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Keywords:

  • streptomycetes;
  • attached growth;
  • glass beads;
  • antibiotic production;
  • Streptomyces granaticolor ;
  • proteome

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

Streptomycetes, soil-dwelling mycelial bacteria, can colonise surface of organic soil debris and soil particles. We analysed the effects of two different inert surfaces, glass and zirconia/silica, on the growth and antibiotic production in Streptomyces granaticolor. The surfaces used were in the form of microbeads and were surrounded by liquid growth media. Following the production of the antibiotic granaticin, more biomass was formed as well as a greater amount of antibiotic per milligram of protein on the glass beads than on the zirconia/silica beads. Comparison of young mycelium (6 h) proteomes, obtained from the cultures attached to the glass and zirconia/silica beads, revealed three proteins with altered expression levels (dihydrolipoamide dehydrogenase, amidophosphoribosyltransferase and cystathionine beta-synthase) and one unique protein (glyceraldehyde-3-phosphate dehydrogenase) that was present only in cells grown on glass beads. All of the identified proteins function primarily as cytoplasmic enzymes involved in different parts of metabolism; however, in several microorganisms, they are exposed on the cell surface and have been shown to be involved in adhesion or biofilm formation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

Streptomycetes are saprophytic filamentous Gram-positive bacteria that inhabit soil ecosystems and marine sediments throughout the world. They excrete extracellular enzymes to hydrolyse polymers such as starch and cellulose and utilise the resulting soluble breakdown products. Their ability to produce many important antibiotics and other useful metabolites has been of interest to academics and has been exploited by many pharmaceutical companies. The streptomycete life cycle is initiated by spore germination, followed by the development of a branched hyphal network covering the surfaces of soil particles or organic debris (Chater et al., 2010; Martin et al., 2011). In response to complex but still poorly defined signals, the substrate mycelium produces aerial hyphae that eventually undergo septation to yield chains of unigenomic spores. This has proven to be a complex process that is linked to primary metabolism (Susstrunk et al., 1998; Hodgson, 2000) and the production of secondary metabolites, including many antibiotics (Manteca et al., 2008; Martin et al., 2011).

As an alternative to agar or cellophane-covered agar, we have developed a two-phase cultivation system (Nguyen et al., 2005), which is based on a solid phase formed by microbeads, representing particles in the natural soil, clay or sand habitats of streptomycetes. The beads are immersed in a liquid medium that allows for easy modification or replacement of nutrients and growth factors, as well as radioactive labelling of proteins and other macromolecules. Cultivation on beads enables studies of differentiation in streptomycetes using molecular biology and global ‘omics’ techniques, along with the analysis of produced secondary metabolites such as undecylprodigiosin in Streptomyces lividans (Holub et al., 2010; Nezbedova et al., 2011).

Streptomycetes are producers of several compounds that inhibit cell-to-cell binding or cell adhesion to extracellular matrices (Amemiya et al., 1994; Zhang et al., 2003). In a liquid-rich environment, the vegetative hyphae have to be able to attach to the surfaces of soil grains or particles of the seabed, which are the natural environments for most streptomycetes species. Atomic force microscopy has been used to demonstrate that Streptomyces coelicolor can attach strongly to hydrophobic surfaces and that the extracellular matrix is involved in attachment (Del Sol et al., 2007). Del Sol et al. concluded that the matrix allows the vegetative hyphae to adhere to glass and other inert silica-based substrates, for example clay particles in soil. In this study, we compared the growth and the production of the antibiotic granaticin by Streptomyces granaticolor in our microbead cultivation system using glass and zirconia/silica beads, which differ in their hydrophobicity and surface roughness. Primary surface adhesion of S. granaticolor vegetative mycelia was followed on the level of proteome-wide changes induced by the different physicochemical properties of the glass and zirconia/silica microbeads surrounded by a liquid cultivation medium.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

Bacterial strains and cultivation conditions

Streptomyces granaticolor (ETH 7437; Czechoslovak Collection of Microorganisms, Brno, Czech Republic), S. coelicolor A3(2) J1501 (John Innes Centre, Norwich, UK) and Streptomyces ambofaciens (OSC2, Raynal et al., 2006) were used in this study.

