Diversity of cultivable actinobacteria in geographically widespread marine sediments.
ABSTRACT Reports describing actinobacteria isolated from marine environments have been dominated by Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown that marine environments contain a high diversity of actinobacterial species that are rarely, if at all, recovered by cultivation-based methods. In this study, it is shown that cultivation-independent methods can be used to guide the application of selective isolation methods. The detection of marine-derived actinobacterial species that have previously only been reported from terrestrial habitats is highlighted. This study provides good evidence that the previously described low diversity of actinobacterial species isolated from marine environments does not reflect an actual low species diversity, and that the use of informed selective isolation procedures can aid in the isolation of members of novel taxa.
- SourceAvailable from: Alan Ward[show abstract] [hide abstract]
ABSTRACT: Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.Applied and Environmental Microbiology 10/2003; 69(10):6189-200. · 3.68 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A large number of mycolate actinomycetes have been recovered from deep-sea sediments in the NW Pacific Ocean using selective isolation methods. The isolates were putatively assigned to the genus Rhodococcus on the basis of colony characteristics and mycolic acid profiles. The diversity among these isolates and their relationship to type strains of Rhodococcus and other mycolate taxa were assessed by Curie point pyrolysis mass spectrometry (PyMS). Three major (A, C, D) and two minor (B, E) groups were defined by PyMS. Cluster A was a large group of isolates recovered from sediment in the Izu Bonin Trench (2679 m); Cluster C comprised isolates from both the Izu Bonin Trench (6390 and 6499 m) and from the Japan Trench (4418, 6048 and 6455 m). These Cluster C isolates showed close similarity to Dietzia maris and this was subsequently confirmed using molecular methods. Cluster D contained isolates recovered from a sediment taken from a depth of 1168m in Sagami Bay and were identified as members of the terrestrial species Rhodococcus luteus. Clusters B and E had close affinities with members of the genera Gordonia and Mycobacterium. The presence of Thermoactinomyces in certain of the deep-sea sediments studied was indicative of the movement of terrestrial material into the ocean depths.16S ribosomal RNA gene sequence analyses produced excellent definition of most genera of the mycolata, and indicated that the among the deep sea isolates (1) were novel species of Corynebacterium, Gordonia and Mycobacterium, and (2) a Sea of Japan isolate the phylogenetic depth of which suggests the possibility of a new genus. Polyphasic taxonomic analysis revealed considerable diversity among the deep sea rhodococci and evidence for recently diverged species or DNA groups.Antonie van Leeuwenhoek 09/1998; 74(1):27-40. · 2.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The search for extraterrestrial life may be facilitated if ecosystems can be found on Earth that exist under conditions analogous to those present on other planets or moons. It has been proposed, on the basis of geochemical and thermodynamic considerations, that geologically derived hydrogen might support subsurface microbial communities on Mars and Europa in which methanogens form the base of the ecosystem. Here we describe a unique subsurface microbial community in which hydrogen-consuming, methane-producing Archaea far outnumber the Bacteria. More than 90% of the 16S ribosomal DNA sequences recovered from hydrothermal waters circulating through deeply buried igneous rocks in Idaho are related to hydrogen-using methanogenic microorganisms. Geochemical characterization indicates that geothermal hydrogen, not organic carbon, is the primary energy source for this methanogen-dominated microbial community. These results demonstrate that hydrogen-based methanogenic communities do occur in Earth's subsurface, providing an analogue for possible subsurface microbial ecosystems on other planets.Nature 02/2002; 415(6869):312-5. · 38.60 Impact Factor
Diversity of cultivable actinobacteria in geographically widespread marine
Luis A. Maldonado1,2,*, James E.M. Stach3, Wasu Pathom-aree1, Alan C. Ward1,
Alan T. Bull3and Michael Goodfellow1
1School of Biology, University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK;2Centro de Investigacio´n y
Asistencia en Tecnologı´a y Disen˜o del Estado de Jalisco (CIATEJ), CP 44270, Guadalajara, Jalisco, Me´xico;
3Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; *Author for corre-
spondence (e-mail: firstname.lastname@example.org; phone: +52-33-33455200; fax: +52-33-33455200)
Received 6 August 2004; accepted in revised form 14 October 2004
Key words: 16S rRNA gene, Actinobacteria, Marine environment, PCR, Selective isolation, Sequencing
Reports describing actinobacteria isolated from marine environments have been dominated by
Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown
that marine environments contain a high diversity of actinobacterial species that are rarely, if at all,
recovered by cultivation-based methods. In this study, it is shown that cultivation-independent methods
can be used to guide the application of selective isolation methods. The detection of marine-derived
actinobacterial species that have previously only been reported from terrestrial habitats is highlighted. This
study provides good evidence that the previously described low diversity of actinobacterial species isolated
from marine environments does not reflect an actual low species diversity, and that the use of informed
selective isolation procedures can aid in the isolation of members of novel taxa.
