FEMS Microbiology Ecology 42 (2002) 419^429

www.fems-microbiology.org

New PCR primers for the selective ampli¢cation of 16S rDNA from
di¡erent groups of actinomycetes1
Paolo Monciardini, Margherita Sosio, Linda Cavaletti, Claudia Chiocchini 2 ,
Stefano Donadio 
Biosearch Italia, via R. Lepetit 34, 21040 Gerenzano (VA), Italy

Received 28 May 2002; received in revised form 2 August 2002; accepted 4 August 2002

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First published online 4 September 2002

Abstract

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive
metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing
unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA
from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from
the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good
sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific
amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to
environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed
to new groups of actinomycetes.
6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Polymerase chain reaction primer ; Environmental clone; Soil DNA; Actinomycetales

1. Introduction tosporangiaceae and Thermomonosporaceae also produce

valuable compounds, including erythromycin, gentamicin,
Actinomycetes are Gram-positive bacteria characterized rifamycin, teicoplanin and vancomycin [3,4]. It is not cur-
by high G+C content in their DNA. They can be found in rently known whether the higher number of streptomycete
a wide range of habitats, and are particularly abundant in metabolites re£ects an increased capacity to produce sec-
soil [1]. They also produce several structurally diverse bio- ondary metabolites, a larger strain diversity, or if it simply
active compounds of pharmaceutical and agricultural im- depends on a higher number of strains screened for bio-
portance. Although the order Actinomycetales encom- active metabolites. This latter possibility may re£ect their
passes more than 80 di¡erent genera [2], the majority of ease of isolation in comparison to many other actinomy-
bioactive metabolites have been isolated from Streptomy- cete genera.
ces species. Nevertheless, microorganisms belonging to the Searching for new microbial metabolites requires strat-
families Micromonosporaceae, Pseudonocardiaceae, Strep- egies directed at decreasing the probability of identifying
known compounds. One approach involves the screening
of metabolites produced by actinomycete groups less ex-
* Corresponding author. Tel. : +39 (0) 96474243;
ploited than Streptomyces in past screening e¡orts [5,6].
Fax : +39 (0) 96474238. Because they are frequently outcompeted by faster grow-
E-mail address : [email protected] (S. Donadio). ing strains, the application of selective methods is a pre-
requisite for the successful isolation of the less common
1
This work is dedicated to the memory of Prof. Franco Tato', former
actinomycete genera [7]. However, the inability to isolate a
president of the Italian Society for General Microbiology and Microbial
Biotechnologies.
particular genus from a given source could depend either
2
Present address: Biochemie-Fachberech Chemie, Philipps-University on the absence of that genus, or on the use of inadequate
of Marburg, Hans-Meerwein Str., D-35032 Marburg, Germany. methods. Knowing a priori the microbial population
0168-6496 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 3 5 3 - 7

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420 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429

present in a source could allow the utilization of proce- 2. Materials and methods
dures appropriate for the isolation of desired genera.
The application of molecular techniques to environmen- 2.1. Bacterial strains and DNAs
tal samples has allowed the analysis of microbial diversity
without the need for cultivation [8], showing that tradi- Bacterial strains used in this study were obtained from
tional cultivation-based techniques greatly underestimate the Biosearch Italia collection and are listed in Table 1. All
the microbial diversity present in environmental samples actinomycete strains were grown in AF/MS medium (per
[8,9]. For this type of analysis, one of the most widely used liter : 20 g glucose, 2 g yeast extract, 8 g soybean £our, 1 g
markers is 16S rDNA, whose sequence also allows bacte- NaCl, 4 g CaCO3 ) at 28‡C for 6 days. Escherichia coli and
rial classi¢cation and the reconstruction of phylogenetic Bacillus subtilis were grown in Luria Broth at 37‡C for
relationships [10]. Taking advantage of the presence in 16 h. Genomic DNA was extracted from bacteria with
the 16S rDNA of small regions diagnostic of phylogeneti- the PUREGENE1 DNA isolation kit (Gentra Systems)
cally correlated groups, speci¢c polymerase chain reaction according to manufacturers instructions and quanti¢ed
(PCR) primers have been designed for the Actinomycetales with the PicoGreen0 dsDNA Quantitation kit (Molecular
genera Pseudonocardia, Saccharomonospora and Saccharo- Probes). Clostridium perfringens DNA was purchased from
polyspora [11,12], as well as for the analysis of community Sigma.
structure by denaturing gradient gel electrophoresis using

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actinomycete-speci¢c primers [13]. 2.2. Primer design
The objective of the present work was the development
of molecular methods for assessing microbial community A total of 423 16S rDNA sequences of di¡erent groups
composition with respect to ¢lamentous actinomycetes. In of Actinomycetales were retrieved from the RDP-II Data-
this paper we describe six new primer sets for the speci¢c base Release 8.0 [14] and aligned using the program PILE-
ampli¢cation of 16S rDNA from di¡erent groups of acti- UP of the Wisconsin Package, Version 9.1 (Genetics Com-
nomycetes. puter Group), following a multi-step approach. First,

