TAXONOMIC IMPLICATIONS OF THE REACTIONS OF
REPRESENTATIVE BACILLUS STRAINS TO THERMOACTINOMYCES-PHAGE
D.I.KURTBOKE and K.SIVASITHAMPARAM
The University of Western Australia, School of Agriculture, Soil
Science and Plant Nutrition, Nedlands, W.A. 6009, Australia
Code Number: AC93001
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ABSTRACT. The sensitivity of representative Bacillus
strains to phage isolated to the members of the genus
Thermoactinomyces and Thermoactinomyces spp. to
Bacillus phage was studied. The phage sensitivity of the
species belonging to the genera Bacillus and
Thermoactinomycetes corresponded closely to those indicated
by chemical and 16S rRNA comparative cataloguing studies and showed
a natural relationship between the two genera. The value of phage
susceptibility as an aid to reliable actinomycete identification
schemes is emphasized.
The genus Thermoactinomyces is among the earliest
known bacteria to have been classified with the actinomycetes
(Tsiklinsky, 1899; Lacey, 1989). However, recent studies have
suggested that genus Thermoactinomyces should no longer be
classified in the Actinomycetales but placed in the family
Bacillaceae (Stackebrandt and Woese, 1981). Not only do 1 6S
rRNA oligonucleotide sequences suggest a close relationship with
the genus Bacillus, but also Thermoactinomyces
species, like Bacillus species, produce endospores
(Cross et al., 1971) and have lower G+C contents than other
actinomycete species (Craveri et al., 1966; Fritzsche,
1967).
On the basis of the 165 rRNA evidence Stackebrandt et al.,
(1983) concluded that Thermoactinomyces vulgaris is
closer to Bacillus subtilis than to Streptomyces
and similar organisms. This finding was also supported by the
plasma membrane menaquinone data (Collins et al., 1982).
Phage to Thermoactinomyces spp. (Prauser and Momirova,
1970; Treuhaft, 1977; Kretschmer, 1982) and to Bacillus spp.
(Saunders and Campbell, 1966; Reanney and Marsh, 1973) have been
reported frequently. However, no phage typing studies were
conducted to examine the cross infectivity of the phage isolated to
members of these two genera. In this study the reactions of
representative Bacillus strains and those of actinomycete
genera with cell-wall chemotype III to Thermoactinomyces-
phage and Thermoactinomyces spp. to Bacillus phage
were examined, in an attempt to provide further evidence in support
of the transfer of the genus Thermoactinomyces in the family
Bacillaceae (Stackebrandt and Woese, 1981).
MATERIALS and METHODS
Bacterial strains. Three Thermoactinomyces
spp., 24 species of Bacillus and 25 actinomycete species
of cell wall chemotype III were used in this study (Tables 1 and
2). Cell wall chemotype III actinomycetes were tested since
Thermoactinomyces belongs to this wall chemotype
(Lechevalier and Lechevalier, 1970). Inoculum was stored as spore
or mycelial macerates in 10% glycerol at 20 C (Wellington and
Williams, 1978).
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Phage Propagation host Strain Activity
spectra code phiTl phiT2
phiT1* Thermoactinomyces candidus ACM 1751 + +
phiT2** Thermoactinomyces candidus ACM 1751 + +
phiT3* Thermoactinomyces candidus ATCC 27868 + +
phiT4** Thermoactinomyces candidus ATCC 27868 + +
phiT5* Thermoactinomyces vulgaris ATCC 15733 + +
phiT6** Thermoactinomyces vulgaris ATCC 15733 + +
Table 1 continued
Phage Activity spectra
phiT3 phiT4 phiT5 phiT6
phiT1* + + + +
phiT2** + + + +
phiT3* + + + +
phiT4** + + + +
phiT5* + + + +
phiT6** + + + +
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Table 1. Activity spectra of Thermoactinomyces phage,
isolated from compost* and from organic mulch** (strain codes as in
Table 2.