Cultivable prokaryotes have been found in ex-
treme environments previously considered hostile
to life (Kristijanson et al. 2000), in association with
hydrothermal vents (Teske et al. 2002), below ba-
salt aquifers (Stevens and McKiney 1995; Chapelle
et al. 2002), in the deep subsurface of oceans
(Krumholz 2000; Parkes and Wellsbury 2004),
inside Antarctic rocks (Banerjee et al. 2000; Hirsch
et al. 2004), and in symbiotic associations with
invertebrates (e.g. Breznak 2004; Hill 2004).
Whitman et al. 1998) based on 16S rRNA gene
sequence data which suggest that more than 99%
of microorganisms in natural habitats are uncul-
tured or unculturable. Clearly this unseen majority
encompasses an enormous genetic diversity for
exploitable biology (Bull et al. 2000; Bull 2004).
actinomycetes in marine habitats even though
these organisms have been studied in more detail
than members of other groups of prokaryotes due
to their biotechnological importance (Goodfellow
et al. 1988; McVeigh et al. 1994; Bull et al. 2000).
Indeed, the marine environment is a virtually
untapped source of novel actinomycete diversity
(Stach et al. 2003a, b) and thereby of new metab-
olites (Fiedler et al. 2005). Bacteria belonging to
the class Actinobacteria (Stackebrandt et al. 1997)
have been recovered frequently from marine hab-
itats but have been assigned mainly to only three
taxa, namely the genera Micromonospora, Rhodo-
coccus and Streptomyces (Weyland 1969, 1981;
Antonie van Leeuwenhoek (2005) 87:11–18
? Springer 2005
Goodfellow and Haynes 1984; Pisano et al. 1989;
Colquhoun et al. 1998).
Representative sampling of members of pro-
karyotic species from natural habitats is not real-
istic at present due to the large numbers and
physiological) involved. Consequently, there is an
urgent need for the development and application
of new strategies for the detection, isolation,
dereplication and subsequent description of novel
organisms, including actinomycetes, from natural
habitats (Zengler et al. 2002).
The strategy adopted in the present investiga-
tion involved the estimation of actinobacterial
diversity based on DNA extracted from marine
sediments and the application of this information
to guide the selective isolation of cultivable
actinomycetes from the same environmental sam-
ples using pre-treatment and selective isolation
procedures. The primary aim of the study was to
address the hypothesis that the low numbers of
genera reported in marine sediments is unlikely to
be due to actual low diversity in these environ-
ments but is due to under sampling and the use of
inappropriate isolation procedures.
Material and methods
Environmental samples were collected from the
Japan Trench (NW Pacific Ocean), the Canary
Basin (Atlantic Ocean), and selected fjords in
Norway, as previously described (Colquhoun et al.
1998; Stach et al. 2003a, b).
DNA extraction from marine sediments
DNA was extracted from the sediment samples as
previously described (Stach et al. 2003a, b).
PCR amplification of actinobacterial 16S rDNA
A nested PCR was carried out in total volumes of
50 ll with slight modifications to the protocol
described in Stach et al. (2003a, b). The first PCR
involved the use of universal primers 27f and 1525r
(Lane 1991), the second PCR used either a 1:50 or
1:100 dilution of the first PCR product and several
sets of primers, as shown in Table 1. The anneal-
ing temperatures used to carry out the second
(‘‘nested’’) PCR are also shown in the table.
