig. 1, the inhibitory action of Gluconacetobacter culture media on X. albilineans growth on solid medium was revealed as a function of the volume of media used in the experiment. Inhibition seemed to be due to the protein secreted from Gluconacetobacter during culture, since an almost identical quantitative effect was achieved by using acetone-precipitated protein instead of the crude medium. Fluorescence emission spectra of FITC-labelled protein from Gluconacetobacter medium before and after incubation with X. albilineans cells did not significantly vary (Fig. 2A). This could imply that fluorescent protein from Gluconacetobacter neither bound to the cell wall nor entered Xanthomonas cells, and thus they were completely recovered after incubation. However, Gluconacetobacter medium contained 14.08 mg protein ml−1 before incubation with Xanthomonas whereas 26.26 mg protein ml−1 were recovered after 6 h of contact with pathogenic bacteria. This could only be explained by assuming that the inhibitory effect of Gluconacetobacter protein on Xanthomonas growth was in
fact a lytic action such that non-labelled protein removed from lysed bacteria were recovered together with those initially contained in Gluconacetobacter medium. This lytic action could explain the continuous decrease in absorbance at 710 nm recorded during the period of Xanthomonas incubation in these media (Fig. 2B). The wall of this Gram-negative microorganism seems to be extremely complex. Three dense layers can be seen in the wall structure overlying the protoplast membrane by TEM (transmission electron microscopy). In some preparations, the innermost of the three dense layers is considerably thicker than the other two layers (Fig. 3A). In some cases, a three-layered structure probably composing the protoplast membrane can be seen immediately inside the innermost layer of the cell wall. When cells of X. albilineans were incubated for 6 h in a solution of protein recovered from media in which Gluconacetobacter was grown, some of those cells showed the disappearance of the wall structure at a polar region, membrane breakage and the cytoplasm was spread outside the cell. The rest of the intact wall preserved its three-layered structure. Moreover, the innermost, thick layer of the cell wall disappeared in ghosts and only
Fig. 3. Transmission electron micrographs of X. albilineans cells immediately removed from the medium on which they were grown (A), or incubated for 6 h in a solution of protein secreted from G. diazotrophicus (B), as specified in Fig. 2B. Black arrow (B) indicates the point at which the cell wall is broken, whereas white arrow indicates a transversal cut of a ghost in which only the outer double track of the cell wall can be seen; cw: cell wall; ic: intact cell; lc: lysed cell.
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Fig. 5. Lytic activity of fractions eluted from Sephadex G-100 showing the highest lytic activity against X. albilineans cells. Values are the means of three replicates. Vertical bars give standard error where larger than the symbols.
Fig. 4. Elution profile of protein from G. diazotrophicus from a column of Sephadex G-100, measuring protein (2) and lytic activity against X. albilineans (") of each fraction, as described in Methods. Inset: Samples of unfractionated culture medium (lane 3) and fraction 67 from a Sephadex G100 column (lane 2) were loaded and resolved in a 15% polyacrylamide gel by standard PAGE/SDS. At least seven bands of molecular mass over 30 kDa in addition to a major band of ∼ 12.5 kDa can be observed in the unfractionated culture medium, whereas only the latter band appeared after fractionation through a Sephadex G-100 column, (lane 2). Lane 1 corresponds to SeeBlue2 plus molecular weight markers. Molecular mass of each band is indicated.