Table 1
Bacterial strains and ampli¢cation results
Straina PCR resultsb
BAC ACT STP STM MMS TMM DCT
Streptomyces coelicolor ATCC 10147 + + ^ + ^ ^ ^
Streptomyces lividans ATCC 19844 + + ^ + ^ ^ ^
Streptomyces cinnamoneus NRRL B-1285 + + ^ + ^ ^ ^
Streptomyces avermitilis ATCC 31267 + + ^ + ^ ^ ^
Streptomyces glaucescens DSM 40716 + + ^ + ^ ^ ^
Micromonospora carbonacea ATCC 27114 + + ^ ^ + ^ ^
Actinoplanes teichomyceticus ATCC 31121 + + ^ ^ + ^ ^
Actinoplanes ianthinogenes ATCC 21884 + + ^ ^ + ^ ^
Dactylosporangium matsuzakiense ATCC 31570 + + ^ ^ + ^ +
Dactylosporangium fulvum ATCC 43301 + + ^ ^ + ^ +
Dactylosporangium sp. ID 83892 + + ^ ^ + ^ +
Kibdelosporangium aridum ATCC 39323 + + ^ ^ ^ ^ ^
Saccharomonospora viridis ATCC 15345 + + ^ ^ ^ ^ ^
Saccharotrix australiensis ATCC 31497 + + ^ ^ ^ ^ ^
Amycolatopsis mediterranei ATCC 13685 + + ^ ^ ^ ^ ^
Planobispora rosea ATCC 53773 + + + ^ ^ ^ ^
Planomonospora sp. ID 77572 + + + ^ ^ ^ ^
Nonomuraea sp. ATCC 39727 + + + ^ ^ ^ ^
Streptosporangium cinnabarinum DSM 44094 + + + ^ ^ ^ ^
Streptosporangium sp. ID 71232 + + + ^ ^ ^ ^
Microbispora sp. ID 75340 + + + ^ ^ ^ ^
Actinomadura madurae DSM 43067 + + ^ ^ ^ + ^
Actinomadura macra ATCC 31286 + + ^ ^ ^ + ^
Actinomadura libanotica ATCC 35576 + + ^ ^ ^ + ^
B. subtilis ATCC 8185 + ^ ^ ^ ^ ^ ^
E. coli ATCC 10536 + ^ ^ ^ ^ ^ ^
C. perfringens + ^ ^ ^ ^ ^ ^
a
Codes for strain collections are: ATCC, American Type Culture Collection (Rockville, MD, USA); DSM, Deutsche Sammlung Von Mikroorganismen
und Zellkulturen GMBH (Braunshweig, Germany); NRRL, Ars Patent Culture Collection (Peoria, IL, USA); ID, Biosearch Italia.
b
Ampli¢cations using 5^10 ng genomic DNA and the primers of Table 2. Symbols + and ^ refer to the presence and absence, respectively, of a product
of the expected size.

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Table 2
PCR primers
Primer Sequence Positiona Reference
F27 5P-AGAGTTTGATCMTGGCTCAG-3P 8^27 [13]
R1492b 5P-TACGGYTACCTTGTTACGACTT-3P 1492^1513 [13]
243F 5P-GGATGAGCCCGCGGCCTA-3P 226^243 [13]
A3Rb 5P-CCAGCCCCACCTTCGAC-3P 1414^1430c This study
21F 5P-GACGAARNTGACGTGTA-3P 407^424c This study
959Rb 5P-CGTTGCGTCGAATTAAGCAA-3P 952^971 This study
M2F 5P-SAGAAGAAGCGCCGGCC-3P 492^508 This study
Sm6F 5P-GGTGGCGAAGGCGGA-3P 721^735 This study
Sm5Rb 5P-GAACTGAGACCGGCTTTTTGA-3P 1283^1303 This study
T3F 5P-GGGAGAATGGAATTCCC-3P 665^681 This study
T8Rb 5P-CCCCACCTTCGACC-3P 1413^1426c This study
D3F 5P-GCGGCTTGTTGCGTCAG-3P 585^601 This study
D2Rb 5P-CCGCTGGCAACATCGAACA-3P 1116^1134 This study
a
Numbering refers to the 16S sequence of E. coli rrnB (GenBank J01695, [29]), unless otherwise indicated.
b
Primer anneals to the complementary strand.
c
Numbering refers to the 16S sequence of Streptosporangium vulgare DSM 43802T (GenBank X89955, [30]).