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Activity spectra
Species Strain phi1 phi2 phi3 phi6 phi8
code
Bacillus brevis NCTC 7577 + - + + -
Bacilluscereus NCTC 9946 + - - - -
Bacillus cereus UWA 1 PH - - - -
Bacillus cereus
var.Mycoides NCTC 926 + - + + -
Bacillus circulans NCTC 7578 - - - - -
Bacillus coagulans NCTC 10334 - - - - -
Bacillus firmus M 2987 - - - - -
Bacillus licheniformis NCTC 6346 - - - - -
Bacillus megaterium NCTC 9848 - - - - -
Bacillus megaterium NCTC 10342 - - - - -
Bacillus polymyxa NCTC 4747 - - - - -
Bacillus pumilis NCTC 8241 - - - - -
Baclllus sphaericus UWA 2 - PH - + +
Bacillus stearothermophilus NCTC 10003 - + - + +
Bacillus stearothermophilus NCTC 10339 - + - + +
Bacillus stearothermophilus NCTC 10007 - + - + +
Bacillus stearothermophilus ATCC 7953 - + - + PH
Bacillus stearothermophilus UWA 6 - + - PH -
Bacillus subtilis NCTC 6276 + - - - -
Bacillus subtilis ATCC 6633 + - - - -
Bacillus subtilis M 708 + - - - -
Bacillus spp. NCTC 5823 + - - - -
Bacillus pp. M 2429 + - + - -
Bacillus thuringiensis UWA 3 + - PH - -
Table 2 continued
Species Activity spectra
phiB1 phiB2 phiB3 phiB4 phiB5 phiB6
Bacillus brevis - - - - - -
Bacilluscereus - - - - - -
Bacillus cereus - - - - - -
Bacillus cereusvar.Mycoides- - - - - -
Bacillus circulans PH - - - - -
Bacillus coagulans - PH - - - -
Bacillus firmus - - PH - - -
Bacillus licheniformis - - - PH - -
Bacillus megaterium - - - - PH -
Bacillus megaterium - - - - + -
Bacillus polymyxa - - + + - -
Bacillus pumilis - - - - - PH
Baclllus sphaericus - - - - - -
Bacillus stearothermophilus- - - - - -
Bacillus stearothermophilus- - - - - -
Bacillus stearothermophilus- - - - - -
Bacillus stearothermophilus- - - - - -
Bacillus stearoihermophilus- - - - - -
Bacillus subtilis - - - - - -
Bacillus subtilis - - - - - -
Bacillus subtilis - - - - - -
Bacillus spp. - - - - - -
Bacillus spp. - - - - - -
Bacillus thuringiensis - - - - - -
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Table 2. Activity spectra of phage isolated to Bacillus
spp. (PH: Propagation host, +: species susceptible to phage
lysis, -: species not susceptible to phage lysis. ACM: Australian
Collection of Microorganisms; ATCC: American Type Culture
Collection, Rockville, Maryland; DSM: Deutsche Sammlung von
Mikroorganismen und Zellkulturen; NCTC: National Collection of Type
Cultures, London; M: Department of Microbiology Culture Collection,
University of Western Australia; UWA: Department of Soil Science
Culture Collection, University of Western Australia)
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Samples of organic mulch and composted eucalyptus bark.
Avocado mulch and composted eucalyptus bark (CEB) were used for
the isolation of phage in this study. These substrates were
selected since they contain a diverse and highly competitive flora
of which Bacillus and actinomycete species form an important
component (Cook and Baker, 1983; Hardy and Sivasithamparam, 1989;
Kurtboke et a1., 1993b).
Mulch was sampled from an avocado plantation in the
experimental plots at the University of Western Australia during
summer (November-December, 1991) when the temperature for the upper
layer of the mulch (10 cm deep) was 20-25 C. The base of the
avocado trees had been previously amended with 40 kg of straw and
25 kg of stabilized chicken manure. The stabilized pH of this mulch
was 7.2 (1:5 water). Samples from the base of 6 avocado trees were
mixed and 2g of this large composite sample were used to isolate
phage.
Composted eucalyptus bark from marri (Eucalyptus
calophylla R. Br. ex Lindl.) and karri (Eucalyptus
diversicolor F.J.Muell) trees was prepared by Western
Australian Chip & Pulp, Manjimup, Western Australia. The details of
this compost have recently been described by Hardy and
Sivasithamparam (1989). They observed that sampling between the
140-160th dd of composting revealed the highest numbers of
thermophilic actinomycetes. This compost was sampled at the 160th.
day of composting and its stabilised pH was 6.5 (1:5 water).
Sampling was done from different positions (corners and middle and
depths of 50, 100 & 150 cm) of bark windrow (width: 3m, height: 1m
and length: 20m). A large composite sample was prepared by mixing
the parts of these subsamples for the isolation of phage .
