Biodiversity of streptomycetes isolated from a succession sequence at a post-mining site and their evidence in Miocene lacustrine sediment

Microbiological Research 165 (2010) 594—608

www.elsevier.de/micres

Biodiversity of streptomycetes isolated from a succession sequence at a post-mining site and their evidence in Miocene lacustrine sediment ˇ´ ´c, Dana Elhottova ˚feka, Martin Tichy Alica Chron akova ´a,b,, Va ´clav Krisˇtu ´a a

Biology Centre of the Academy of Sciences of the Czech Republic, v. v. i. – Institute of Soil Biology, Na Sa´dka´ch 7, 370 ˇ eske´ Bude ˇjovice, Czech Republic 05 C b ˇ eske´ Bude ˇjovice, Czech Republic Faculty of Sciences, University of South Bohemia, Branisˇovska´ 31, 370 05 C c ˇ, Czech Institute of Microbiology, Academy of Sciences of the Czech Republic, v. v. i., Opatovicky´ Mly´n, 379 81 Trˇebon Republic Received 22 May 2009; received in revised form 5 October 2009; accepted 31 October 2009

KEYWORDS Actinomycetes; Antibiotics; Primary succession; Subsurface; 16S rDNA-ITS

Summary The genetic diversity of streptomycetes in colliery spoil heaps (Sokolov, Czech Republic) was investigated by restriction pattern analysis of 16S-internal transcribed spacer rDNA and 16S sequences. We sampled freshly excavated Miocene sediment (17–19-million-year-old) and four sites of primary succession (initial, early, middle, and late stages; aged 1–44 years) on the same sediment. Active bacteria were present even in fresh Miocene sediment, and the relative proportion of actinomycetes among total bacterial and their genetic diversity increased significantly with the age of the sampling site. The replacement of pioneer species by late succession species during succession was observed. Plate assays of Streptomyces strains revealed 27% antibiotic-producing strains. Screening for nonribosomal peptide synthases and type I polyketide synthases systems suggested that 90% and 55% streptomycetes, respectively, are putative producers of biologically active compounds. The frequencies of tetracycline-, amoxicillin-, and chloramphenicol-resistant streptomycetes were 6%, 9%, and 15%, respectively. These findings document the occurrence of genetic elements encoding antibiotic resistance genes and the production of antibiotics by streptomycetes located in pristine environments. Our results indicate key roles for ancient streptomycetes related to S. microflavus, S. spororaveus, and S. flavofuscus in pioneering community development in freshly excavated substrates. & 2009 Elsevier GmbH. All rights reserved.

Corresponding author at: Biology Centre of the Academy of Sciences of the Czech Republic, v. v. i. – Institute of Soil Biology, Na

ˇeske Sa ´dka ´ch 7, 370 05 C ´ Bude ˇjovice, Czech Republic. Tel.: +420 387 775 770. E-mail address: [email protected] (A. Chron ´kova ´). ˇa 0944-5013/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2009.10.002

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment

Introduction During open cast mining, newly exposed earth (terra nova) becomes the site of genesis of soil and ecosystems. Colliery spoil heaps composed of material excavated from brown coal mining sites occupy a large area near Sokolov (North-West Bohemia, Czech Republic; for more details see Frouz et al. 2006) and serve as models for studying the primary succession of microbial communities in soil. Miocene sediment (formerly the Cypris Formations; 17–19-million-yearold; Krˇ´bek ı et al. 1998) has been used as the basal material for heapings since the beginning of mining activities at the studied sites (early 1950s). Vegetation developed during the different stages of succession. Miocene lacustrine sediment excavated from a depth of 200 m represents a very different environment as compared to other environments in which the primary succession of microbial communities has been studied. Miocene lacustrine sediment lacks recent organic carbon, but may contain some fossil carbon (Krˇ´bek ı et al. 1998). This fossil carbon could be an important source of organic matter for aerobic heterotrophic bacteria, as has been described for shale-derived dissolved organic matter (Schillawski and Petsch 2008). Because they are able to fix carbon, autotrophic colonizers are expected to be important in the initial stage of primary community assembly; however, Hodkinson et al. (2002) proposed an important role for heterotrophs during early colonization of terra nova. Heterotrophic organisms, including microflora, are responsible for accessing a range of essential nutrients, nitrogen and phosphorus in particular, that can be limiting factors for further community development. In addition, heterotrophic organisms usually occur much earlier than plants and precede autotrophs, and this phase might facilitate the establishment of green plants and consolidate the process of community development (Hodkinson et al. 2002). Moreover, the role of actinomycetes and arbuscular mycorrhizal fungi (AMF) as plant growth promoters and promoters of the establishment and survival of colonizing plants has already been demonstrated (Kurtbo ¨ke et al. 2007). Changes in the diversity of heterotrophic bacteria during plant succession were studied by Schipper et al. (2001) using a catabolic response profile method; they found differences in microbial heterotrophic evenness, which increased with increasing age and vegetation establishment. In contrast, they did not find differences in richness, which they explained by the fact that newly exposed and undeveloped soils are colonized rapidly by a wide range of heterotrophic microbes growing on very small amounts of organic matter. Therefore, heterotrophic microflora can first occupy the inert substrate and may play a dominant