Streptomyces granaticolor spores were produced on soya flour mannitol (SFM) medium (Kieser et al., 2000). For morphological and proteomic studies, S. granaticolor was cultivated on glass or zirconia/silica beads immersed in complex liquid medium 8 containing 0.4% yeast extract, 1% malt extract, 0.4% glucose (pH 7.2). Liquid MG medium (Doull & Vining, 1989) was used for growth and measuring granaticin production. The glass beads ‘Balotina’ no. 10 (Ornela a.s., Czech Republic) had diameters in the range of 265–325 μm and were treated as described previously (Nguyen et al., 2005). Zirconia/silica beads (BioSpec Inc.) had a diameter of 500 μm and were treated in the same way. For proteomic studies, 30 mL of sterile glass/zirconia beads were poured into each Petri dish (9 cm diameter). Subsequently, 11 mL of a threefold diluted culture of S. granaticolor that had grown for 24 h were added to the surface of the beads and incubated at 30 °C for 6 h in plastic boxes with water-soaked cotton wool to prevent drying of the cultures. For scanning electron microscopy (SEM) analysis, 2 mL of sterile glass or zirconia/silica beads were poured into a glass micro-Petri dish (1 cm high and 2 cm in diameter). Subsequently, 850 μL of a threefold diluted S. granaticolor culture that had grown for 24 h were added to the surface of the beads and incubated at 30 °C for 6 h.

Preparation of protein samples

Streptomyces granaticolor cultures grown on glass or zirconia/silica beads were frozen for at least 2 h at −70 °C and then disrupted by grinding in a precooled mortar with lysis buffer (50 mM Tris–HCl pH 8.0, 10 mM MgCl2) containing a protease inhibitor cocktail (Complete Mini; Roche) and Benzonase Nuclease (2.5 U mL−1; Novagen). The beads and cell debris were removed by centrifugation (two times for 5 min at 5000 g at 4 °C). The samples were precipitated with 6% TCA for 1 h on ice and then centrifuged for 25 min at 10 000 g at 4 °C. The pellet was washed with ice-cold ethanol. Protein pellets were resuspended in sample buffer containing 7 M Urea, 2 M thiourea, 4% (w/v) CHAPS, 1% Pharmalyte 3-10, 65 mM DTT and bromophenol blue. A 2-D Cleanup Kit (GE Healthcare) was used to purify samples for 2-D gel electrophoresis. The protein concentrations were measured with a 2-D Quant kit (GE Healthcare).

2-D polyacrylamide gel electrophoresis and protein visualisation

Samples (200 and 300 μg of protein per sample, respectively) were soaked into IPG strips (Immobiline DryStrips, 18 cm, pH 4–7; Amersham Biosciences) and rehydrated overnight. In the first dimension, isoelectric focusing (IEF) was performed using a voltage that linearly increased to the steady state (voltage was set to 150 V for 2 h, 300 V for 2 h, 3500 V for 5 h) and then stabilised at 3500 V for 31 h (Multiphor II; Amersham Pharmacia Biotech). After IEF, the strips were treated for 10 min with equilibration solution (50 mM Tris–HCl pH 6.8, 6 M urea, 30% glycerol, 2% SDS) containing 0.02 g mL−1 DTT and followed by a second 10-min wash in equilibration solution containing 0.025 g mL−1 iodoacetamide and a few crystals of bromophenol blue. SDS-PAGE was carried out on 12% polyacrylamide slab gels (Investigator; Genomic Solutions). Proteins were visualised using a SYPRO-Ruby Protein Gel Stain (Invitrogen) and a Colloidal Blue Staining Kit (Invitrogen) to obtain reference maps for MS identification. The gels were scanned at a 100-μm resolution using a Molecular Imager FX (Bio-Rad, Hercules, CA).

Digitalisation of gel images and data analysis

Two-dimensional electrophoresis (2-DE) image analysis was carried out using the PDQuest™ 7.3.1 software (Bio-Rad). For matching and quantification, raw images were smoothed to remove noise, background was subtracted, and spots were detected in noncalibrated quantification mode.

