STRATEGY FOR THE USE OF PRETREATMENTS IN THE ISOLATION
OF NON-STREPTOMYCETE ACTINOMYCETES FROM SOIL
P. F. LONG and H. G. WILDMAN
Natural Products Discovery Department, Glaxo Group Research
Limited, Greenford, Middlesex
UB6 OHE, U.K.
Code Number: AC93008
File Size:
Text: 20K
No associated graphic files.
ABSTRACT. A strategy for the isolation of a diverse range of
non-streptomycete actinomycetes is proposed using chemical
pretreatments before depletion of streptomycete numbers using
bacteriophage. The two pretreatments, 0.05% sodium dodecyl sulphate
(SDS) and O.1M tetrasodium pyrophosphate (Na4P0207), had differing
effects on the total actinomycetes recovered. The physicochemical
characteristics of the soil may have been responsible for this.
Although the number of non-streptomycetes isolated was not
significantly altered by each pretreatment, the use of the
pretreatments significantly changed the diversity of the
non-streptomycete isolates in some soils. The use of the two
chemicals is therefore recommended in addition to air drying soil
at room temperature when isolating non-streptomycete actinomycetes
from soil.
Soils have been the predominant reservoir for the isolation of
actinomycetes, particularly the genus Streptomyces which is
well known as a prolific producer of antibiotics and other
biologically active secondary metabolites (Okami and Hotta, 1988).
However, it has become increasingly apparent that other
soil-inhabiting actinomycetes also share the same ability to
produce metabolites of potential use to the pharmaceutical industry
(Goodfellow and O'Donnell, 1989). Increasing effort therefore has
focused on the isolation of these organisms (Labeda and Shearer,
1990). It has been estimated that close to 95% of all actinomycetes
recovered from soil are Streptomyces species (Lechevalier
and Lechevalier, 1967).
Selective techniques for the recovery of non-streptomycete
genera can still result in the isolation of a substantial number of
streptomycetes (Goodfellow and Williams, 1983). A novel technique
to reduce the numbers of streptomycetes in soil samples has been
the incorporation of Streptomyces-specific bacteriophage prior to
soil dilution plating (Kurtboke et al.,1992). It was
envisaged that a reduction in streptomycete numbers should be
accompanied by an increase in diversity of other non-streptomycete
actinomycetes, since the presence of streptomycetes on isolation
plates can prevent the development and detection of
non-streptomycete taxa (Williams and Vickers, 1988). However,
evidence for this is vague and at best might only be observed when
soil samples have been physically pretreated (Kurtboke et al.,
1992). Here we investigate further the role of two
pretreatments in the recovery of non-streptomycete actinomycetes
from soil.
MATERIALS and METHODS
Soils. Five soil samples were collected from a number
of locations in Sierra Leone: #7276 from a riverbank, #7278 from a
forest, #7279 from farmland, #7284 from a garden, and #7286 from
the roots of a Enterolobium tree. The soils were stored at 4
C prior to use.
Bacteriophage. Sixteen Streptomyces-specific
bacteriophage (Wellington and Williams, 1981) were obtained from
the University of Liverpool and handled according to the methods of
Kurtboke et al. (1992).
Soil Treatments. All soils were air dried at room
temperature to constant weight before two pretreatments were
tested. One gram of soil was suspended in lOOml sterile distilled
water at (10^-2 dilution) by shaking at maximum speed on a Griffin
flask shaker (Griffin and George Ltd., Manchester, UK) at room
temperature for 30min - this acted as the control. One gram of soil
was suspended in lOOml of 6% yeast extract broth supplemented with
0.05% (w/v) sodium dodecyl sulphate (SDS, Sigma) by shaking on a
Griffin shaker at maximum speed for 30min at 37 C (Nonomura and
Hayakawa, 1988). One gram of soil was suspended in 0.1M tetrasodium
pyrophosphate (Na4PO207, Sigma) by shaking at maximum speed on a
Griffin shaker at room temperature for 30min.