Isolation of phage. Phage were isolated to
Thermoactinomyces candidus (ATCC 27868), T.candidus
(ACM 1751), T.vulgaris (ATCC 15733). Flasks containing
20ml of sterile peptone-yeast extract calcium (PYCa) broth (Bradley
et a1., 1961) were inoculated with 1ml glycerol suspension
of the prospective host bacteria and 2g of the organic mulch or
CEB. These were then incubated in a gyratory shaker (Model G76, New
Brunswick Scientific-Edison, NJ, USA) at 200rpm overnight at 37 C.
After incubation, suspensions from each flask were centrifuged for
1h at 2000g and supernatants filtered through sterile Millipore
(Millipore Corp.) membranes of 0.221um pore size and collected in
sterile tubes. 0.2ml of filtrates were spotted on plates of PYCa
agar (Bradley et a1., 1961) on which 0.3ml of a glycerol
suspension of the prospective host had been spread and dried for
30min in a laminar flow. The plates were then incubated at 37 C for
96h and examined for plaques. Single plaques were removed from the
agar plate and resuspended in 1ml of PYCa broth for 36hrs. A sample
of this broth was then filtered and spotted on the plates
previously inoculated with the prospective hosts as before. Single
plaques developing on these plates were removed, resuspended,
filtered and the resulting phage suspensions were stored at 4 C.
Phage were also isolated to Bacillus circulans (NCTC
7578), B.coagulans (NCTC 10334), B.firmus (M 2987),
B.licheniformis (NCTC 6346), B.megaterium (NCTC
9848), B.pumilis (NCTC 8241) from the organic mulch, using
the procedure described above. Other Bacillus phage used in
this study had been previously isolated by Kurtboke et a1.
(1993a) from the CEB (Table 2).
Phage isolated to Thermoactinomyces spp. were mixed to
prepare a stock suspension of phage set 1 (Table 1). Phage isolated
to Bacillus spp. were also mixed for phage set 2 (Table 2).
Phage sets 1 and 2 were used to test the susceptibility of
Thermoactinomyces and Bacillus spp. to each others
phage.
Phage activity spectra. Phage cross infections were
studied by spotting 0.2ml of stock phage suspensions of the phage
sets containing 10^6 pfu/ml onto PYCa agar plates each seeded with
a spore suspension (x10^6 cfu/ml) of the strain being studied
(Table 2). The routine test dilution of 10^6 pfu/ml was the
ten-fold dilutions of the clear spots in PYCa broth (Wellington and
Williams, 1981). The plates were then incubated for 48h at
appropriate temperatures (Claus and Berkeley, 1989; Lacey, 1989)
and examined for lysis.
RESULTS and DISCUSSION
The phage sensitivity of the species belonging to the genera
Bacillus and Thermoactinomyces in this study
corresponded closely to those indicated by chemical and 16S rRNA
comparative cataloguing studies (Stackebrandt and Woese, 1981).
Thermoactinomyces phage used in this study did not
show any cross infectivity with other cell wall chemotype III
actinomycetes (Table 3) and our findings agreed with those of
Prauser (1983) and Kempf et al. (1987).
The cross infectivity observed between the members of the
genus Bacillus and Thermoactinomyces (Table 4)
supports the transfer of the genus into the family Bacillaceae
(Stackebrandt and Woese, 1981). Thermoactinomyces
continue to be included with other actinomycetes, by many
workers, due to their morphological characteristics (Lacey,
1989).
However, RNA oligonucleotide sequencing studies showed that
the possession of branched hyphae as in the members of the genus
Thermoactinomyces does not warrant the placement of a
bacterium within the order Actinomycetales, nor should the
inability of an organism to form branching filaments such as
Arthrobacter, Cellulomonas and Rothia, necessarily
exclude them from this taxon (Goodfellow and Cross, 1984). Indeed,
the exact composition and boundaries of the order
Actinomycetales and its genera (Buchanan, 1918) has remained
open to question and modification from continued application of new
taxonomic methods (Stackebrandt and Woese, 1981; Goodfellow and
Cross, 1984).
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Actinomadura citrea, ATCC 27887
A.pusilla, ATCC 27296
A.spadix, ATCC 27298
A.verrucosospora, ATCC 27299
A.viridis, ATCC 27103
Brevibacterium linens, ATCC 9172
Dermatophilus congolensis, ATCC 14640
Geodermatophilus obscurus, ATCC 27100
Kitasatosporia setae, ATCC 33774
Microbispora aerata, ATCC 15448
M.rosea, ATCC 12950
M.thermodiastatica, ATCC 27098
M.thermorosea, ATCC 27099
Microtetraspora fusca, ATCC 23058
M.helvata, ATCC 27295
M.roseoviolacea, ATCC 27297
Nocardiopsis flava, ATCC 29533
Planomonospora parontospora, ATCC 23863
P.parontospora ssp. antibiotica, ATCC 23864
P.parontospora ssp. parontospora, ATCC 23864
Spirillospora albida, ATCC 23863
Streptosporangium albidum, ATCC 15331
S.pseudovulgare, ATCC 25243
S.roseum, ATCC 27100
Thermomonospora curvata, DSM 43183
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Table 3. Actinomycete species with cell wall chemotype III
not susceptible to Thermoactinomyces phage.