Sediment samples (1 g fresh weight) were asepti-
cally added to 9 ml sterile 0.25 · 1/4 strength
Ringer’s solution (Oxoid) and shaken on a reci-
procal flask shaker then diluted to 10?3. Sediment
samples (1–2 g fresh weight) were also treated
using the dispersion and differential centrifugation
technique (DDC; Hopkins et al. 1991). The three
supernatant fractions and the residue fraction de-
rived by the application of the procedure to each
of the sampleswere
(12,000 · g at 4 ?C), and the resultant pellets sus-
pended in 1 ml sterile water. In addition a pooled
sample was prepared from the four fractions.
Aliquots of the various preparations (75 ll) were
spread over the surfaces of glucose–yeast extract
agar supplemented with rifampicin and strepto-
mycin (GYRS; Athalye et al. 1981), humic acid
vitamins agar (HA; Hayakawa and Nonomura
1987), M3 agar (Rowbotham and Cross 1977),
raffinose–histidine agar (RH; Vickers et al. 1984),
SM3 agar (Tan 2002) and starch–casein–nitrate
agar (SCN; Ku ¨ ster and Williams 1964). The agar
media were dried for 15 min prior to inoculation,
as recommended by Vickers et al. (1984). All of the
(50 lg ml?1) to suppress fungal growth. The sets
of isolation plates, three per dilution, were incu-
bated at 28 ?C for 3 weeks.
Maintenance, culture conditions and morphological
Actinomycete colonies were taken from the M3,
RH and SC selective isolation plates using sterile
toothpicks and inoculated onto freshly prepared
agar media and the inoculated plates incubated for
2 to 3 weeks at 28 ?C. Isolates were maintained on
the plates for short-term storage and as suspen-
sions in 20% (v/v) glycerol at ?20 ?C for
long-term maintenance. Representatives of the
different colony types wereinoculatedonto
Table 1. Specific primers used for identification of members of the class Actinobacteria
Pisano et al. (1989)
Monciardini et al. (2002)
Hayakawa and Nonomura 1987
Athalye et al. (1981)
N/A, Not applicable.
aThis set of primers are universal and not specific for the order Actinomycetales.
bComplementary sequences are shown for the reverse primer.
modified Bennett’s (Jones 1949), glucose–yeast
extract agar (GYEA; Gordon and Mihm 1962) or
oatmeal (ISP 3; Ku ¨ ster 1959) agar plates. Purified
isolates were then assigned to artificial groups
based on aerial spore mass colour, reverse pigment
colours and the colour of any diffusible pigments.
Representatives of the resulting colour groups
were chosen for DNA extraction and 16S rRNA
gene sequencing as described below.
DNA was extracted from the representative iso-
lates by using DNAceTMSpin Plant kits (Bioline
Ltd; London, UK), following the manufacturer’s
instructions, but using higher concentrations of
lysozyme and proteinase-K (50 mg ml?1).
PCR amplification and determination of 16S rRNA
The primers S-C-Act-0235-a-S-20 and S-C-Act-
0878-a-A-19 were used to check whether or not the
isolates belonged to the class Actinobacteria using
the procedure described previously (Stach et al.
2003b). Similarly, the primer sets shown in Table 1
were used to assign representative isolates to spe-
cific genera and families. Representatives of the
resultant taxa were chosen randomly and their 16S
rRNA gene sequences obtained using oligonucle-
otides and PCR conditions described previously
(Chun and Goodfellow 1995). In some cases,
only partial 16S rRNA gene sequences (approxi-
mately 600 base pairs) were obtained by using
S-C-Act-0235-a-S-20 as the sequencing primer.