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sequences were aligned within each genus to obtain genus- version 3.01 (http://www.basic.nwu.edu/biotools/oligo-
speci¢c consensi. These consensi were then aligned within calc.html) and primer lengths were adjusted in order to
families, giving a family level consensus for Micromono- minimize Tm di¡erences within each primer pair. The
sporaceae, Streptomycetaceae, Streptosporangiaceae and primers used are listed in Table 2.
Thermomonosporaceae. The family level consensi were
aligned with each other and with consensi obtained in 2.3. PCR ampli¢cations
the same way for each of the suborders Actinomycineae,
Corynebacterinae, Frankineae, Micrococcineae, Propioni- All ampli¢cation reactions were performed in a ¢nal
bacterinae and Pseudonocardineae. Aligned sequences volume of 50 Wl, containing 50 mM KCl, 10 mM Tris^
were then visually compared for the identi¢cation of re- HCl, pH 8.3, each primer at 500 nM, each dNTP at
gions showing a high degree of conservation within the 0.2 mM, 1.5^3.5 mM MgCl2 , 1.5^2.5 U of AmpliTaq or
selected family, but having at their 3P-end one or more AmpliTaq Gold DNA Polymerase (Applied Biosystems)
mismatches with the consensi of other groups. Possible and 3 Wl of 10UDenhardt’s reagent (per liter : 2 g Ficoll
primers were evaluated by probing their sequences against 400, 2 g polyvinylpyrrolidone, 2 g bovine serum albumin)
the whole RDP-II database through the Probe Match soft- [15]. Optimized cycling parameters, enzyme and MgCl2
ware (http://rdp.cme.msu.edu/html/index.html), ensuring concentration for each primer pair are shown in Table 3.
that at least 90% of the sequences belonging to the target Each ampli¢cation program was initiated by denaturation
groups were recognized by the primer. Non-target groups for 5 min at 95‡C (10 min when using AmpliTaq Gold)
of sequences that could potentially yield a non-speci¢c and ¢nished with an extension step of 10 min at 72‡C.
extension product with one primer were taken into ac- Reactions were performed either in a PE 9600 (Applied
count when choosing the other primer of the pair. The Biosystems) or in a PCRExpress thermal cycler (Hybaid
Tm of each primer was evaluated through Oligo Calculator Ltd.) equipped with a gradient block for optimization of

Table 3
PCR conditions
Target Code Primers Ampli¢ed Enzyme unitsa MgCl2 (mM) Cycling parametersb Detection limit
fragment (kb) (pg DNA)
Bacteria BAC F27^R1492 1.5 1.5 1.5 45/94^45/61^120/72 NDc
Actinomycetales ACT 243F^A3R 1.25 1.5 3 45/94^120/68 2
Streptomycetaceae STM Sm6F^Sm5R 0.6 1.5 1.5 45/94^45/65^60/72 91
Micromonosporaceae MMS M2F^A3R 1.0 2.5d 1.5 45/94^120/68 1
Streptosporangiaceae STP 21F^959R 0.5 1.5 2.5 45/94^45/58^60/72 1^5
Thermomonosporaceae TMM T3F^T8R 0.8 2d 1.5 45/94^45/59^60/72 1
Dactylosporangium DCT D3F^D2R 0.57 1.5 1.5 30/94^90/68 91
a
AmpliTaq DNA Polymerase, except as noted.
b
Numbers before dash refer to time (in s), after dash refer to temperature (in ‡C) for each step in cycle. PCR comprised 30 cycles, except for primers
TMM (35 cycles).
c
ND: Not determined.
d
AmpliTaq Gold DNA Polymerase.

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the annealing temperature. As a positive control, all DNA was extracted from 250 mg soil using the Ultra
DNAs yielded the expected ampli¢cation fragment of Clean1 Soil DNA Isolation kit (MoBio Laboratories
about 1.5 kb when tested with the BAC primers (F27 Inc.), and resuspended in 50 Wl of the elution solution
and R1492, Table 2), speci¢c for all bacteria [13]. provided. Soil DNA was quanti¢ed with the PicoGreen0
dsDNA Quantitation kit.
2.4. Sequencing of PCR products

PCR products were cloned in the pCR0 2.1-TOPO0 3. Results

vector using the TopoTA cloning0 kit (Invitrogen). The
ligation mixture was used to transform E. coli JM109 us- 3.1. Development of selective methods
ing standard techniques [16]. About 40^200 colonies were
obtained in each cloning experiment. After boiling single The actinomycete families described by Stackebrandt
colonies in 40 Wl sterile H2 O for 5 min and removing cell and colleagues [2] were used as the starting point for de-
debris by centrifugation, 5 Wl of the supernatant was used veloping PCR-based methods. PCR primers were designed
as template for PCR. PCR products, puri¢ed with the and validated in silico as described under Section 2. Even
GFX1 PCR DNA and Gel Band Puri¢cation kit (Amer- if primers for all actinomycetes were already available
sham Pharmacia Biotech), were directly sequenced with [13,19], the ampli¢ed fragment did not encompass all the