Two biological replicates and four technical replicates were performed for each sample. Statistical analysis was performed using Student's t-test integrated in the PDQuest™ software.

Protein identification using MALDI-TOF mass spectrometry

CBB-stained protein spots were excised from the gel, cut into small pieces and distained using 50 mM 4-ethylmorpholine acetate (pH 8.1) in 50% acetonitrile (MeCN). After complete distaining, the gel was washed with water, shrunk by dehydration in MeCN and reswelled again in water. The supernatant was removed, and the gel was partly dried in a SpeedVac concentrator. The gel pieces were then incubated overnight at 37 °C in a cleavage buffer containing 25 mM 4-ethylmorpholine acetate, 5% MeCN and trypsin (100 ng; Promega). The resulting peptides were extracted using 40% MeCN/0.1% TFA. An aqueous 50% MeCN/0.1% TFA solution of α-cyano-4-hydroxycinnamic acid (5 mg mL−1; Sigma) was used as a MALDI matrix. One microlitre of the peptide mixture was deposited onto the MALDI plate, allowed to air-dry at room temperature and overlaid with 0.4 μL of the matrix.

Mass spectra were measured on an Ultraflex III MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) in the mass range of 700–4000 Da and calibrated internally using the monoisotopic [M+H]+ ions of trypsin autoproteolytic fragments (842.5 and 2211.1 Da). The peak lists, created using the flexAnalysis 3.3 program, were searched in-house using the MASCOT search engine against the NCBInr20130120 database subset of bacterial proteins with the following search settings: peptide tolerance of 30 ppm, missed cleavage site value set to one, variable carbamidomethylation of cysteine, oxidation of methionine and protein N-terminal acetylation. The identity of the protein candidate was confirmed by MS/MS analysis.

Scanning electron microscopy

The samples were prepared according to the procedure described by Kofronova et al. (2002). The glass and zirconia/silica bead cultures were fixed in 2% OsO4 vapour in a desiccator for at least 1 week with subsequent alcohol dehydration. Samples were analysed with an AQUASEM scanning electron microscope (TESCAN Ltd, Czech Republic) at 15 kV. It was possible to observe the whole glass micro-Petri dish in the microscope, and therefore, it was not necessary to use low-melting agarose for sample fixation (Kofronova et al., 2002).

Optical microscopy

A Digital Stereo microscope (National, Motic Group Co. Ltd, China) was used to study biofilm morphology after 72 or 96 h of cultivation.

Analysis of granaticin production

Streptomyces granaticolor was grown on glass or zirconia/silica beads as described above for 72 h at 30 °C in MG medium. The culture from the small Petri dish (10 mL of beads) was transferred to a 50-mL Erlenmeyer flask, 10 mL of sterile distilled water was added, and the flask was shaken on a rotary shaker at 160 r.p.m. at room temperature for 24 h. The culture was then centrifuged for 10 m at 10 000 g, and the absorbance of the supernatant was measured at 572 nm. An extinction coefficient of 22 800 M−1 cm−1 was used to quantify the antibiotic (James & Edwards, 1989).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

Growth and differentiation on glass and zirconia beads

In this work, we aimed to determine whether the hydrophobicity and roughness of a surface on which streptomycetes may attach in their natural environments influences their growth and differentiation. We have chosen S. granaticolor as a model organism because, in addition to the fact that it produces the easily quantified antibiotic granaticin, it is one of the few streptomycetes that grows planktonically without forming clumps. This was important for the analysis of the adhesion process of individual hyphae on the surface of the beads.

The microbeads we used differed in hydrophobicity, which was quite apparent during our manipulation of them. Streptomyces granaticolor mycelia inoculated onto the surface of wetted microbeads formed a continuous lawn of completely differentiated culture on glass, while on zirconia/silica beads distinct but also completely differentiated colonies were formed (Fig. 1). We made the same observation in the cases of S. coelicolor A3(2) and Sambofaciens (Supporting Information, Fig. S1). The difference in hydrophobicity between the two types of beads also caused different levels of surface hydration. Thus, mycelia on glass, which is more hydrophilic, spread more readily on the surface of the bead. In contrast, on zirconia/silica, where there is less hydration, the mycelia soon cease to grow and start to differentiate, leading to the formation of smaller distinct colonies (Figs 1 and S1).

image

Figure 1. Cultivation of Streptomyces granaticolor on glass (a) and zirconia/silica beads (b) beads for 96 h at 30 °C. The beads were surrounded by medium 8 supporting the differentiation of streptomycetes. In the inset, the detailed shapes of the colonies are shown.