Putative species identification
Isolates were putatively classified to the genus level
based on 16S rDNA similarity data. 16S rRNA
gene nucleotide sequences of the isolates were
compared using the BLAST option on the
GenBank public database. The sequences were
aligned using CLUSTAL X software (Thompson
et al. 1997) together with their corresponding
closest neighbours highlighted from the BLAST
search. Evolutionary distance matrices for the
neighbor-joining method (Saitou and Nei 1987)
were generated after Jukes and Cantor (1969). The
resultant tree topologies were evaluated by boot-
strap analyses (Felsenstein 1985) of neighbor-
joining data sets based on 1000 resamplings using
the SEQBOOT and CONSENSE programs from
the PHYLIP suite of programs (Felsenstein 1993).
Results and discussion
It was evident from the community DNA analyses
that the marine sediments contained a diverse
range of mycelial forming actinomycetes belonging
to the genera Amycolatopsis and Pseudonocardia
Thermomonosporaceae (Figure 1). These results
not only influenced the choice of selective isolation
procedures used in the cultivation studies but
provide further evidence that marine sediments are
a rich source of taxonomically diverse actinomy-
cetes (Mincer et al. 2002; Stach et al. 2003a, b).
Over 800 actinomycetes were isolated from the
GYRS, HA, M3, RH. SM3 and SCN isolation
plates following the detection of specific 16S
rRNA gene signatures in the marine sediments.
It is clear from Figure 2a that much larger
numbers of mycelial actinomycetes were isolated
onthe M3,RH and
inoculated with suspensions of sediment samples
prepared from Canary Basin, Japan Trench and
Norwegian samples using DDC procedure com-
pared with corresponding numbers derived from
reciprocal shaking technique. These results are in
line with those from previous studies which
showed that the DDC procedure was more effec-
tive in extracting actinomycete propagules from
soil samples than classical shaking techniques
(Atalan et al. 2000; Sembiring et al. 2000). It is
apparent from Figure 2b that the highest actino-
mycete counts were derived from the supernatant
A fraction and the lowest from the supernatant D
suspensions, similar results were recorded for rhi-
zosphere and rhizoplane samples by Sembiring
et al. (2000). The available data suggest that asso-
ciations between taxonomically different mycelial
actinomycetes and particulate material in natural
habitats may be the major factor in limiting rep-
resentative sampling of sporoactinomycets from
environmental substrates. The multistage DDC
procedure, which entails the use of mild detergent
(sodium cholate), buffering (Tris buffer), attenu-
ated physical disruption (mild ultrasonication) and
ionic shock (distilled water), may be effective in
breaking down such associations.
The strains cultured from the selective media
were assigned to colour groups based either on the
morphology of the colonies or on their ability to
glucose–yeast extract, and oatmeal agar plates.
Representatives of randomly chosen isolates sent
for partial or complete 16S rRNA gene sequencing
provided evidence that the marine isolates can be
assigned to several different taxa in the class
randomly chosen isolates suggest a higher degree
of cultivable actinomycete diversity in marine
sediments than reported hitherto. In particular,
myces’’ grouping as the dominant and ubiquitous
Figure 1. (a) Nucleic acid extracts from marine sediments. Lanes m: DNA marker, 1: Negative control (no sediment), 2: Fana Fjord, 3:
Canary Basin, 4: Japan Trench. (b) Family-specific PCR for the Streptosporangiaceae and Thermomonosporaceae. Lanes 1 and 2:
Negative controls (no template) 3: By Fjord/Hjelte Fjord, 4: Fana Fjord, 5: Oster Fjord/Sør Fjord, 6: Canary Basin, 7: Japan Trench,
8: Sagami Bay, m: DNA Marker. (c) Genus-specific PCR for Amycolatopsis and Pseudonocardia. Lanes 1–8 as above, Lane 9:
Pseudonocardia species positive control.
Isolates belonging to genera previously only
isolated from terrestrial habitats were clearly
evident in the present survey. It was particularly
Streptosporangiaceae (i.e. the genus Streptospo-
genus Actinomadura) were isolated from the
Japan Trench where the PCR primers yielded a
positive band. However, Amycolatopsis isolates
were not recovered from any of the marine
samples that yielded a positive band despite the
use of a proven selective isolation procedure
(Tan 2002). These findings suggest that it is not
a lack of species diversity in marine sediments
that limits the ability to isolate members of no-
vel species, but rather a need to improve selec-
tive procedures for the isolation of representative
The actinomycetes recovered in this study were
assigned to the following genera on the basis of
either partial or complete 16S rRNA gene
Figure 2. (a) Total number of actinomycetes recovered from Fana Fjord sediment. The graph shows the number of actinomycetes
isolated using the DDC procedure vs. the reciprocal shaking technique. (b) The graph indicates the distribution of actinomycetes
recovered in the different fractions from the DDC procedure.