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the BigDye1 Cycle Sequencing kit (Applied Biosystems) taxon-speci¢c primers described here. We thus combined
according to manufacturer’s instructions, and run on an primer 243F, used for selective ampli¢cation of actinomy-
ABI Prism 310 automatic sequencer (Applied Biosystems). cete rDNA [13], with primer A3R (Table 2). The resulting
All sequences were read once with one primer (the forward ACT primer set should allow ampli¢cation of a 1.25-kb
primer utilized for the ampli¢cation), for a total length of fragment, which includes all the positions where the genus-
400^500 nt. and family-speci¢c primers are located. While tested on
puri¢ed DNAs from members of a limited number of ac-
2.5. Sequence analysis tinomycete families (Table 1), the ACT primers ampli¢ed
a wide range of actinomycete DNAs from soil (see below).
Sequences were analyzed with the Sequence Match tool However, these primers might not amplify all actinomy-
available through the RDP-II web site, which calculates cete families, since some Corynebacterinae, the family No-
similarity values (Sab score) with all 16S rRNA sequences cardiopsaceae and several Micrococcineae are predicted to
present in the database. All sequences were also inspected be poor targets for the ACT primers.
with the program Chimera Check of the RDP-II web site Serial dilutions of genomic DNA isolated from target
to test for their possible derivation by PCR-mediated re- organisms were used to determine the lowest amount of
combination of two di¡erent sequences. For the construc- target DNA yielding a visible PCR product (see Fig. 1).
tion of phylogenetic trees, sequences were aligned with For all primers, 5 pg DNA or less was su⁄cient to yield a
PILEUP and analyzed with programs included in the distinct PCR product in a gel. No ampli¢cation product
PHYLIP package [17]. Distance matrices were calculated was obtained from members of non-target groups (Table
with DNADIST, using the Maximum Likelihood method 1), with the exception of the STM primers, which yielded a
implemented in the program and the method of Jukes and very faint band from Saccharomonospora viridis, but only
Cantor [18]. Trees were derived from the distance matrices when using 60 ng DNA (Fig. 1a, lane 10). Details of the
using neighbor-joining, UPGMA and the Fitch^Margo- PCR protocols and of the sensitivity of each primer pair
liash least squares method with contemporary tips (all are summarized in Table 3.
methods are included in the PHYLIP package). All anal-
yses were performed on a bootstrapped data set contain- 3.2. Analysis of soil DNA
ing 100 replicates (generated by the program SEQBOOT).
The primer pairs and ampli¢cation conditions devised
2.6. DNA extraction from soil samples with a limited number of puri¢ed DNAs were further
evaluated against DNA extracted from three soil samples
Soil A (from a temperate forest near Siena, Italy) and (A, B and C). Since soil contains a large number of diverse
soil B (from an unspeci¢ed location in Niger) were ran- bacteria, sequence analysis of the cloned PCR products
domly chosen from the Natural Source Collection of Bio- should allow their phylogenetic classi¢cation, thus estab-
search Italia, which contains soils collected since the mid lishing whether they derive from the target groups. Each
1970s. Soil C was collected from a temperate forest in primer set yielded a distinct band using 5^10 ng of soil
Gerenzano, Italy, in August, 1999. After collection, all DNA as template, as exempli¢ed in Fig. 2. For each prim-
soils were air-dried at 30‡C for 2 days and stored in a er set, the ampli¢cation products obtained from soil DNA
sealed container at room temperature (this is a common were cloned and a number of clones (between 18 and 30)
procedure for the isolation of sporogenic actinomycetes). partially sequenced. As some sequence di¡erences could

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Fig. 1. Ampli¢cation of genomic DNA. a: STM Primers. Lanes contain PCR products from DNA of: 1, Streptomyces coelicolor ATCC 10147 (10 ng);
2, Streptomyces glaucescens DSM 40716 (10 ng); 3, Streptomyces cinnamoneus NRRL B-1285 (10 ng); 4^9, Streptomyces lividans ATCC 19844 (10 ng,
1 ng, 100 pg, 10 pg, 2 pg, 1 pg, respectively) ; 10, Saccharomonospora viridis ATCC 15345 (60 ng); 11, Actinomadura madurae DSM 43067 (20 ng);
12, Micromonospora carbonacea ATCC 27114 (5 ng); 13, Streptosporangium cinnabarinum DSM 44094 (20 ng); 14, no DNA; X, molecular mass
marker X (Roche Molecular Biochemicals, Mannheim, Germany). b: DCT Primers. Lanes contain PCR products from DNA of: 1, Dactylosporangium
matsuzakiense ATCC 31570 (100 pg); 2^7, Dactylosporangium sp. ID 83892 (5 ng, 500 pg, 50 pg, 5 pg, 1 pg, 0.5 pg, respectively) ; 8, S. lividans ATCC
19844 (10 ng); 9, Sts. cinnabarinum DSM 44094 (20 ng); 10, Actinoplanes teichomyceticus ATCC 31121 (5 ng); 11, M. carbonacea ATCC 27114 (5 ng);
12, Saccharomonospora viridis ATCC 15345 (60 ng); 13, A. madurae DSM 43067 (20 ng); 14, no DNA ; X, molecular mass marker X. PCR conditions
were as described in Table 3.

derive from nucleotide misincorporation, we have conser-

vatively treated as identical those sequences displaying up
to 1% di¡erence. Phylogenetic analyses yielded trees con-
sistent with each other by di¡erent methods. The complete
list of sequences, their accession numbers and the results
of similarity searches are summarized in Table 4. The re-
sults obtained with each primer set are described below.