Each soil was then diluted in peptone-yeast extract-calcium
(PYCa) broth (Bradley et al., 1961) to 10^-4. The diluent at
10^-4 contained the 16 phage stock suspension of Kurtboke et
al., (1992) at 40% (v/v) of the final soil dilution. The
dilutions were left to stand at room temperature for 2hrs with
intermittent shaking. Aliquots of 0.1ml were then spread inoculated
onto the surface of ammonium chloride glycerol agar (Okazaki et
al., 1987). Cyclohexamide (Sigma) and nystatin (Sigma) were
added to the media at 60 ug/ml each to inhibit fungal growth. The
plates were left to dry in a laminar flow cabinet for 1hr (Vickers
and Williams, 1987) before incubation at 28 C for 8 weeks.
Identification of Isolates. Total viable counts of
actinomycetes were estimated directly from the isolation plates
before morphologically representative colonies were subcultured for
each soil and pretreatment. Isolates were subcultured onto malt
yeast extract agar (MYA, Pridham et al.,1956/57) and grown
to maturity (14-21dd) at 28 C. Streptomyces species were
differentiated from non-streptomycete genera by analysis of the
cell wall isomeric form of diaminopimelic acid (DAP) following the
whole cell method of Hasegawa et al. (1983). Cellulose F
(Merck 5718) thin layer chromatography (TLC) plates were used.
Non-streptomycete actinomycetes were grouped superficially
according to gross morphology between each soil and
pretreatment.
Statistics. Analysis of variance and Chi-square tests
were calculated using the MINITAB statistical package (Minitab
Statistical Software, Pennsylvania State University).
RESULTS
There were significant differences (P<0.01) between the
number of actinomycetes recovered from each soil before the two
pretreatments were used (Table 1). A significant interaction
(P<0.001) between soils and pretreatments indicated an
inconsistent relationship between the effects of the two
pretreatments on the total actinomycetes recovered. The total
actinomycete count was greater than the control with either
pretreatment only in soil #7286.
Over 50% of the actinomycete populations in all but two
soil/pretreatment combinations were non-streptomycetes (i.e.,
contained the meso-isomer of DAP rather than the LL-)
(Table 2). The use of pretreatments had no significant effect on
the proportion of actinomycetes to streptomycetes.
----------------------------------------------------------------
--- Air Dried Soil Pretreated with
Soil Air Dried Soil -------------------------------------
0.05% SDS 0.1M Na4P2O7
----------------------------------------------------------------
---
7276 24.7 +/- 6.5 62.3 +/- 6.6 34.0 +/- 8.9
7278 43.3 +/- 17.0 26.6 +/- 3.8 49.3 +/- 9.2
7279 31.0 +/- 11.0 22.7 +/- 9.2 50.3 +/- 6.6
7284 31.7 +/- 8.6 40.7 +/- 3.2 22.7 +/- 3.0
7286 25.3 +/- 7.0 40.3 +/- 3.0 80.7 +/- 19.2
----------------------------------------------------------------
---
Two Way Analysis of Variance
df SS MS Fs
-------------------------------------------------------------------
Soils 4 1534.8 383.7 4.340 **
Pretreatments 2 1974.2 987.1 11.166 ***
Interaction 8 7770.3 971.4 10.988 ***
Error 30 2650.7 88.4
TOTAL 44 13929.9
---------------------------------------------------------------
---
Table 1. Effect of two different pretreatments on the total
viable counts of actinomycetes recovered from five soils (10^4 x
total counts of actinomycetes). Average number of colony forming
units/g air dried wt soil; standard error, 3 plates per
pretreatment. ** = P<0.01, *** = P<0.001.
----------------------------------------------------------------
---
-------------------------------------------------------------------
Air Dried Soil Pretreated with
Soil Air Dried Soil ---------------------------------
0.05% SDS 0.1M Na4P2O7
---------------------------------------------------------------
--
7276 64.3 84.6 76.5
7278 77.8 60.0 87.5
7279 75.0 87.5 90.0
7284 75.0 88.2 44.4
7286 75.0 50.0 73.3
---------------------------------------------------------------
---
Two Way Analysis of Variance
df SS MS Fc
-------------------------------------------------------------------
Soils 4 574 143 0.538 NS
Pretreatments 2 2 1 0.0036 NS
Error 8 2125 266
TOTAL 14 2701
---------------------------------------------------------------
---
Table 2. Effect of two different pretreatments on the
percentage of non-streptomycete actinomycetes recovered from five
soils.