__________________________________________________________________
Phage typing together with these new taxonomic methods may be
used to define the boundaries of a taxon. To determine the place of
phage typing in reliable actinomycete identification schemes more
information is clearly needed on the specificity of phage and
host-phage interactions in their natural substrates.
___________________________________________________________________
Species Code Phage set Phage set 1
1 2
_______________________________________________________________
___
Thermoactinomyces candidus ATCC 27868 +
Thermoactinomyces candidus ACM 1751 +
Thermoactinomyces vulgaris ATCC 15733 +
Bacillus brevis NCTC 7577 -
Bacillus cereus NCTC 9946 -
Bacillus cereus UWA 1 -
Bacillus cereus var. Mycoides NCTC 926 -
Bacillus circulans NCTC 7578 -
Bacillus coagulans NCTC 10334 -
Bacillus firmus M2987 -
Bacillus licheniformis NCTC 6346 -
Bacillus megaterium NCTC 9848 +
Bacillus megaterium NCTC 10342 +
Bacillus polymyxa NCTC 4747 -
Bacillus pumilis NCTC 8241 +
Bacillus sphaericus UWA 2 +
Bacillus stearothermophilus NCTC 10003 -
Bacillus stearothermophilus NCTC 10339 -
Bacillus stearothermophilus NCTC 10007 -
Bacillus stearothermophilus ATCC 7953 -
Bacillus stearothermophilus UWA 6 -
Bacillus subtilis NCTC 6276 +
Bacillus subtilis ATCC 6633 +
Bacillus subtilis M 708 +
Bacillus spp. NCTC 5823 +
Bacillus spp. M 2429 +
Bacillus thuringiensis UWA 3 +
__________________________________________________________________<
Table 4. Reactions of Thermoactinomyces to phage set
2 and of Bacillus spp. to phage set 1. (Species susceptible
(+) and not ()) susceptible to phage lysis)
__________________________________________________________________<
P>
Also for the determination of the natural relationships of genera,
the members of a host specific phage set must be selected after
intensive cross-infection studies using as many strains and phage
as possible (Prauser, 1984; Kurtboke and Williams, 1991). In this
study we only used three Thermoactinomyces spp. the other
members of this genus were not accessible due to quarantine
restrictions in Australia imposed mainly because of their
implication in hypersensitivity pneumonitis (Lacey, 1988). Further
studies using phage with other members of the two genera are
certainly warranted. Korn-Wendish and Schneider (1992) after
extensive studies on 905 strains from several actinomycete genera
using species specific and genus specific phage concluded that
phage typing is a useful aid for the identification of
actinomycetes. At the genus level, the specificity of actinophage
has led them to correct the placement of falsely identified strains
(Korn-Wendish et a1., 1978; Korn-Wendish and Schneider,
1992). Williams et a1., (1980) also used phage, in an
attempt to provide supplementary information on the classification
of the genus Nocardia.
At species level, the acceptance of phage typing as a useful
tool in actinomycete systematics requires extensive studies
including many phage and host strains, since almost no phage is
virulent for all strains of the taxon under study (Prauser,
1970).
Detailed studies in actinophage ecology and phage-host
interactions in their natural substrates is likely to provide more
information about actinophage specificity (Williams et a1., 1987).
This in turn would enhance the use phage typing as a taxonomical
tool (Prauser, 1984; Kurtboke and Williams, 1991; Korn-Wendish and
Schneider, 1992). This study emphasizes the value of phage
susceptibility as an aid to reliable identification schemes and for
the detection of natural relationships among taxa.
ACKNOWLEDGEMENTS. We would like to thank Dr.L.I.Sly and
Ms.S.Ben Dekhil of the Australian Collection of Microorganisms,
University of Queensland, Australia, Dr B.Mee and Mr.C.Manensis,
The University of Western Australia, Department of Microbiology,
Perth, Australia for supplying the type strains.
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Copyright 1993 C.E.T.A.