3.2.1. Actinomycetales
The 1.25-kb products obtained with the ACT primers
from the three soil DNAs were independently cloned and
30 clones partially sequenced. Database searches indicated
that 22 out of the 30 clones correspond to 20 di¡erent
sequences highly similar to 16S rDNA of cultured mem-
bers of the order Actinomycetales (Table 4). Seven other
clones, corresponding to six di¡erent sequences, also clus- Fig. 2. Ampli¢cation products obtained from soil DNA. Lane 1 con-
ter with actinomycetes by phylogenetic analysis (not tains the ampli¢cation product obtained with the TMM primers from
DNA extracted from soil B. Lanes 2^6 contain the ampli¢cation prod-
shown), and most likely derive from uncultured actinomy-
ucts obtained with primers ACT, STM, MMS, DCT and STP, respec-
cetes, as has also been observed by others [19]. The only tively, using DNA from soil C as template. X refers to molecular mass
sequence diverging from Actinomycetales is that of clone marker X (as Fig. 1); sizes of selected fragments (bp) are reported on
ACT-A-4, which is correlated to that of an uncultured the right. PCR conditions were as described in Table 3.

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Table 4
Soil clones
Sequence IDa Acc. No.d Clonese Best match with RDP-II entries Sab scoref
Primersb Soilc No. Acc. No.d Description
ACT A 1 AJ427635 1 Y08536 Pseudonocardia asaccharolytica 0.879
ACT A 2 AJ427636 1 AF005021 Aeromicrobium erythreum 0.921
ACT A 3 AJ427637 1 AB020031 Saccharotrix tangerinus 0.970
ACT A 4 AJ427638 1 X92708 Clone TM226 0.660
ACT A 5 AJ427639 1 AB006160 ‘Sarraceniospora aurea’ 0.905
ACT A 6 AJ427640 1 AF054278 Mycobacterium str. CH-1 0.976
ACT A 7 AJ427641 1 L40617 Frankia sp. str. HR27-14 0.856
ACT B 1 AJ427642 1 AB028653 Planomonospora parontospora 0.987
ACT B 2 AJ427643 2 D87974 Nocardioides sp. str. KP7 0.944
ACT B 3 AJ427644 1 D87974 Nocardioides sp. str. KP7 0.880
ACT B 4 AJ427645 1 X92612 Micromonospora rhodorangea 0.925
ACT B 5 AJ427646 2 X90830 Nocardioides sp. str. EL-17 0.880
ACT B 6 AJ427647 1 AF005020 Kribbella sandramycini 0.923
ACT B 7 AJ427648 1 Z78210 Nocardioides jensenii 0.842
ACT B 8 AJ427649 1 Z78210 Nocardioides jensenii 0.839