Williamsia. This is the first study in which such an
extreme degree of actinomycete diversity has been
reported actinobacteria isolated from the marine
environment. Previous reports have mainly high-
lighted the presence of members of the mycolic acid
subclade (Colquhoun et al. 1998). It is also evident
from the 16S phylogenetic trees that many of the
isolates in this study represent novel species and,
hence, prospective novel sources of bioactive
compounds. The taxonomic descriptions of mem-
bers of these novel taxa will be published elsewhere.
In contrast to some previous studies (Mincer et
al. 2002) none of the isolates recovered in this
study required salt for growth. Nevertheless, the
significance of finding such an extensive actino-
mycete diversity in marine samples lies in the
intrinsic economic importance that this particular
group of bacteria have from a biotechnological
perspective (Bull et al. 2000). It is clear, therefore,
that the present study reinforces the view that the
deep-seas constitute an unexplored (and still
unexploited) environment for the bioprospecting
of actinomycetes and that cultivation-independent
techniques can be used to inform cultivation
strategies to improve the recovery of novel ac-
tinobacteria from marine environments.
The authors gratefully acknowledge the support of
the UK Natural Environmental Research Council
(grants NER/T/S/2000/00614 and NER/T/S/2000/
00616). We thank Professor Gjert Knutsen, Uni-
versity of Bergen, and the crew of the F/F Hans
Brattstro ¨ m for the collection of fjord sediments;
Professor Koki Horikoshi, Japan Marine Science
& Technology Center, for providing deep-sea se-
diments from Pacific Ocean sites; and the crew of
the RRS Charles Darwin, for providing Canary
Basin samples. Wasu Pathom-aree is grateful to
the Royal Thai Government DPST programme
for finanicial support.
Atalan E., Manfio G.P., Ward A.C., Kroppenstedt R.M. and
Goodfellow M. 2000. Biosystematic studies on novel strep-
tomycetes from soil. Antonie van Leeuwenhoek 77: 337–353.
Athalye M., Lacey J. and Goodfellow M. 1981. Selective iso-
lation and enumeration of actinomycetes using rifampicin. J.
Appl. Microbiol. 51: 289–297.
Banerjee M., Whitton B.A. and Wynn-Williams D.D. 2000.
Phosphatase activities of endolithic communities in rocks of
the Antarctic Dry Valleys. Microbial. Ecol. 39: 80–91.
Breznak J.A. 2004. Invertebrates-insects. In: Bull A.T. (ed.),
Microbial Diversity and Bioprospecting. ASM Press, Wash-
ington, DC, pp. 191–203.
Bull A.T. (ed.) 2004. Microbial Diversity and Bioprospecting.
ASM Press, Washington, DC, pp. 1–496.
Bull A.T., Ward A.C. and Goodfellow M. 2000. Search and
discovery strategies for biotechnology: the paradigm shift.
Microbiol. Mol. Biol. Rev. 64: 573–606.
Chapelle F.H., O’Neill K., Bradley P.M., Methe B.A., Ciufo
S.A., Knobel L.L. and Lovley D.R. 2002. A hydrogen-based
subsurface microbial community dominated by methano-
gens. Nature 415: 312–315.
Chun J. and Goodfellow M. 1995. A phylogenetic analysis of
the genus Nocardia with 16S rRNA gene sequences. Int. J.
Syst. Bacteriol. 45: 240–245.
Colquhoun J.A., Mexson J., Goodfellow M., Ward A.C.,
Horikoshi K. and Bull A.T. 1998. Novel rhodococci and
other mycolate actinomycetes from the deep sea. Antonie van
Leeuwenhoek 74: 27–40.