the outer double track can be seen in a similar way to that observed after lysozyme treatment (Fig. 3B). This evidence suggested that the thick layer is composed at
least partially of mucopeptides. Quantitative analysis of six different replicates, similar to that shown in Fig. 3B, showed that about 75% of total bacteria were lysed during incubation whereas the cell wall of the rest (25%) remained unaltered. Gluconacetobacter medium was then filtered through a column of Sephadex G-100 equilibrated with 10 mM phosphate buffer, pH 6.24. Protein was eluted by using the same buffer by collecting fractions of 5.0-ml volume. Protein was recovered from 270 to 345 ml of eluate with a maximum at 290 ml of elution volume. However, lytic activity against X. albilineans was restricted to a narrow peak at 335 ml (fraction 67) although fractions 66 and 68 showed low values of lytic activity (Fig. 4). Analysis by PAGE/SDS of this active fraction revealed only one band corresponding to a protein with a molecular mass of about 12.5 kDa (inset in Fig. 4). The same analysis of the medium containing secreted proteins from G. diazotrophicus revealed three major bands, in addition to that of 12.5 kDa, of 150, 70, and 55 kDa, respectively, and several minor bands with molecular masses ranging from 50 to 33 kDa. In addition, fractions 66 to 68 were active against a suspension of cell walls isolated from M. luteus (Fig. 5). The most active fraction, as expected, was that which showed the highest lytic activity against the living microorganism. Lytic activity was completely lost after 1 h at 60 ◦ C or by incubation with trypsin for 1 h at 25 ◦ C (data not shown).
4. Discussion G. diazotrophicus colonize the rhizosphere and the surface of roots. However, the maximal efficiency at improving the growth of sugar cane plants is achieved by bacteria colonizing the interior of roots and shoots [25,33], although G. diazotrophicus cells are also found in the apoplast, i.e., intercellular spaces and xylem vessels of sugar cane stalks [11]. Since this endophyte occupies spaces through which pathogens such as X. albilineans develop an infection path-
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way, [31], the hypothesis exists as to possible competition between these two bacteria, in a similar way to that found for the antagonistic competition between G. diazotrophicus and the fungus Colletotrichum falcatum, the causal organism of red-rot disease of sugar cane [28]. In fact, G. diazotrophicus in liquid culture produce a factor, probably a secreted protein (Fig. 1), able to impede the growth of X. albilineans on solid medium. This factor rapidly diffuses in agar medium, a characteristic of lactobacillus-synthesized bacteriocin [16]. Many other bacteria produce bacteriocins involved in the biological control of some plant diseases. For example, excellent biological control of crown gall is obtained by soaking germinated seeds or dipping nursery seedlings in a suspension of a particular strain of Agrobacterium radiobacter, antagonistic to most strains of A. tumefaciens. The antagonist controls crown gall initiation by establishing itself on the surface of the plant tissues where it produces the bacteriocin agrocin 84, an inhibitor of the growth of most virulent A. tumefaciens strains [15]. There is no binding of G. diazotrophicus bacteriocin to the cell wall of Xanthomonas cells (Fig. 2) but lysis of these cells is achieved by destroying the innermost thick layer of the cell wall and producing the loss of cytoplasmic content to the medium (Fig. 3B). Lytic protein eluted from the size-exclusion chromatography column in a volume fraction in which cytochrome c (Mr . 14 kDa) was recovered from the standard mixture (Fig. 4). However, its electrophoretic mobility in SDS/PAGE gives a molecular mass of about 12.5 kDa, lower than other lysozymes the molecular mass of which is 14.6 kDa [6]. The lytic activity is in reality supported by a proteinic structure since this fraction is compeltely inactivated at 60 ◦ C for 1 h or after tryptic digestion. By considering that the Gluconacetobacter protein was able to lyse isolated cells walls of X. albilineans in in vitro conditions (Fig. 5), this lytic protein could thus be defined as a lysozyme-like bacteriocin, similar to pyocines described for several species of Pseudomonas [30].
Acknowledgements We wish to thank Dr. M. Vicente-Manzanares for help and criticism and Miss Raquel Alonso for her excellent technical assistance. This work was supported by a grant from the Dirección General de Investigación Científica y Tecnológica (Ministerio de Educación y Cultura, Spain) BFI2000-0610 and a grant from the Subdirección General de Cooperación Internacional (Ministerio de Educación y Cultura, Spain) PR77/00-9027.
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