595

role in pioneering microbial community assembly during the heterotrophic phase, as has been proposed for soil invertebrates (Hodkinson et al. 2002). The role of autochthonous microorganisms in community assembly has not yet been explained satisfactorily, but several studies have described bacterial and fungal surviving in vegetative forms, persisters, or as spores, even in granite (Fajardo-Cavazos and Nicholson 2006), old Siberian permafrost sediments (Mindlin et al. 2008), amber (Cano and Borucki 1995), and brine samples (Vreeland et al. 2000) for a very long time (Lavelle and Spain 2001). In addition, adsorption of microorganisms on to clay particles may help to protect microbial cells under natural conditions, as proposed by Kilbertus and Mangenot (1981). Recent reports have evaluated whether ancient bacteria could persist in old samples in the form of spores, vegetative forms, or persisters (Johnson et al. 2007; Lewis et al. 2008). The presence and release of biodegradable dissolved organic matter in ancient sedimentary rocks (Schillawski and Petsch 2008) supports the idea that aerobic heterotrophic bacteria can survive in such a habitat. Therefore, they can be present in the spoil and, after the surrounding conditions change, they might be able to proliferate. The first evidence of viable microbes and actinobacterial markers in virgin Miocene lacustrine sediment from the Sokolov coal basin was provided by Elhottova ´ et al. (2006) using a phospholipid fatty acid (PLFA) survey. They showed that actinomycetes and microscopic fungi predominated in the core of the substrate taken from a depth of 200 m. Organic matter and an alkaline pH (7.8) in the material may favor actinomycete survival. The dominance of Gram-positive bacteria in Sokolov spontaneous succession plots was shown by Krisˇtu ˚fek et al. (2005) using PLFA biomarkers. The ability of streptomycetes to form spores and bridge soil spaces using mycelium, together with their ability to use a broad spectrum of suitable carbon sources and inorganic nitrogen, enable them to survive long term under extreme conditions, such as drought, frost, hydrostatic pressure, or anaerobiosis induced by water saturation in soil (Ka ¨mpfer 2006). The complex metabolic machinery of streptomycetes can produce an infinite variety of secondary metabolites, including dark-brown to black melanoid pigments, that play important ecological roles in the immediate environment (Challis and Hopwood 2003; Fajardo and Martı´nez 2008). The competitive superiority of some Streptomyces species in the rhizosphere protects plants against root pathogens (Madigan and Martinko 2006; Schrey and Tarkka 2008), which could be important in the early stages of plant establishment (Kurtbo ¨ke et al. 2007). In addition, streptomycetes can facilitate mycorrhiza

596 and are beneficial to AMF development (Ames 1989; Riedlinger et al. 2006), which can lead to the establishment and survival of colonizing plants by aiding in nutrition (Gemma et al. 1989). In turn, the rhizosphere of plants influences the species composition and diversity of isolated Streptomyces strains in diverse Greek soils, as shown by Katsifas et al. (1999). There is little information about actinomycete development during natural colonization and primary succession of new substrates; however, Kurtbo ¨ke et al. (2007) showed that mesophilic actinomycetes developed from simple micromonosporae species through Streptomycetes to a complex actinomycete community in natural coastal dunes of Fraser Island in Australia. However, this work did not describe the diversity of isolated actinomycetes in detail. In the present study, we evaluated the diversity of streptomycete isolates from freshly excavated Miocene lacustrine sediment and from a spontaneous succession line developing on colliery spoil heaps. The study of sites of different ages, socalled chronosequence, provided insights about ecological succession within a manageable timeframe, whereas long-term observation of an ecosystem over decades is an impractical and complex task. However, our approach has some pitfalls, namely the variability of the local conditions among the sites. We tried to minimize the effects of this variability by selecting geologically similar sites and selecting relatively homogeneous tertiary clays (Frouz et al. 2009). We also evaluated the biological activity of streptomycetes as producers of antibiotics, which led to the isolation of strains with potential economic value. Sensitivity to antibiotics was screened to estimate frequencies of genetic determinants for resistance that developed in a pristine environment before intensive antibiotic loading and in the succession sequence that developed without any antibiotic loading interference. The primary aim was to compare characteristics of habitats in freshly excavated substrate with the characteristics of habitats that developed during subsequent succession stages. We hypothesized that (i) streptomycetes inhabiting the Miocene sediment and those prevalent in the pioneer stage would differ from the dominant streptomycetes in more developed soil; (ii) the richness of isolated Streptomyces species would be greater in the middle and late stages, when biologically available carbon and the diversity of higher plants and animals were increasing; and (iii) antibiotic and melanoid production potential and antibiotic resistance in isolated streptomycete strains would increase along the primary succession line as a result of strong microbial competition for nutrients. To address these hypotheses, we performed 16S-

A. Chron ´kova ´ et al. ˇa internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) analysis and 16S rDNA sequencing to estimate genetic diversity among the strains. Antibiotic and melanoid production tests, as well as tests for sensitivity to three antibiotics, were performed for phenotype screening.

Material and methods The sites Substrates and soils used in this study were collected from colliery spoil heaps (North-West Bohemia, Sokolov Coal Basin; see Krˇ´bek ı et al. 1998) that had been undergoing spontaneous succession for different amounts of time. The sites represented a succession sequence of four different stages left non-reclaimed: the initial stage (1–2 years after heaping), the early stage (11–12 years), the mid stage (21–22 years), and the late stage (43–44 years). Excavated spoil substrate originating from Miocene lacustrine clay sediments (pH 7.8) was heaped by the same technology. Details about the sites and soil chemical and microbial properties were summarized by Krisˇtu ˚fek et al. (2005). For a full description of the Miocene sediment, see Krˇ´bek ı et al. (1998).

Sampling Samples (three independent replicates from 13 sub-sampling sites) were taken using a cylindrical soil corer (cross Section 3 630 mm2) from the top (0–50 mm) and the mineral layers (100–150 mm) in depressions of heap ribs at the end of vegetation dormancy in March 2004 and 2005. Samples were homogenized by sieving through a 5-mm screen sieve and stored for 1–2 d at 41C before analysis. The Miocene sediment was sampled as an intact solid block of clay-stone (cca 0.5  0.3  0.3 m) from 200 m depth in the open cast mine ‘‘Jirˇ´’’ ı (Sokolov coal mining district, Czech Republic lat 501120 4200 N, long 121410 0000 E) using open cast mining machinery. Sterile conditions and aseptic sampling procedures were utilized according to previous protocols (Elhottova ´ et al. 2006). Three mixed 50-g samples were acquired from the core and processed immediately for microbiological analysis.