Felsenstein J. 1985. Confidence limits on phylogenies: an ap-
proach using the bootstrap. Evolution 39: 783–791.
Felsenstein J. 1993. PHYLIP (phylogenetic inference package).
version 3.5c. Department of Genetics, University of Wash-
ington, Seattle, USA.
Fiedler H.-P., Bruntner C., Bull A.T., Ward A.C., Goodfellow
M. and Mihm G. 2004. Marine actinomycetes as a source of
novel secondary metabolites. Antonie van Leeuwenhoek 87:
37–42 (This issue).
Goodfellow M. and Haynes J.A. 1984. Actinomycetes in mar-
ine sediments. In: Ortiz-Ortiz L., Bojalil L.F. and Yakoleff V.
(eds), Biological, Biochemical and Biomedical Aspects of
Actinomycetes. Academic Press, Orlando, pp. 453–472.
Goodfellow M., Williams S.T. and Mordarski M. (eds) 1988.
Actinomycetes in Biotechnology. Academic Press Ltd, Lon-
don, pp. 1–501.
Gordon R.E. and Mihm J.M. 1962. Identification of Nocardia
caviae (Erikson) nov. comb. Ann. N.Y. Acad. Sci. 98: 628–
Hayakawa M. and Nonomura H. 1987. Humic-acid vitamins
agar, a new medium for the selective isolation of soil ac-
tinomycetes. J. Ferm. Tech. 65: 501–509.
Hill R.T. 2004. Microbes from marine sponges: a treasure trove
of biodiversity for natural products. In: Bull AT. (ed.),
Microbial Diversity and Bioprospecting. ASM Press, Wash-
ington, DC, pp. 191–203.
Hirsch P., Mevs U., Kroppenstedt R.M., Schumann P. and
Stackebrandt E. 2004. Cryptoendolithic actinomycetes from
antarctic sandstone rock samples: Micromonospora endolith-
ica sp. nov. and two isolates related to Micromonospora co-
erulea Jensen 1932. Syst. Appl. Microbiol. 27: 166–174.
Hopkins D.W., MacNaughton S.J. and O’Donnell A.G. 1991.
A dispersion and differential centrifugation technique for
representatively sampling microorganisms from soil. Soil
Biol. Biochem. 23: 217–225.
Jones K.L. 1949. Fresh isolates of actinomycetes in which the
presence of sporogenous aerial mycelia is a fluctuating
characteristic. J. Bacteriol. 57: 141–145.
Jukes T.H. and Cantor C.R. 1969. Evolution of protein mole-
cules. In: Munro H.N. (ed.), Mammalian Protein Metabo-
lism, Vol. 3. Academic Press, New York, pp. 21–132.
Kristijanson J.K., Hreggvidson G.O. and Grant W.D. 2000.
Taxonomy of extremophiles. In: Priest F.G. and Goodfellow
M. (eds), Applied Microbial Systematics. Kluwer Academic
Publishers, Dordrecht, pp. 231–291.
Krumholz L.R. 2000. Microbial communities in the deep sea
subsurface. Hydrogeol. J. 8: 4–10.
Ku ¨ ster E. 1959. Outline of a comparative study of criteria used
in characterization of the actinomycetes. Int. Bull. Bact.
Nom. Tax. 9: 97–104.
Ku ¨ ster E. and Williams S.T. 1964. Selective media for isolation
of streptomycetes. Nature 202: 928–929.
Lane D.J. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E.
and Goodfellow M. (eds), Nucleic Acid Techniques in Bac-
terial Systematics. John Wiley and Sons, Chichester, United
Kingdom, pp. 115–175.
McVeigh H.P., Divers M., Warwick S., Munro J. and Embley
T.M. 1994. Exploration of actinomycete diversity using
ribosomal RNA sequences. Biotechnologia 7–8: 253–260.
Mincer T.J., Jensen P.R., Kauffman C.A. and Fenical W. 2002.
Widespread and persistent populations of a major new
marine actinomycete taxon in ocean sediments. Appl. Env.