----------------------------------------------------------------
---
-------------------------------------------------------------------
Morphological Type
Soil Pretreatment ---------------------- Chi-Square Value
A B C D
Air dried 3 3 0 3
7276 0.05% SDS 2 7 1 1 16.382, df= 6;*
0.1M Na4P2O7 0 2 6 5
Air dried 1 3 0 3
7278 0.05% SDS 0 1 1 1 8.269; df = 6;NS
0.1M Na4P2O7 0 3 4 0
Air dried 3 0 2 7
7279 0.05% SDS 6 1 0 0 15.707; df = 6;*
0.1M Na4P2O7 5 3 6 4
Air dried 1 3 0 2
7284 0.05%SDS 6 4 2 3 5.503,df = 6;NS
0.1M Na4P2O7 0 2 0 2
Air dried 4 2 0 3
7286 0.05%SDS 4 3 0 3 10.204, df =6; NS
0.1M Na4P2O7 2 5 6 9
------------------------------------------------------------------
---
Table 3. Effect of two different pretreatments on the
distribution and numbers of different morphological types of
non-streptomycete actinomycetes recovered from five soils (* =
P< 0.05, NS = Not significant;
A = Fragmenting substrate mycelia, no aerial hyphae;
B = Fragmenting substrate mycelia, aerial hyphae present;
C = Stable substrate mycelia, tough colony texture, aerial hyphae
absent;
D = Stable substrate mycelia, tough colony texture, aerial hyphae
present with either dry or wet spore mass).
----------------------------------------------------------------
---
The superficial grouping of the non-streptomycetes based on
morphology allowed an assessment of changes in diversity with
pretreatments. Chi-square tests showed some effect of the
pretreatments on the distribution of the actinomycetes within the
four morphological groups chosen (Table 3). That is, although the
percentage recovery of non-streptomycetes was not significantly
affected by the pretreatments, the distribution between types of
those actinomycetes recovered was. The effects of the pretreatments
on the distribution of non-streptomycetes was not consistent for
all soils however. Significant changes (P < 0.05) in diversity
using pretreatments were observed in two soils (#7276 and #7279)
and non-significant, but nevertheless noticeable, changes were seen
in the other soils also. A comparison of the change in diversity
between the two pretreatments was not possible because the number
of organisms sampled was low.
DISCUSSION
The isolation of a diverse range of actinomycetes to enter
industrial screening programmes has become an increasingly
important part of natural product discovery. However, development
of a technique selective for non-streptomycete taxa to the
detriment of Streptomyces species has been difficult. The
use of Streptomyces-specific bacteriophage has proved an important
and effective technique to reduce streptomycete numbers on
isolation plates (Kurtboke et al., 1992). In our study
bacteriophage pretreatment shifted the actinomycete population in
favour of non-streptomycetes, with greater than 50% of the
actinomycetes recovered belonging to non-streptomycete taxa.
Despite this success, the factors affecting phage/host interactions
and the role of soil and the actinomycete populations themselves in
these interactions is poorly understood. The technique thus remains
empirical with varying rates of recovery of non-streptomycete
genera from each soil.
Tetrasodium pyrophosphate has previously been used to remove
fungi and inorganic matter during nucleic acid extraction from soil
(Hahn et al., 1990). While investigating Na4PO207 as a
possible alternative to costly anti-fungal agents, it was initially
observed that the recovery of actinomycetes was greater on
isolation plates for soils pretreated with Na4PO207. Sodium dodecyl
sulphate in combination with yeast extract has previously been
described as an actinomycete spore activator (Nonomura and
Hayakawa, 1988). However, there is no evidence from this study to
suggest this, or that Na4PO207 acts in a similar manner.
Examination of shake flasks containing soils with pretreatments
showed the soils to be better dispersed in the pretreated flasks
compared to the water controls. It has been suggested that the soil
microflora are in close association with soil particles, hence any
technique that dissociates the microflora from the soil could
result in a greater recovery on isolation plates (Hopkins et
al., 1991). The action of Na4PO207 and SDS as dispersion agents
may explain the increase in total viable counts observed in some
instances in this study. The role of the physico-chemical
characteristics of the soil also may have exerted an effect since a
significant interaction between soil and the pretreatment applied
was observed. Investigating soil effects would require detailed
physical and chemical analyses of larger numbers of soils and
improved techniques to group or characterise greater numbers of
isolates. The mechanism of the observed Na4PO207- and SDS - induced
changes in diversity of the non-streptomycete populations is as yet
unknown.