Soil analysis Soil pH was measured according to standard techniques (ISO/DIS 10390 1992). Microbial biomass (Cmic) was determined by the chloroform fumigation-extraction method (Vance et al. 1987) and

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment expressed as mg C g1 dw. Vegetation cover (herbs, grasses, shrubs, and tree cover) on 1–44year old plots was described by Frouz and Nova ´kova ´ (2005) and Krisˇtu ˚fek et al. (2005). The 1-year accumulation of litter (leaf/grasses/herbs) produced on the plots was measured by collecting three replicates from surface areas of 0.36 m2. Material was dried at room temperature, weighed, and expressed as kg m2.

Colony forming unit counts and isolation of actinomycete strains Soil samples (5 g) were transferred to Erlenmeyer flasks containing 45 ml of sterile 0.1% sodium pyrophosphate solution and placed in an ultrasonication bath (50 kHz, 4 m, room temperature). Then, serial 10-fold dilutions were made in sterile tap water, and 200 ml of each dilution was spread on to McBeth Scale agar (MBS) containing (g/l) starch (10), CaCO3 (3), K2HPO4 (1), (NH4)2SO4 (2), MgSO4  7H2O (1), NaCl (1), and agar (25), pH 7.0. Plates were incubated at 28 1C in the dark for 7 d and colonies of bacteria and actinomycetes were enumerated after 7 and 14 d. The ratio of colony forming units (CFUs) of actinomycetes to CFUs of culturable bacteria (CFUact/CFUbact) was calculated. Actinomycete colonies were randomly isolated from MBS after 14 d of incubation. All strains were grown on MBS at 28 1C in the dark for approximately 14 d. A total of 366 isolates (169 from the top layer, 178 from the mineral layer, and 19 from the Miocene sediment) were obtained and grouped according to their morphological–physiological properties. Test strains were maintained as suspensions of spores and mycelial fragments in glycerol (15% v/v) at 80 1C.

Melanoid pigment production and antibiotic activity assays The excretion of a melanoid pigment into solid media was evaluated on tryptone-yeast extract agar (Shirling and Gottlieb 1966). Dark-brown pigmentation of the media was considered a positive signal. Antibiotic activity of solid cultures was assayed by the agar plug method using Bacillus subtilis CCM 1718 (BS), Escherichia coli CCM 2024 (EC), and Saccharomyces cerevisiae CCM 8191 (SC) as test organisms. The cultures were grown at 28 1C for 4 d in a medium containing (%) glucose (1.0), soybean meal (1.5), NaCl (0.3), CaCO3 (0.3), agar Oxoid No. 3 (2.5), and tap water, pH 6.1–6.2. The size of inhibition zone was measured after 18 h incubation at 28 1C.

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Susceptibility/resistance test to antibiotics The standard procedure (Kirby–Bauer method; Madigan and Martinko 2006) was used to assess susceptibility to amoxicillin, chloramphenicol, and tetracycline. MH agar medium, containing in (g/l) yeast extract (4), malt extract (10), and glucose (4), pH 7.3, was inoculated by evenly spreading an actinomycete spore suspension of defined density obtained from pure cultures onto the agar surface. Filter paper discs containing 25 mg of amoxicillin and 30 mg of both chloramphenicol and tetracycline (Bio-Rad Laboratories, USA) were placed on the surface of the inoculated medium. The size of the inhibition zone was measured after 48 h at 28 1C. The mean size of the inhibition zones was calculated for all antibiotics. Data were interpreted according to Madigan and Martinko (2006).

DNA extraction Actinomycete strains were cultivated as submerged cultures in MH liquid medium incubated at 28 1C until the appropriate biomass grew and checked for bacterial contamination by light microscopy. Genomic DNA was isolated using the Wizard Genomic DNA Isolation Kit (Promega, USA) according to the manufacturer’s instructions. DNA samples were resolved by 1% agarose gel electrophoresis to estimate the quality and quantity of the template for downstream polymerase chain reaction (PCR) amplification. Genomic DNA was diluted 1:10 or 1:100 for PCR assays.

Amplification of 16S rDNA and 16S rDNA-ITS region A stretch of 16S rDNA gene was amplified using a semi-specific actinomycete primer set, 243F and 1378R (Heuer et al. 1997). PCR was performed in a 50-ml volume containing 75 mM Tris–Cl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 200 mM each dNTPs, 40 pmol of both primers, and 1.5 U of Taq DNA polymerase (all MBI Fermentas, Lithuania). The thermal cycle was 95 1C/5 m for initial denaturation followed by 35 cycles of 94 1C/ 60 s, 64 1C/60 s, 72 1C/90 s, and a final extension 72 1C/10 m. The ITS and the 16S rDNA were amplified using universal bacterial PCR primers pA and BL235R (Lanoot et al. 2005). PCR products were resolved by 1% agarose gel electrophoresis and visualized on an ultraviolet transilluminator after ethidium bromide staining.

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Restriction fragment length polymorphism analysis PCR products were precipitated in ethanol (96% v/v) using potassium acetate (0.3 M, pH 5.2); DNA was collected by centrifugation (18 000 rpm, 5 m). Pellets were air dried and resuspended in the appropriate restriction buffer (MBI Fermentas). Ten units of restriction endonuclease were used per reaction (10 ml); samples were incubated at the recommended temperature for 3 h. RFLP analysis of partial 16S rDNA was performed using AluI, TaqI, and Hin6I (MBI Fermentas). RFLP of the 16S-ITS region was performed using BsuRI and Bsh1236. All samples were resolved by 1.5% SynerGel agarose electrophoresis (80 V for 1 h). Restriction patterns were analyzed using the Photo-doc system (Vilber Lourmat, France).

Screening for nonribosomal peptide synthetase and type I polyketide synthases genes Genes for nonribosomal peptide synthetases (NRPS) and type I polyketide synthase (PKS-I) were detected using specific PCR-targeted actinomycete sequences (Ayuso-Sacido and Genilloud 2004). Multiple PCR amplification of target genes was performed using the primer pairs A3F/A7R (NRPS) and KSF/M6R (PKS-I), using genomic DNA as a template. Amplification and temperature cycling conditions were as described in the original protocol.