Microbiol. 68: 5005–5011.
Monciardini P., Sosio M., Cavaletti L., Chiocchini C. and
Donadio S. 2002. New PCR primers for the selective ampli-
fication of 16S rDNA from different groups of actinomycetes.
FEMS Microb. Ecol. 42: 419–429.
Moro ´ n R., Gonzalez I. and Genilloud O. 1999. New genus-
specific primers for the PCR identification of members of the
genera Pseudonocardia and Saccharopolyspora. Int. J. Syst.
Bacteriol. 49: 149–162.
Parkes R.J. and Wellsbury P. 2004. Deep biospheres. In: Bull
A.T. (ed.), Microbial Diversity and Bioprospecting. ASM
Press, Washington, DC, pp. 120–129.
Pisano M.A., Sommer M.J. and Brancaccio L. 1989. Isolation
of bioactive actinomycetes from marine sediments using rif-
ampicin. Appl. Microbiol. Biotechnol. 31: 609–612.
Rowbotham T.J. and Cross T. 1977. Ecology of Rhodococcus
coprophilus and associated actinomycetes in freshwater and
agricultural habitats. J. Gen. Microbiol. 100: 231–240.
Saitou N. and Nei M. 1987. The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol. Biol.
Evol. 4: 406–425.
Sembiring L., Ward A.C. and Goodfellow M. 2000. Selective
isolation and characterisation of members of the Streptomy-
ces violaceusniger clade associated with the roots of Parase-
rianthes falcataria. Antonie van Leeuwenhoek 78: 353–366.
Stach J.E., Maldonado L.A., Masson D.G., Ward A.C.,
Goodfellow M. and Bull A.T. 2003a. Statistical approaches
to estimating bacterial diversity in marine sediments. Appl.
Env. Microbiol. 69: 6189–6200.
Stach J.E., Maldonado L.A., Ward A.C., Goodfellow M. and
Bull A.T. 2003b. New primers for the class Actinobacteria:
application to marine and terrestrial environments. Env.
Microbiol. 5: 828–841.
Stackebrandt E., Rainey F.A. and Ward-Rainey N.L. 1997.
Proposal for a new hierarchic classification system, Actino-
bacteria classis nov. Int. J. Syst. Bacteriol. 47: 479–491.
Stevens T.O. and McKinley J.P. 1995. Lithotrophic microbial
ecosystems in deep basalt aquifers. Science 270: 450–454.
Tan G.Y.A. 2002. PhD thesis. Amycolatopsis Systematics: A
New Beginning. University of Newcastle upon Tyne, New-
castle upon Tyne, UK.
Teske A., Hinrichs K.U., Edgcomb V., Gomez A.D., Kysela
D., Sylva S.P., Sogin M.L. and Jannasch H.W. 2002.
Microbial diversity of hydrothermal sediments in the Guay-
mas Basin: evidence for anaerobic methanotrophic commu-
nities. Appl. Env. Microbiol. 68: 1994–2007.
Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F. and
Higgins D.G. 1997. The CLUSTAL X windows interface:
flexible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Res. 25: 4876–4882.
Vickers J.C., Williams S.T. and Ross G.W. 1984. A taxonomic
approach to selective isolation of streptomycetes from soil.
In: Ortiz-Ortiz L., Bojalil L.F. and Yakoleff V. (eds), Bio-
logical, Biochemical and Biomedical Aspects of Actinomy-
cetes. Academic Press, London, pp. 553–551.
Weyland H. 1969. Actinomycetes in North Sea and Atlantic
Ocean sediment. Nature 223: 858.
Weyland H. 1981. Distribution of actinomycetes on the sea
floor. Zentral Bakteriol. supp. 11: 185–193.
Whitman W.B., Coleman D.C. and Wiebe W.J. 1998. Prok-
aryotes: the unseen majority. Proc. Nat. Acad. Sci. USA 95:
Zengler K., Toledo G., Rappe M., Elkins J., Mathur E.J., Short
J.M. and Keller M. 2002. Cultivating the uncultured. Proc.
Nat. Acad. Sci. USA 99: 15681–15686.