A suitable strategy for the use of the pretreatments in an
isolation campaign would be to use both the pretreatments
independently in conjunction with an air dried control. In this
way, particularly if bacteriophage were used to control the
streptomycete population, an increased number of diverse
non-streptomycete actinomycetes could be obtained.
ACKNOWLEDGEMENTS. The authors would like to thank Miss J.S.Whybrew
for typing the manuscript. The soils used in this study were
imported into the UK under licences issued by the Ministry of
Agriculture, Fisheries and Food.
REFERENCES
Bradley, S.G., D.L Anderson & L.A.Jones (1961). Phylogeny of
actinomycetes as revealed by susceptibility to actinophage.
Dev.Ind.Microbiol., a 223-237
Goodfellow, M. & A.G.O'Donnell ( 1989). Search and discovery
of industrially significant actinomycetes In: S.Baumberg, I.S.
Hunter & P.M.Rhodes (eds.) Microbial Products: New Approaches.
Society for General Microbiology Symposium No. 44. Cambridge
University Press, Cambridge, pp. 343-383
Goodfellow, M. & S.T.Williams (1983). Ecology of
actinomycetes. Ann.Rev.Microbiol., 37: 189-216
Hahn, D., R.Kester, M.J.C.Starrenburg & A.D.L.Akkermans
(1990). Extraction of ribosomal RNA from soil for detection of
Frankia with oligonucleotide probes. Arch.Microbiol.,
154: 329-335
Hasegawa, T., M.Takizawa & S.Tanikda (1983). A rapid
analysis for chemical grouping aerobic actinomycetes.
J.Gen.Appl.Microbiol., 29: 319-322
Hopkins, D.W., S.J.MacNaughton & A.G. O'Donnell (1991). A
dispersion and differential centrifugation technique for
representatively sampling microorganisms from soil. Soil Biol.
Biochem., 23: 217-225
Kurtboke, D.I., C.F.Chen & S.T.Williams (1992). Use
of polyvalent phage for reduction of streptomycetes on soil
dilution plates. J.Appl. Bacteriol., 72: 103-111
Labeda, D.P. & M.Shearer (1990). Isolation of actinomycetes
for biotechnological applications. In: D.P.Labeda (ed.)
Isolation of Biotechnology Organisms from Nature. McGraw
Hill Inc., New York, pp. 1-19
Lechevalier, H.A. & M.P.Lechevalier (1967). Biology
of actinomycetes. Ann.Rev.Microbiol. 21: 71-100
Nonomura, H. & M.Hayakawa (1988). New methods for the
selective isolation of soil actinomycetes. In: Y.Okami, T.Beppu &
H.Ogawara (eds.) Biology of Actinomycetes '88. Japan
Scientific Societies Press, Tokyo, pp. 288-293
Okami, Y. & K.Hotta (1988). Search and discovery of
new antibiotics. In: M.Goodfellow S.T.Williams & M.Mordarski (eds.)
Actinomycetes in Biotechnology. Academic Press,
London, pp. 33-67
Okazaki, T., R.Enokita, H.Miyaoka, T.Takatsu & A.Torikata
(1987). Chloropolysporins A, B and C, novel glycopeptide
antibiotics from Faenia interjecton sp. nov. 1. Taxonomy of
producing organism. J.Antib., 40: 917-923.
Pridham, T.G., P.Anderson, C.Foley, H.A. Lindenfelser,
C.W.Hesseltine & R.G.Benedict (1956/57). A selection of
media for maintenance and taxonomic study of Streptomyces.
Antibiotics Annual 1956-1957, pp. 947-953
Vickers, J.C. & S.T.Williams (1987). An assessment of
plate inoculation procedures for the enumeration and isolation of
streptomycetes. Microbios Lett., 36: 113-117.
Wellington, E.M.H. & S.T.Williams (1981). Host ranges
of phage isolated to Streptomyces and other genera.
Z.Bakteriol.Mikrobiol.Hyg., Suppl., 11: 93-98
Williams, S.T. & J.C.Vickers (1988). Detection of
actinomycetes in the natural environment. In: Y.Okami, T.Beppu &
H.Ogawara (eds.) Biology of Actinomycetes ,88. Japan
Scientific Societies Press, Tokyo, pp. 265-270
Copyright 1993 C.E.T.A.