Sequencing A gene stretch between positions 27 and 1522 (E. coli numbering) of the 16S rDNA were amplified using a primer set previously reported by Edwards et al. (1989). The PCR products were separated by preparative agarose gels electrophoresis (1.5%), excised, and extracted from the gel using QIAquick Gel Extraction Kit (Qiagen, USA). The DNA concentration was estimated by absorbance at 260 nm. Sequencing reactions were performed using Big Dye Terminator chemistry (ABI PRISM DYE Terminator Cycle Sequencing Kit, Perkin Elmer v3.1, Applied Biosystems, USA). Sequencing was performed on an ABI 3130xl Genetic Analyzer (Perkin Elmer, USA).

Phylogenetic analysis and sequence accession numbers Sequence fragments were visualized and edited using Bioedit 7.0.4.1 (Hall 1999) software and assembled using SeqMAN (Swindell and Plasterer

A. Chron ´kova ´ et al. ˇa 1997; DNAStar, Inc.). Contigs of 1400-kb length were tested against all DNA sequences available in GenBank (www.ncbi.nml.nih.gov) using the programs blastn and RDP 10 (http://rdp.cme.msu. edu/). The 16S rRNA sequences were aligned with a collection of actinomycete sequences retrieved from GenBank (40). Multiple alignments were created and edited using Mafft 5.861 (Katoh et al. 2002) and Bioedit. The 16S rRNA data sets were analyzed by maximum likelihood (ML) analysis using Phyml 2.4.4. software (Guindon and Gascuel 2003) under the GTR+I+G8 model of evolution, chosen according to the Akaike information criterion value as implemented in Modeltest 3.7 (Posada and Crandall 1998). Bootstrap support was assessed from 1000 replications by the ML algorithm using the above described model and software, and by maximum parsimony (MP) using PAUP 4.0b10 (Swofford 2002). The nucleotide sequences of partial 16S rDNA of the isolates were deposited in GenBank under Accession Nos. EU098010–EU098045.

Statistical analysis Analysis of variance (ANOVA) followed by post hoc comparison between means (Tukey HSD test) were used to determine the significant differences in chemical and microbiological properties between studied sites in each layer separately. All statistical tests were performed using STATISTICA (v7.1, StatSoft, Inc. 2005). The possible relationships among the frequencies of resistance phenotypes and bacterial or actinomycete densities were tested using Pearsons’ correlation coefficient. A P-value o0.05 was considered statistically significant.

Results Soil analysis Generally, pH values in the top and mineral layers were significantly higher in the initial and early stages of the succession sequence as compared to the middle and late stages (One-way Anova, Tukey HSD, Po0.05; Table 1). Microbial carbon (Cmic) content was significantly greater in the top layer than in the mineral layer for the middle and late stages in both years of sampling, while there were no differences observed between the top and mineral layers in the initial and early stages (Table 1). As expected, Cmic content in both layers correlated with site age, reaching the highest values in the middle stage for the top layer (663.5 mg g1 dw) and in the late stage

Stage

Years after deposition

Cmicx(lg g 1 dw)

pH/H2Oy

Litterz(g m2)

CFUbactw(x108 g1 dw) CFUactq(x106 g1 dw) CFUact/ CFUbacto(%)

Top layer (0–50 mm) Initial 1–2 Early 11–12 Middle 21–22 Late 43–44

94.7 210.1 663.5 586.1

(56.3–133.1)a (119.9–300.2)b (459.3–867.8)a,b (553.9–618.3)a,b

8.39 7.95 7.19 7.27

(8.1–8.7)a (8.1–7.8)b (7.6–6.8)a,b (7.5–7.1)a,b

0.0a 191.4 (95.8–286.9)b 765.6 (384.2–1147.1)a,b,c 514.5 (492.4–536.5)a,b,c

0.28 0.15 1.55 1.20

(0.51–0.05) (0.11–0.19) (1.11–1.99) (1.06–1.34)

0.05 0.39 12.77 10.14

(0.09–0.01)a (0.39–0.40) (7.37–18.17)a (6.31–13.97)a

0.2 2.6 11.5 8.5

Mineral layer (100–150 mm) Initial 1–2 Early 11–12 Middle 21–22 Late 43–44

103.1 144.7 200.6 269.0

(74.7–131.5)a,c (77.0–212.4)b (188.8–200.1)a,c (257.1–280.9)a,b

8.60 8.15 7.83 7.65

(8.7–8.5)a (8.3–8.0) (8.2–7.5)a (7.9–7.4)a

– – – –

0.15 0.09 0.72 0.42

(0.24–0.06) (0.09–0.08) (0.36–1.09) (0.42–0.41)

0.04 0.56 3.88 6.31

(0.02–0.07)a (0.56–0.57) (1.49–6.27)a (2.82–9.80)a

0.3 6.2 5.4 15

A same superscript letter means significant difference. x Microbial biomass was determined by the chloroform fumigation-extraction method (Vance et al. 1987) and expressed as mg C g1 dw. y For pH measurement 5 g of dry soil was suspended in 12.5 ml distilled water for 1 d. z An accumulation of 1-year-old litter (leaf/grasses/herbs) produced on plots was measured by collecting it from an area of 0.36 m2 (three repeats). w The number of bacteria as colony forming units per gram of dry soil. q The number of actinomycetes as colony forming units per gram of dry soil. o The relative proportion of actinomycetes (CFUact/CFUbact) calculated from means.

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment

Table 1. Means of investigated environmental and microbiological parameters of the primary succession on spoil of brown coal colliery substrate. The mean over year values are given in the brackets (sampling years 2004 and 2005, respectively).