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ACT B 9 AJ427650 1 U27857 Clone Nb 0.936
ACT C 1 AJ427651 1 AF016407 Mycobacterium sp. IMVS B76676 0.963
ACT C 2 AJ427652 1 Y07606 Clone DA016 0.784
ACT C 3 AJ427653 1 AF074412 Kitasatospora sp. str. JL 415 0.967
ACT C 4 AJ427654 1 U72718 Frankia sp. Cea 5.1 0.832
ACT C 5 AJ427655 1 Z73391 Clone Ep_T1.240 0.745
ACT C 6 AJ427656 1 Y07606 Clone DA016 0.825
ACT C 7 AJ427657 1 Z73396 Clone Ep_T1.79 0.867
ACT C 8 AJ427658 1 L40617 Frankia sp. str. HR27-14 0.830
ACT C 9 AJ427659 1 Z73396 Clone Ep_T1.79 0.774
ACT C 10 AJ427660 1 AJ005005 Mycobacterium shimoidei 0.901
ACT C 11 AJ427661 2 Z73385 Clone Ep_T1.12 0.803
STM A 1 AJ427676 1 D63868 Streptomyces bottropensis 0.951
STM A 2 AJ427677 1 Z76683 Streptomyces albido£avus 1.000
STM A 3 AJ427678 1 D63865 Streptomyces acidiscabies 0.964
STM A 4 AJ427679 1 U94490 Streptomyces thermocaboxydus 0.903
STM A 5 AJ427680 1 Z76683 Streptomyces albido£avus 0.942
STM A 6 AJ427681 1 Y15502 Streptomyces griseus 0.990
STM A 7 AJ427682 1 X87320 Streptomycetaceae str. SR70 0.946
STM A 8 AJ427683 2 AJ007425 Streptomyces sp. str. CFBP4508 0.938
STM A 9 AJ427684 1 AB018092 Streptomyces armeniacus 0.886
STM A 10 AJ427685 1 D63868 Streptomyces bottropensis 0.957
STM A 11 AJ427686 1 AF186411 Clone UC32f 0.774
STM C 1 AJ427668 1 AB024442 Streptomyces kasugaensis 0.928
STM C 2 AJ427669 1 X99943 Streptomyces griseocarneus 0.867
STM C 3 AJ427670 1 X81573 Amycolatopsis orientalis subsp. lurida 0.945
STM C 4 AJ427671 1 AB024443 Streptomyces albulus 0.913
STM C 5 AJ427672 3 AF101415 Streptomyces sp. CN 732 0.926
STM C 6 AJ427673 1 AB024440 Streptomyces albulus 0.935
STM C 7 AJ427674 2 AF101415 Streptomyces sp. CN 732 0.893
STM C 8 AJ427675 1 Y15502 Streptomyces griseus 0.990
STP C 1 AJ427662 3 X97892 Nonomuraea salmonea 0.967
STP C 2 AJ427663 1 X97892 Nonomuraea salmonea 0.965
STP C 3 AJ427664 1 AB018046 Microbispora corallina 0.977
STP C 4 AJ427665 3 D85496 Planotetraspora mira 0.959
STP C 5 AJ427666 2 D85496 Planotetraspora mira 0.971
STP C 6 AJ427667 12 X89955 Streptosporangium vulgare 1.000
MMS A 1 AJ427703 1 U58525 Actinoplanes philippinensis 0.942
MMS A 2 AJ427704 2 X93199 Catellatospora ferruginea 0.955
MMS A 3 AJ427705 1 AB024701 Catenuloplanes crispus 0.939
MMS A 4 AJ427706 1 X92631 Micromonospora rosaria 0.978
MMS A 5 AJ427707 1 X93188 Actinoplanes regularis 0.936
MMS A 6 AJ427708 2 U58525 Actinoplanes philippinensis 0.916
MMS A 7 AJ427709 3 U58526 Actinoplanes auranticolor 0.959
MMS A 8 AJ427710 1 X93202 Catenuloplanes caeruleus 0.935
MMS A 9 AJ427711 1 X93187 Actinoplanes philippinensis 0.918

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Table 4 (Continued).
Sequence IDa Acc. No.d Clonese Best match with RDP-II entries Sab scoref
b c d
Primers Soil No. Acc. No. Description
MMS A 10 AJ427712 1 X92631 Micromonospora rosaria 0.913
MMS B 1 AJ427713 1 X92599 Micromonospora carbonacea 0.970
MMS B 2 AJ427714 1 X93201 Catenuloplanes japonicus 0.979
MMS B 3 AJ427715 2 X92612 Micromonospora rhodorangea 0.954
MMS B 4 AJ427716 2 X92631 Micromonospora rosaria 1.000
MMS C 1 AJ427695 2 X92601 Micromonospora halophytica 0.908
MMS C 2 AJ427696 1 D85479 Couchioplanes caeruleus 0.912
MMS C 3 AJ427697 1 X92631 Micromonospora rosaria 0.929
MMS C 4 AJ427698 1 X93188 Actinoplanes regularis 0.895
MMS C 5 AJ427699 1 X92612 Micromonospora rhodorangea 0.916
MMS C 6 AJ427700 1 X92613 Micromonospora olivasterospora 0.890
MMS C 7 AJ427701 1 X92599 Micromonospora carbonacea 0.948
MMS C 8 AJ427702 1 X92612 Micromonospora rhodorangea 0.933
TMM B 1 AJ427687 2 D85473 Actinocorallia herbida 0.946
TMM B 2 AJ427688 1 X97889 Actinomadura madurae 0.941
TMM B 3 AJ427689 1 AB006155 ‘Streptomycoides glauco£avus’ 0.964

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TMM B 4 AJ427690 2 U49003 Actinomadura cremea 0.926
TMM B 5 AJ427691 6 X89953 Streptosporangium viridialbum 0.826
TMM B 6 AJ427692 2 X89953 Streptosporangium viridialbum 0.806
TMM B 7 AJ427693 2 X89953 Streptosporangium viridialbum 0.822
TMM B 8 AJ427694 2 X89953 Streptosporangium viridialbum 0.808
DCT A 1 AJ427719 7 X93191 Dactylosporangium aurantiacum 0.946
DCT A 2 AJ427720 4 X93196 Dactylosporangium vinaceum 0.946
DCT A 3 AJ427721 1 X93191 Dactylosporangium aurantiacum 0.899
DCT C 1 AJ427717 8 X93196 Dactylosporangium vinaceum 0.956
DCT C 2 AJ427718 2 X93193 Dactylosporangium matsuzakiense 0.960
a
Clone ID. Each clone is denoted by primer set, soil and a progressive number.
b
For primers, refer to Table 2.
c
Soil DNA used for ampli¢cation.
d
EMBL accession number of the corresponding sequence.
e
Number of identical sequences obtained from independent clones from same soil DNA.
f
Sab scores obtained with the Sequence Match program.