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There is no simple correlation between the hydrophobicity of the support surface to which the bacteria adhere and the strength of adhesion. It was shown previously that microbial adhesion is closely associated with the overall surface hydrophobicity of the microorganisms (Zita & Hermansson, 1997). To describe these interactions, Liu et al. (2004) developed a mathematical model that demonstrates that a high cellular hydrophobicity strongly facilitates microbial adhesion on both hydrophobic and hydrophilic support surfaces.

Surface roughness, which is also one of the differences between the types of beads that we used (Fig. 3), has an influence on biofilm formation and stability but appears to be a minor factor during initial adhesion (Bos et al., 1999). In the case of Streptococcus thermophilus, it was shown previously that biofilm structures do not adhere preferentially to scratches or grooves on their support surface (Van Hoogmoed et al., 1997).

Granaticin production on glass and zirconia/silica beads

In our previous work (Nguyen et al., 2005), we showed that S. coelicolor A3(2) produces a larger amount of the antibiotic actinorhodin on glass beads than in liquid culture (Fig. S1). In this study, we quantified and compared the production of granaticin on glass and zirconia/silica beads to determine whether the quality of the bead surfaces influences the production of this pigmented antibiotic.

We compared biomass and granaticin production after 72 h of stationary cultivation, using the antibiotic production-supporting medium, MG. Figure 2 shows more intense blue pigmentation on the plate containing glass beads (a) than was observed on the plate containing zirconia/silica beads (b). It is important to note that the white colour of the zirconia/silica beads, which are not transparent like the glass beads, contributes to this observed colour difference.

image

Figure 2. Cultivation of Streptomyces granaticolor in minimal MG medium, which supports antibiotic production on glass (a) and zirconia/silica (b) beads for 72 h at 30 °C. In the inset, the produced pigmented antibiotic granaticin on the backgrounds of the glass and zirconia/silica beads is shown.

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However, the measurement of cell biomass, as assessed by protein content, for the whole plate showed that there was more biomass produced on glass beads as well as more granaticin per milligram of protein (Table 1). Comparison with liquid culture showed that there was more biomass and antibiotic production on glass microbeads than in liquid culture (Table 1).

Table 1. Production of biomass and granaticin by Streptomyces granaticolor in minimal MG medium, which supports antibiotic production, on glass and zirconia/silica beads and in a liquid medium incubated for 72 h at 30 °C. Production of granaticin was measured spectrophotometrically and expressed relative to the milligrams of protein in each sample
Cultivation systemProtein mg mL−1Granaticin μg mg−1 protein
Glass63.09.1
Zirconia34.76.4
Liquid13.47.0

Our results show that the qualities of the support surface used during attached growth, along with the cultivation conditions and media composition can influence antibiotic biosynthesis.

Adhesion to glass and zirconia/silica beads

The process of bacterial attachment and the subsequent development of a biofilm can be divided into two stages: the primary or docking stage and the secondary or locking phase. The second stage of adhesion employs molecularly mediated binding between specific adhesins and the surface (Dunne, 2002). In the mature biofilm, cells are encased in an extracellular matrix composed of proteins, exopolysaccharides and extracellular DNA (Flemming & Wingender, 2010; McDougald et al., 2012).

Because we have shown that differences in the physicochemical properties of microbeads can influence both the growth and secondary metabolism of streptomycetes, we focused our attention on protein expression during adhesion and recognition of these surfaces by streptomycetes.

The development of streptomycetes is accompanied by a change in cell surface properties: vegetative hyphae growing in moist substrates have hydrophilic cell surfaces, whereas the aerial hyphae and spores are hydrophobic (Elliot et al., 2003; Del Sol et al., 2007). Del Sol et al. (2007) studied the cell surface of S. coelicolor using atomic force microscopy and showed that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix.