599

600 Table 2. Comparison of certain physiological characteristics and biosynthetic systems of Streptomyces strains isolated from the Miocene deep subsurface sediment core and substrates of the primary succession process on colliery spoil heaps. Sitesa

Layer (mm) Nb total Nb melanoid Nb actibiotic Nb strains with antibiotic activity againstc producing active EC BS SC

Miocene sediment – Initial stage 0–50 100–150 Early stage 0–50 100–150 Middle stage 0–50 100–150 Late stage 0–50 100–150 Top layer Mineral layer Total

0–50 100–150

Nb strains resistant tod

Biosynthetic systems

AMO

Nb examined NRPS PKS-I

CHLO

TET

19 37 46 41 43 41 45 50 44

0 0 6 11 11 9 24 16 8

10 7 15 11 13 11 14 11 6

0 0 0 0 2 2 4 0 1

8 7 14 11 12 11 14 10 6

5 1 1 4 1 1 0 4 0

7 4 1 6 2 2 3 2 0

2 0 3 15 5 4 7 6 4

1 2 0 0 3 1 1 7 4

14 6 10 5 7 16 16 12 12

14 6 9 5 7 15 13 10 9

10 3 7 2 5 10 9 4 4

169 178

36 49

40 48

2 7

39 46

10 2

14 6

25 19

10 8

39 45

36 38

19 25

347

85

98

9

93

17

27

46

19

98

88

54

a

Sampling years 2004 and 2005. N – number of strains. c EC – Escherichia coli, BS – Bacillus subtilis, SC – Saccharomyces cerevisiae. d AMO – amoxicillin, CHLO – chloramphenicol, TET – tetracycline; strains were concluded to be resistant to AMO and CHLO if the width of the zone was higher than 12 mm and resistant to TET if the width of the zone was higher than 14 mm (Madigan and Martinko 2006). b

A. Chron ´kova ´ et al. ˇa

Phylotypes

OTU1 OTU2 OTU3 OTU4 OTU5 OTU6 OTU7 OTU8 OTU9 OTU10 OTU11 OTU12 OTU13 OTU14 OTU15 OTU16 OTU17 OTU18 OTU19 OTU20 OTU21 OTU22 OTU23 OTU24 OTU25 OTU26 OTU27 OTU28 OTU29 OTU30 Richness

RFLP

Sequence affiliation

16S

16S-ITS

S1 S1 S1 S1 S1 S2 S3 S3 S3 S3 S3 S3 S3 S3 S4 S4 S6 S6 S8 S9 S12 S12 S12 S14 S14 S15 S16 S16 – –

ITS1 ITS6 ITS3 ITS7 ITS5 ITS8 ITS1 ITS5 ITS8 ITS3 ITS10 ITS12 ITS13 ITS7 ITS4 ITS9 ITS3 ITS7 ITS1 ITS10 ITS7 ITS3 ITS5 ITS3 ITS2 ITS11 – ITS5 ITS14 ITS15

S. microflavus S. sp. P5, S. avidinii S. griseochromogenes S. phaeochromogenes S. avidinii S. californicus S. atratus, S. albidoflavus S. exfoliatus S. sp. 3471, S. aureus S. atratus S. tauricus S. griseochromogenes S. scabieia S. spinicoumarensisa S. turgidiscabiesa S. sp. 65, S. turgidiscabies – S. californicusa S. atratus Kitasatospora gansuensis S. phaeochromogenes – S. venezulae S. prunicolora S. prunicolor Amycolatopsis rifamycina Amycolatopsis umgenii S. sp. P5, S. avidinii S. champavatii S. sp. 445, S. hygroscopicus

Miocene sediment

x

Initial stage

Early stage

Mid stage

Late stage

Top

Mineral

Top

Mineral

Top

Mineral

Top

X X X

x

x x x

x x

x

x

x

Mineral

x

x

x

x x

x x

x

x

x x x x x

x x x x x

x x

x x x x x

x

x

x x x x x x x

x

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment

Table 3. Distribution of phylotypes (OTU) of Streptomyces strains isolated from the Miocene deep subsurface sediment core and substrates of the primary succession process on colliery spoil substrates from the top layer (0–50 mm) and mineral layer (100–150 mm).

x x 4

4

2

6

6

9

9

7

4

Sequence affiliation of retrieved 16S rDNA sequences is based on BLAST and GenBank entries. Richness represents the number of different phylotypes in the habitat. a Affiliation based on shorter sequence fragment of 968–1401 (E. coli numbering) position.

601

602 for the mineral layer (269.0 mg g1 dw). Differences among sites were significant. The middle and late stages were characterized by increased litter input of 765.67453.1 and 514.57108.3 g m2, respectively, as compared to 191.47131.7 g m2 in the early stage. There was an insignificant amount of litter in the initial stage of the succession gradient.

Bacterial and actinomycete CFU counts and the ratio of actinomycetes to culturable bacteria In freshly excavated Miocene lacustrine sediment, the density of culturable bacterial cells was estimated at 0.71  106 g1 dw and CFUact was estimated at o1.0  104 g1 dw. The estimated ratio of culturable actinomycetes among bacteria was roughly 1% or less. CFUbact ranged from 106 to 108 units per gram of dry soil. CFUbact counts from the mineral layer of all stages of the succession sequence were lower as compared with those in the top layer, but the differences were not significant (Two-way ANOVA, Tukey HSD, Po0.05; Table 1). The highest CFUbact counts in the succession sequence were detected in the middle succession stage, and this observation was repeated in both layers and for both sampling seasons. The average number of CFUact ranged from 104 to106 units per gram of dry soil (Table 1). CFUact was higher in the top layer than in the mineral layer, with the largest differences in mid and late stages. CFUact increased from 0.05  106 g1 of dry soil in the initial stage of the top layer to 0.39  106, 12.77  106, and 10.14  106 in the early, middle, and late stages, respectively. The highest counts were detected in the middle and the late succession stages in both layers and sampling periods. Differences among actinomycete counts were significant among sites after logarithmic transformation of data, but were not significant for total bacterial counts (Table 1). The CFUact/CFUbact ratio increased with the age of deposition from the initial to the late stage (1–44 years; Table 1). Over 44 years, the maximum CFUact/CFUbact increased from 0.2% to 11.5% in the top layer, and from 0.3% to 15% in the mineral layer.