member of the class Actinobacteria (clone TM226 [20] ; with low Sab scores (0.774; Table 4), suggesting that STM-
Table 4). Phylogenetic analysis (not shown) suggests that A-11 probably derives from some as yet unclassi¢ed acti-
ACT-A-4, like clone TM226, derives from the order Acidi- nobacterium.
microbiales [2]. In summary, 29 of the 30 clones, corre-
sponding to 26 distinct sequences, group with 16S rDNA 3.2.3. Streptosporangiaceae
of Actinomycetales, indicating a speci¢city of ampli¢cation The STP primers yielded a faint 0.5-kb band from the
of about 97%. three soil samples. Analysis of 22 random clones from soil
C gave highest similarity scores with sequences derived
3.2.2. Streptomycetaceae from genera of the family Streptosporangiaceae, and phy-
We sequenced a total of 23 clones obtained from soils A logenetic analysis con¢rmed their a⁄liation to the family
and C with the STM primers. Of these, 22 (representing 17 (not shown). The cloned PCR products correspond to
di¡erent sequences) are highly related to members of the three groups. A group of four clones, comprising two dif-
genus Streptomyces (Sab values between 0.867 and 1.000; ferent sequences, is highly related to Nonomuraea. The
Table 4). Clone STM-C-3 actually shows as best match second group consists of six clones, representing three dis-
Amycolatopsis orientalis subsp. lurida, but this strain may tinct sequences related to Planotetraspora or Microbispora
be a Streptomyces species [21]. Sequence STM-C-7 may (these two genera have similar 16S sequences in the ana-
derive from the recombination of PCR products from lyzed region). The last 12 clones correspond to a single
two Streptomyces strains, as suggested by the Chimera sequence, related to the 16S rDNA of several Streptospo-
Check program. The a⁄liation of all these sequences to rangium species. It is worth mentioning that the sequenced
the family Streptomycetaceae is supported by high boot- region is particularly conserved within the genus Strepto-
strap values (exceeding 98%) with all treeing methods (not sporangium. In fact, the RDP database includes nine
shown). Only clone STM-A-11 appeared to be unrelated Streptosporangium species that show 97% sequence iden-
to Streptomycetaceae. This clone gave the highest match tity in a 1450-nt span. However, these same sequences are
with sequences from uncultured actinomycetes, although identical to STP-C-6 over the considered 450 nt. The se-

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426 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429

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Fig. 3. Phylogenetic analysis of soil-ampli¢ed Micromonosporaceae. Neighbor-joining tree showing the phylogenetic position of environmental clones ob-
tained from three soil samples with MMS primers. Genetic distances were calculated based on 451 unambiguously aligned positions. Numbers at the
nodes are bootstrap values based on 100 resamplings; only values higher than 60 are shown. Scale bar represents 10 inferred substitutions per 100 nu-
cleotides. The tree was rooted using E. coli 16S rDNA as outgroup. Environmental sequences are indicated as in Table 4, e.g. MMS-C-2 refers to clone
2 from soil C, ampli¢ed with the MMS primers.

quenced region is therefore of little help in appreciating Micromonosporaceae in all 100 resampled data sets. With-
the diversity of Streptosporangium strains. in the family, however, bootstrap values are low for most
nodes, so that only a few clones can be unambiguously
3.2.4. Micromonosporaceae attributed to one genus (e.g. clone MMS-B-1 appears to
A total of 29 clones, representing at least 22 di¡erent derive from a member of the genus Micromonospora).
sequences, was analyzed. Database searches con¢rmed From this ¢nding, it appears that the sequenced region
that all clones were strictly related to members of the (about 450 bp) is of low value for genus di¡erentiation
family Micromonosporaceae (Table 4). Fig. 3 shows the within Micromonosporaceae.
phylogenetic relationships among the cloned sequences
and some 16S sequences from known species. Clones 3.2.5. Thermomonosporaceae
from the same soil tend to cluster together, but it was Only soil B gave an ampli¢cation product with the
possible to identify similar sequences in di¡erent soils, TMM primers. At least eight di¡erent sequences were ob-
such as clone MMS-A-3 and clone MMS-B-2, both related served from 18 clones analyzed. Four sequences, cor-
to members of the genus Catenuloplanes. Bootstrap anal- responding to six clones, showed high similarity to 16S
ysis con¢rmed the a⁄liation of all soil clones with known sequences from Actinocorallia, Actinomadura and ‘Strepto-

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 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429 427

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Fig. 4. Phylogenetic analysis of soil-ampli¢ed Thermomonosporaceae. Neighbor-joining tree showing the phylogenetic placement of soil clones ampli¢ed
with the TMM primers. Genetic distances were calculated based on 696 unambiguously aligned positions. The tree was rooted using E. coli as out-
group. Legend is otherwise as for Fig. 3.