Our SEM observations showed that young mycelia (6 h) adhere more readily to the surface of glass beads, and we also observed more massive growth on glass than on zirconia/silica (Fig. 3). This is in agreement with our observations using a light stereo microscope (Fig. 1) and our biomass measurements (Table 1).

image

Figure 3. SEM images of Streptomyces granaticolor growing on glass (a, b) and zirconia/silica (c, d) surfaces for 6 h. Scale bars are indicated in each panel.

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Proteome comparison of streptomycetes grown on glass vs. zirconia/silica beads

There are a number of proteomic studies of streptomycetes grown in liquid culture (Hesketh et al., 2002, 2007; Manteca et al., 2011; Zhou et al., 2011); however, there are only a very limited number of studies of streptomycetes proteomes obtained during their sessile growth and differentiation phases (Manteca et al., 2006, 2010).

Because we used the beads to disintegrate the mycelia growing on them, there was a good chance of also recovering the soluble proteins secreted by the cells from the surface of the beads.

Comparison of proteomes from 6 h S. granaticolor mycelia growing on glass and zirconia/silica beads did not reveal many differences (a total of 4 spots with different densities out of 300–400 protein spots detected in each gel). We identified one unique protein present in the glass bead proteome, spot no. 3303, and three proteins differing in expression (different by a factor of 2 and a statistical confidence level of 95%). Spot no. 7506 was up-regulated in the glass bead proteome, and spots no. 3504 and 6604 were up-regulated in the zirconia/silica bead proteome (Fig. 4).

image

Figure 4. Representative reference 2-D gel of the zirconia/silica proteome sample of Streptomyces granaticolor (300 μg of proteins were loaded). Proteins were stained with fluorescent dye SYPRO® Ruby and scanned at 100-μm resolution using a Molecular Imager FX (Bio-Rad) and 300–400 protein spots were detected in each gel. The numbered spots representing differences between glass and zirconia/silica beads were cut out, and the proteins were identified by MALDI-TOF (the results are shown in Table 2). Spot no. 3303 was unique and spot no. 7506 was up-regulated in the glass bead proteome (solid circles). Spots no. 3504 and 6604 were up-regulated in the zirconia/silica bead proteome (dashed circles). The experimentally obtained MW (kDa) scale is indicated. The inserted graphs show the quantitative differences between the proteins of interest in the glass (solid columns) and zirconia/silica (striped columns) proteomes, as determined using the PDQuest™ software. The expansion shows the detail of the area where unique protein no. 3303 is located.

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The identification of these proteins by mass spectrometry revealed that they do not have very interesting functions at first glance (Table 2); however, a search of the literature revealed that all of these enzymes are multifunctional and that orthologs were described to be involved in adhesion or biofilm formation in different microorganisms.

Table 2. MALDI-TOF peptide mass mapping identification of different proteins selected from glass and zirconia/silica beads proteomes as analysed by 2-D gel electrophoresis. The positions of selected spots are marked on the gel in Fig. 4
Spot no.ProteinNCBI DTB no.DescriptionReferences
3303Glyceraldehyde-3-phosphate dehydrogenase, type I302542762A tetrameric NAD-binding enzyme common to both the glycolytic and gluconeogenic pathways http://www.ncbi.nlm.nih.gov/protein/302542762
3504Cystathionine beta-synthase12082815A multifunctional enzyme: catalyses beta-replacement reaction between l-serine, l-cysteine, cysteine thioethers or some other beta- substituted alpha-l-amino acids and a variety of mercaptans http://www.ncbi.nlm.nih.gov/protein/12082815
6604Amidophosphoribosyl-transferase408679559An enzyme in the de novo pathway of purine ribonucleotide synthesis and is regulated by feedback inhibition by AMP and GMP http://www.ncbi.nlm.nih.gov/protein/408679559
7506Dihydrolipoamide dehydrogenase408679449A component of the multienzyme 2-oxo acid dehydrogenase complexes; catalyses oxidation of its dihydrolipoyl groups http://www.ncbi.nlm.nih.gov/protein/408679449

The most interesting of the proteins identified is dihydrolipoamide dehydrogenase (DLDH, spot no. 7506), the enzyme classically involved in the reoxidation of dihydrolipoamide during the conversion of 2-oxo acids, such as pyruvate, in several multienzyme complexes in central metabolism (Perham et al., 1987). However, it was shown previously that DLDH is not involved in metabolising 2-oxo acids in the Pneumococcus, indicating that DLDH may serve other functions (Smith et al., 2002), which is supported by evidence that organisms that lack 2-oxo acid dehydrogenases still express a DLDH protein (Danson, 1988).