Antibiotic activity, melanoid pigment production, and genetic potential for secondary metabolite production Most of the antibiotic substances produced were effective against BS (93 positive strains), followed by 17 and nine strains displaying antimicrobial

A. Chron ´kova ´ et al. ˇa activity against SC and EC, respectively (Table 2). Twelve strains inhibited the growth of both EC and BC, another 12 strains were active against BC and SC, and one strain was active against all three strains (data not shown). An extraordinarily high proportion of antibiotic-producing strains were observed in Miocene sediment, where 80% of isolated strains displayed activity against BS and 50% against SC. Twenty-three percent of the isolated strains produced melanoid pigments during in vitro incubation. Streptomycete isolates from Miocene sediment and from the initial succession stage (top layer) did not produce melanoid pigments, but the proportion of organisms that produced the pigments increased with the succession sequence, with the highest number of positive strains (33) observed in the middle stage. Significant differences in the number of melanoid pigment-positive actinomycete strains were found between the top and mineral layers of the initial, middle, and late stages of the succession sequence, with the highest number occurring in the mineral layers (Table 2). Ninety-eight strains were screened for the presence of genes involved in secondary metabolite production. Genes coding for NRPS were detected in most Streptomyces strains tested (90%), and their occurrence was uniformly distributed among strains from all environments. Genes coding for type I PKS were less abundant (54%), with a higher occurrence in strains isolated from the mineral layer (Table 2).

Susceptibility test of isolates against amoxicillin, chloramphenicol, and tetracycline Susceptibility testing was performed on 313 strains (Table 2). Forty-six strains were resistant to chloramphenicol (ChloR), 27 strains to amoxicillin (AmoR), and 19 strains to tetracycline (TetR). Two multiresistant phenotypes were observed: AmoRChloR and ChloRTetR, with frequencies of 2.5% and 0.3%, respectively. The proportion of ChloR strains was the highest in the early stage in the top layer (37%), not exceeding 15% at other locations. The proportion of TetR strains was the greatest in the late stage in the top layer (16%), with the proportion tending to increase with the succession stage. In contrast, the proportion of AmoR strains was highest in the Miocene lacustrine sediment (37%) and decreased with the succession stage. These frequencies were not correlated to bacterial or actinomycete density (CFU counts), which would indicate more intensive spread of resistance determinants in the community under density pressure (P40.1).

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment

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Figure 1. Phylogeny of streptomycete 16S rDNA sequences (accession numbers are given in brackets). The maximum likelihood (ML) tree was constructed under the GTR+I+G8 model of evolution using PhyML 2.4.4. (Guindon and Gascuel 2003). The bootstrap support was calculated from 1000 replications using PhyML 3.0 for ML (before dash) and Paup 4.0b10 (Swofford 2002) for maximum parsimony (after dash). The tree was rooted with 16S rDNA sequence of Kitasatospora gansuensis HKI 0314. The scale bar indicates 0.1 changes/site.

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Genetic diversity of culturable actinomycetes Thirty different operational taxonomic units (OTUs), according to rDNA and ITS profiling, were obtained. The compositions of soil actinomycete communities based on 16S rDNA and 16S rDNA-ITS RFLPs are summarized in Table 3. Richness (R), representing the number of phylotypes, was evaluated for each particular environment and layer. The greatest richness was detected in the middle stage, where nine different phylotypes were detected in both layers. The least richness was detected in the mineral layer of the initial stage, where only two phylotypes were described, followed by communities in the Miocene sediment and top layer of initial stage, where four phylotypes were identified. A shift in community composition was observed: the pioneer representatives were characterized by OTU1–3 phylotypes of the S1 group, which were very rare in the late stage. OTU1 was also present in the Miocene sediment. In contrast, the late succession community was dominated by the S3 group (OTU7–14). These S1 and S3 clusters comprised the majority of the actinomycete collection, with 52% of isolates belonging to the S1 group and 44% to the S3 group (data shown for isolates from 2005). Remaining phylotypes were rare, appearing mostly in the middle stage, thereby increasing its richness. Richness of actinomycete communities in mineral layers of early and late stages was lower than richness in the top layers.

A. Chron ´kova ´ et al. ˇa of Streptomyces microflavus, S. flavofuscus, S. anulatus (all OTU1), S. atratus (OTU7, OTU10, OTU19), S. exfoliatus (OTU8), and S. avidinii (OTU2, 5 and 28), was dominated by isolates from the initial and early stages of the succession sequence. The Middle-Late (ML) cluster, dominated by isolates from the middle and late stages, represented relatives of S. aureus (OTU9), S. tauricus (OTU11), S. prunicolor (OTU25), S. phaeochromogenes (OTU4, 21), and S. griseochromogenes (OTU3, 12). The third cluster, named the S. turgidiscabies cluster, was populated by isolates from the early and late stages (OTU13, 15, 16). Finally, the Miocene cluster (MIO), harboring sequences of Miocene isolates only (Miocene 4, 9 and 15), showed close relationships with Streptomyces gougerotii, S. champavatii (both OTU29), and S. sp. 445 (OTU30). These strains were present only in this ancient sediment and were not retrieved from soils of the primary succession gradient. Two other sequences retrieved from Miocene sediment (Miocene 5 and 11) clustered with sequences of S. microflavus (OTU1), S. flavofuscus (OTU1), and S. avidinii (OTU5; IE cluster).The greater diversities in the middle and late stages revealed by RFLP profiling were confirmed by a wider variability of sequences in the phylogenetic tree. While most sequences characteristic of initial and early stages were grouped together and were more similar to each other, sequences characteristic of middle and late stages were more diverse and did not cluster together. A few isolates of Kitasatospora and Amycolatopsis genera were identified among strains from the middle and late stages (Table 3).