mycoides glauco£avus’ (Table 4). The remaining four se- sequences constitute a monophyletic lineage supported by
quences, representing 12 clones, showed Sab values be- a 100% bootstrap value (Fig. 4). For this primer pair too,
tween 0.800 and 0.836 with other Thermomonosporaceae therefore, the ampli¢cation can be considered speci¢c,
(not shown). Phylogenetic trees consistently divided the even if part of the ampli¢ed sequences are divergent
eight di¡erent sequences into two groups: the ¢rst four from known Thermomonosporaceae.
sequences strictly associated with known Thermomono-
sporaceae, while the remaining four constituted a separate 3.2.6. Dactylosporangium
clade. In order to gather more information about this The PCR products from soils A and C were cloned.
group of clones, the sequence of the entire 0.8-kb segment Sequence di¡erences among the 22 clones analyzed were
was used for database searches. Streptosporangium viridial- low, and altogether four di¡erent sequences were identi¢ed
bum was found as the closest relative for all sequences, (Table 4). All clones correspond to Dactylosporangium se-
albeit with low Sab values (between 0.806 and 0.822; Table quences with Sab values between 0.899 and 0.960. The low
4). From a phylogenetic analysis based on 696 unambig- variability found in the sequenced clones was expected, as
uously aligned positions, this group of clones clusters to- the sequenced region is highly conserved in most Dactylo-
gether with members of the family Thermomonosporaceae. sporangium strains. Phylogenetic analyses consistently
Even if they are clearly di¡erent from known genera, these placed the clones together with Dactylosporangium strains,

FEMSEC 1414 7-11-02

428 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429

with bootstrap values of 100% for all methods. Since the con¢rming the worldwide distribution of many actinomy-
sequenced region has a good overlap (over 300 nt) with cete families [26^28]. 1 pg of puri¢ed DNA corresponds to
that obtained with the MMS primers, the sequences about 100 genomes, and a typical PCR reaction employed
obtained with the two primer sets were compared over DNA extracted from approximately 5 mg of soil. Assum-
332 unambiguously aligned positions. Dactylosporangium ing that the e⁄ciency of ampli¢cation remains the same in
clones cluster in a single group within the Micromonospo- the presence of a large excess of non-target DNA, we can
raceae, but none of the 23 clones (14 from soil A and nine estimate that positive soils contained at least 20 genomes
from soil C) obtained with the MMS primers is a⁄liated from Dactylosporangium species per mg. Thus, even a ge-
to this group (Table 4). nus like Dactylosporangium seems to be widely distributed
and relatively abundant in soil.
In conclusion, the primers described here show good
4. Discussion speci¢city and sensitivity. They work well in complex
DNA mixtures extracted directly from soil, and recognize
PCR ampli¢cation of 16S sequences has proven to be a di¡erent members of the target group, including new se-
valuable tool for the detection of bacteria in environmen- quences from uncultured bacteria, giving an accurate in-
tal samples [13,22], and speci¢c PCR primers for some sight into microbial diversity in an environmental sample.
genera of actinomycetes have been described [11,12,21]. These primers can therefore ¢nd a useful application in

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Since the use of partial 16S rDNA sequences in phyloge- ecological studies, even if we are mainly interested in their
netic studies can be misleading [9,23,24], we tried to design use for actinomycete isolation. One possible application
primers allowing the ampli¢cation of as large a portion of might be in the prescreening of soils, followed by the re-
the 16S gene as possible. Validation of the primer sets with trieval of desired strains through the application of selec-
soil DNA was an important con¢rmation of their speci¢c- tive isolation procedures. With additional tools in hand, a
ity, since the primers were screened against a limited num- better understanding of the distribution and role of ¢la-
ber of puri¢ed DNAs. A rich source such as soil might mentous actinomycetes in soil microbial communities can
have contained good templates originating from non-tar- be obtained. In addition, unusual actinomycetes might be
get groups of bacteria. However, this was not the case, at isolated with high frequency, thus exploiting their biosyn-
least for the three soils analyzed. Only for two clones did thetic potential for discovering novel bioactive metabo-
the ampli¢cation products not correspond to sequences lites.
from target groups. However, both clones showed a low
similarity to known 16S rDNA sequences, suggesting ei-
ther their ampli¢cation from the DNA of uncultured Acknowledgements
groups or derivation by the PCR-mediated recombination
of di¡erent 16S RNA gene fragments. We wish to thank Elena Bossi and Emiliana Corti for
Considering only the four family-speci¢c primers, 54 their early contributions to the project, and Elena Maestri
distinct sequences were obtained from 92 clones examined. for helpful advice. We are also grateful to all our col-
While the existence of di¡erent 16S rRNA sequences with- leagues at Biosearch Italia for stimulating discussions.
in the same strain has been reported [25], it is not currently This work was partially supported by a grant from the
known how widespread this phenomenon is within actino- Italian MURST, legge 451. P.M. and C.C. were supported
mycetes. Although this might be the case for some of our by a fellowship from the Italian MURST.
sequences, our data suggest the existence of a large diver-
sity for most of the considered groups of actinomycetes.
Furthermore, even when the soil clones were highly related
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