Siegmann et al. (2009) identified DLDH in Rhodococcus ruber as an exocellular protein involved in binding titanium dioxide particles by nonelectrostatic forces.

Rhodococcus is a genus closely related to Mycobacteria, belonging to the Corynebacteria, which together with Streptomyces belong to Actinomycetales.

These findings and our results support the hypothesis that DLDH serves as an adhesion or binding protein in certain bacteria.

The unique protein we found on glass beads was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is known as a classical glycolysis enzyme, but is also known to be a cell surface-exposed protein that can act as an adhesin and is involved in bacterial pathogenicity (Bergmann et al., 2004). Minor density of GAPDH spot which is just next to a major spot of the same molecular weight (see Fig. 4) might indicate posttranslation modification of this essential enzyme. Phosphorylation of GAPDH by protein kinase Cι/λ was found in rabbit muscle (Tisdale, 2002) or in wheat endosperm by SnRK1 kinase (Piattoni et al., 2011), in both cases at serine residues. It was demonstrated previously that GAPDH possesses plasmin- and plasminogen-binding activities in Streptococcus pneumoniae (Bergmann et al., 2004), and in a proteomic study of Streptococcus pyogenes surface-associated proteins, GAPDH was identified among other 15 proteins that were shown to have dual localisation in bacterial cells, both in the cytoplasm and on the bacterial surface (Severin et al., 2007).

Two up-regulated proteins in the proteome from S. granaticolor grown on zirconia/silica beads were amidophosphoribosyltransferase, which is also known as glutamine phosphoribosylpyrophosphate amidotransferase and is involved in purine metabolism, and cystathionine beta-synthase, which is a cytoplasmic enzyme that functions in cysteine biosynthesis.

Amidophosphoribosyltransferase was found in a biofilm formed by Staphylococcus aureus on human keratinocytes. Staphylococcus aureus is prevalent in cutaneous infections, such as chronic wounds, and is an important human pathogen (Secor et al., 2011).

Cystathionine beta-synthase was found to be highly up-regulated in Neisseria gonorrhoeae, the causative agent of gonorrhoea, in the proteome from a biofilm produced in a continuous-flow apparatus in comparison with the proteome of cells grown in planktonic culture (Phillips et al., 2012).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

Streptomycetes spend the longest part of their lives in the form of aerial mycelia and spores, when their surface is highly hydrophobic and they are not attached to any surface (Elliot et al., 2003; Claessen et al., 2006; de Jong et al., 2012). However, the early stages of attached growth determine not only the invasivity of the species but also the spectrum and amount of secondary metabolites they produce. We have demonstrated here that even small differences in the physicochemical properties of the surface on which attached growth takes place can influence the amount of biomass and antibiotic produced. It is noteworthy that there are proteins involved in the process of adhesion in streptomycetes that are also involved in adhesion in a number of other bacteria, including several important pathogens. The finding, that the quality of the surface influences quantitatively the production of secondary metabolites during attached growth in streptomycetes, could be used in antibiotic screening programmes. The use of microbeads would allow miniaturisation and automation of cultivation process in combination with the on-line metabolomic analyses.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information

This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (Grant No. IAA500200913 to J.W.) and by the Institutional Research Concept RVO 61388971 of the Czech Academy of Sciences.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement
  8. Authors' contribution
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
fml12129-sup-0001-FigS1.pdfapplication/PDF2994KFig. S1. Cultivation of S. coelicolor (A, B) and S. ambofaciens (C, D) on glass (A, C) and zirconia/silica (B, D) beads for 96 h at 30 °C.

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