Phylogenetic analysis The 16S rDNA sequences of 36 isolates were aligned with 40 actinomycete sequences obtained from GenBank, generating a 1393-bp-long alignment containing 130 parsimony-informative positions. High similarity (497%) with known sequences of Streptomyces, Kitasatospora (OTU20) or Amycolatopsis spp. (OTU26, 27) was observed, with the majority of analyzed strains belonging to Streptomyces spp. (93%, all other OTUs; Table 3). Based on previous analyses with more general taxon sampling (data not shown), the dataset was rooted with Kitasatospora gansuensis; simultaneously, Amycolatopsis spp. and related sequences were excluded from analysis. Maximum likelihood (ML) and maximum parsimony (MP) methods produced similar topologies (only the ML tree is shown; Figure 1). Phylogenetic analysis revealed four distinct clusters (Figure 1) corresponding to the stages of succession. The Initial-Early (IE) cluster, representing relatives

Discussion Occurrence of viable streptomycetes in Miocene lacustrine sediment Here we report the isolation of viable streptomycetes (19 strains) from Miocene lacustrine sediment in the Cypris formation (17–19 million years old; see Krˇ´bek ı et al. 1998). There are several studies describing the isolation of viable bacteria from deep sediments (permafrost in eastern Siberia) about 3 million years old (Mindlin et al. 2008), from 25- to 40-million-year-old Dominican amber (Cano and Borucki 1995), from a 250-millionyear-old primary salt crystal (Vreeland et al. 2000), and deep terrestrial subsurface (Fliermans and Balkwill 1989). All of these studies reported the prevalence of aerobic or facultative anaerobic heterotrophic bacteria in ancient samples, with

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment oxidative rather than fermentative metabolism; in addition, some reported that a larger proportion of ancient bacteria in their samples were able to grow in the laboratory on rich medium (Fliermans and Balkwill 1989). In their report, Fliermans and Balkwill (1989) showed that the diversity of bacteria was not related to depth, but fluctuated from one stratum to another, with the lowest bacterial density and diversity occurring in the nontransmissive zones where clay content exceeded 20%. In our study, the density of culturable bacterial cells was estimated at 0.71  106 g1 dw and CFUact was estimated at o1.0  104 g1 dw for Miocene lacustrine sediment. These values are likely 100-times lower than that for the initial stage in the top layer, which had undergone microbial succession for 1 year only. Previously reported PLFA-based evidence of an actinobacterial pristine community in Miocene lacustrine sediment (Elhottova ´ et al. 2006) was supported by our isolation of viable streptomycetes from this sediment. Remarkably, some strains originally present in the Miocene sediment could survive the changes associated with excavation of the original material and appeared to contribute to the colonization of the bare substrate. These strains were related to S. microflavus, S. spororaverus, and S. flavofuscus. Some of the species disappeared when they could no longer adapt to the changing conditions of the substrate and new colonizers replaced them (Lavelle and Spain 2001). In our study, these noncolonizing species were related to S. gougerotii, S. champavatii, and S. sp. 445 (S. aureus).

Microbial biomass, abundance of culturable bacteria, abundance of actinomycetes Microbial biomass increased during succession, in accordance with the findings of Frouz and Nova ´kova ´ (2005) and Baldrian et al. (2008), who studied the development of soil microbial properties during spontaneous succession on the same plots. Sigler and Zeyer (2002) investigated the development of microbial populations that increased in number and activity along the forefields of receding glaciers, and they found different modes of bacterial community establishment between two studied succession sequences. The increase in bacterial abundance during community development in the newly formed soils of receding glaciers was also reported by Kandeler et al. (2006). The development of both top and mineral layers in our studied sequence showed similar trends; however, the difference between the top and mineral layers was greatest during the middle and late stages, which seems to

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be connected with greater differences in the overall microbial biomass between layers in the middle and late stages (Table 1). As concluded by Frouz and Nova ´kova ´ (2005), low input of organic matter, winter freezing, summer desiccation, and high pH may limit development of the microbial community in the initial stage. The input of organic matter by sedimentation from the air may occur in the top layer, but the mineral layer is not affected due to the absence of macrofauna, mainly earthworms; so, mixing of top (organic) and mineral layers does not occur until the late stage (Frouz et al. 2002). Further increases in CFU counts were coupled with earthworm-mediated soil mixing and changes in soil organic carbon availability (Krisˇtu ˚fek et al. 2005). In general, smaller numbers of bacterial counts in the mineral layer are explained by a smaller amount of organic matter relative to the top layer, so the population densities of most microorganisms decrease regularly with depth (Lavelle and Spain 2001). The increase in the relative proportion of actinomycetes with age of succession is in good agreement with the findings of Krisˇtu ˚fek et al. (2005) and Kandeler et al. (2006), who showed a prevalence of K-strategists during older succession stages or vice versa. Krisˇtu ˚fek et al. (2005) indicated that the greatest numbers and activity of bacteria, including actinomycetes, during substrate colonization is positively influenced by the age of the primary succession stage, manifested by changes in pH, the amount of litter, the activity of fauna, and the development of plant roots. We found the greatest proportion of actinomycetes (30%) in the late stage, in agreement with primary succession theory, which postulates that opportunistic bacteria develop in the pioneer stage and K-strategists prevail in the late succession community where soil has already been developed (Shrestha et al. 2007). These results could be affected by both succession changes and variability in site conditions. However, previous studies of other microbial parameters on the same sites (Frouz and Nova ´kova ´ 2005) that evaluated numerous replications of similarly aged sites suggest that, for most parameters, variability between sites of the same age is smaller than variability between sites of different ages. This indicates that succession drivers contribute substantially to the observed community changes.

Physiological properties: antibiotic and melanoid production and resistance to tested antibiotics Streptomycetes isolated from primary succession plots and Miocene sediment varied in their antibiotic

606 production and resistance abilities. In general, there was a very small difference between the frequency of antibiotic production (28.2%, 98 strains) and resistance (26.2%, 82 strains) abilities. This observation differs from that made by Davelos et al. (2004) in prairie soils. They found a greater frequency of resistance phenotypes than antibiotic-producing abilities (67% and 17%, respectively). Our findings might be explained by lower or absent antibiotic load (veterinary or other) expected in succession plots originating from spoil material originally deposited 200 m below the surface. These data might indicate a low frequency of resistance genes and their spread by horizontal gene transfer under such conditions. Surprisingly large proportions of strains producing antibiotics and strains harboring NRPS and type I PKS genes were found in the Miocene sediment sample and in the pioneer stages of the primary succession gradient. The relatively high proportion of AmoR streptomycetes was registered in Miocene sediment. Presumably, AmoR is not rare, because the occurrence of antibiotic resistance determinants in ancient bacteria from Siberian sediments has been reported by Mindlin et al. (2008). We predicted greater antibiotic production by streptomycetes coming from the middle and late stages, where strong competition from rhizosphere bacteria and microscopic fungi could be expected; however, our results did not supported this prediction. Nonetheless, melanoid production increased with succession. At this time, we are unable to explain this phenomenon. These results support the conclusions of Bell and Wheeler (1986), who found that melanoid pigments are not essential for growth and development but that they enhance the survival and competence of species in certain environments.

Phenotype and genetic diversity of actinomycetes Based on phenotypic characteristics such as melanoid pigment production and antibiotic activity of isolated strains, the diversity of actinomycetes increased from the initial to the middle stage. These observations corresponded well to the molecular data, which indicated the greatest diversity (estimated as richness) of RFLP profiles in the middle stage in both layers. Development of a pioneer actinomycete community on a freshly excavated substrate was supported by the presence and activity of ancient streptomycetes related to the S. champavatii, S. sp. 445, S. microflavus, and S. flavofuscus groups. While relatives of S. microflavus and S. flavofuscus can be proposed as pioneer species in the studied environment, relatives of

A. Chron ´kova ´ et al. ˇa S. champavatii and S. sp. 445 were not found in any stage of the primary succession sequence and were present only in the core of the Miocene sediment. The shift in streptomycete community composition during succession was documented by the replacement of pioneer species by late succession species, which grouped with the ML cluster. Community shifting has already been demonstrated by Krisˇtu ˚fek et al. (2005), who showed shifting associated with plant root development and increasing availability of organic matter. Moreover, the feeding activity of earthworms may favor some Streptomyces species in the surrounding soil and affect their community composition (Krisˇtu ˚fek et al. 1993). The significant shift in the composition of the actinomycete community associated with primary succession in natural coastal dunes was reported by Kurtbo ¨ke et al. (2007). These authors indicated that beneficial actinomycete species could play a crucial role in early plant development via plant root protection and/or facilitation of mycorrhiza. The primary succession of the microbial community on colliery spoil heaps progressed more effectively once plants were established and offered a favorable niche rich in root exudates (Elhottova ´ et al. 2009). This event could happen about 5 years after excavating the Miocene sediment; the actinomycete community in the early stage (11–12 years) was strongly affected by plants and was more diverse than that characteristic of the initial stage (1–2 years). Further development occurs between early and middle stages, when richness of phylotypes reached the maximum. This is in agreement with the assumption of Baldrian et al. (2008) that the rate of nutrient accumulation and development of soil under primary succession is largely regulated by the vegetation succession. The switch from grassland to a forest-dominated ecosystem that occurs between 11 and 21 years might be of great importance in our sites, because these ecosystems differ in soil properties and annual dead plant biomass production.

Conclusions The survival of ancient Streptomyces spp. strains in the Miocene lacustrine sediment and the colonization of excavated substrate (1-year old) by the same species, together with species of unknown origin, support the idea that ancient streptomycetes can play an important role in facilitating spontaneous succession in post-mining colliery site. During succession, the streptomycete community developed dynamically, and diversity reached maximum after 20 years of development. The shift in streptomycete community

Biodiversity of streptomycetes and their evidence in Miocene lacustrine sediment composition was suggested by replacement of pioneer species by the late succession species, followed by changes at the functional level. For example, antibiotic production potential and antibiotic resistance changed, which could reflect changes in the interactions between microbes and their microenvironments. The production of antibiotics and resistance abilities seem to be interlinked and their genetic determinants were detected in strains from ancient samples. We conclude that the discrimination power of 16S-ITS fingerprinting is greater than that of 16S rRNA sequencing (1.4-kb long) and blastn alignment, due to greater variability in the ITS sequences. Environments such as colliery spoil heaps and other disturbed areas offer the opportunity to study pristine microbial communities, which have not been accomplished before. Streptomycete isolates from unusual environments can produce many bioactive compounds. This study contributed to the establishment of the ˇeske Culture Collection of Actinomycetes C ´ Bude ˇjovice (CCACB). The CCACB was founded in the Biology Centre AS CR, v. v. i. – Institute of Soil Biology in 2007 and serves as a depository for cultures of soil actinomycetes (www.actinomycetes.cz).

Acknowledgements The authors would like to acknowledge M. Obornı´k and A. Hora ´k (Biology Centre of AS CR, v. v. i., Institute of Parasitology) for helpful recommendations on phylogenetic data analysis and J. Moravcova ´ (Biology Centre of AS CR, v. v. i., Institute of Plant Molecular Biology) for sequencing. We thank L. Kodytkova ´, E. Zada ´kova ´, and M. Jorda ´kova ´ for their laboratory help. This research was funded by projects of Grant Agency of the Academy of Sciences of the Czech Republic (IAA600660607), Czech Science Foundation (526/03/1259), Grant Agency of University of South Bohemia (54/2004/P-BF), Research Plan of the Institute of Soil Biology ASCR (AV0Z60660521), and by Ministry of Education of the Czech Republic (LC 06066, 2B06154). The authors thank Professor D. Hopwood, J. Frouz and J. Hynsˇt for critical reading of the manuscript. Manuscript was edited by San Francisco Edit.

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