New thermal inducible Streptomyces phages isolated from tropical soils

New thermal inducible Streptomyces phages isolated from tropical soils

Andrea Balan and Gabriel Padilla

Departamento de Microbiologia, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil. Fax: [phone]. E-mail: [e-mail]. Send correspondence to G.P.

ABSTRACT

Two new Streptomyces phages, øBP1 and øBP2, were isolated from tropical soil samples. These phages presented a large host range and developed both lytic and lysogenic responses in different Streptomyces species tested. Variations in the incubation temperature showed to be important in the development of the replication cycle. Increasing incubation temperature from 30^oC to 42^oC induced the lytic response of øBP2 and lysogenic of øBP1 in the host strain Streptomyces sp. WL6. øBP1 and øBP2 have icosahedral heads with long tails and were characterized in relation to morphology, G + C content, genome size and adsorption curve.

INTRODUCTION

Streptomyces phages have been isolated mainly from temperate soil samples and investigated for a variety of reasons, including: interest in controlling the problems they cause in fermentation processes, use as taxonomic tools for typing strains in screening programs (Anné et al., 1984; Kurtboke et al., 1992; Long et al., 1993), and studies of fundamental biological processes (Lomovskaya et al., 1980; Long and Amphlett, 1996). Actinophages are also of interest in Streptomyces genetics because of their potential use in the construction of cloning vectors (Kuhstoss et al., 1991). Genetic studies of Streptomyces phages were initiated with phages R4, øC31 and S10 (Lomovskaya et al., 1980). øC31 is the best characterized actinophage, and several cloning vectors have been derived from it (Suarez and Chater, 1980; Kobler et al., 1991). Ecological studies of tropical phages are important for obtaining information about interactions between hosts and parasites, and the control exerted by phages on several chemical transformations. The aim of the present study was to screen tropical soil samples in search of new actinophages which could develop lytic and lysogenic cycles on Streptomyces species. Interestingly, many of these phages were different from Streptomyces phages isolated from temperate environments, mainly in their lytic response, that was thermoinducible.

MATERIAL AND METHODS

Phages were isolated from tropical soil samples from different regions: Colombian Amazon basin and Minas Gerais and São Paulo States, Brazil. The soil pH was between 4.6 to 6.5 and soil temperature from 18^oC to 23^oC. Streptomyces sp. WL6 and S. lydicus were chosen as hosts because they grow easily at high temperatures. The Streptomyces strains used in this work are listed in Table I.

Streptomyces strainsPhagesøBP1øBP2p.f.u*Plaque typeSize (mm)p.f.u.Plaque typeSize (mm)S. albocyaneus 51391+turbid1+clear2S. alni M66152+ +clear2+turbid1S. aureofaciens M30312+turbid2+turbid1S. bobili 33103+ +turbid2---S. capoamus M31232+ + +clear3+ + +clear3S. clavuligerus 270643------S. coelicolor A3(2)4+ +turbid2---S. coelicolor 15014+ +turbid1---S. felleus M30792+ + +clear3+ + +clear1S. felleus M31822+ + +turbid1+ +turbid1S. griseus M30592------S. lavenduluae M30032------S. lydicus 54611+ + +turbid1+ + +turbid2S. lividans TK244+clear3+ +turbid1S. lividans TK544+turbid1+turbid2S. melanochromogenes31823------S. nigellus 54901------S. novaecasareae 30743+clear3+ + +clear4S. olindensis M56222+ + +clear3+ + +clear3S. parvulus M30422+ + +turbid3+ + +turbid4S. plicatus M31662------S. recifenci M30642+clear2---S. rimosus M31982+clear1+turbid1Streptomyces sp. WL65+ + +clear1+ + +turbid2

Streptomyces strains	Phages
	øBP1			øBP2
	p.f.u*	Plaque type	Size (mm)	p.f.u.	Plaque type	Size (mm)
S. albocyaneus 51391	+	turbid	1	+	clear	2
S. alni M66152	+ +	clear	2	+	turbid	1
S. aureofaciens M30312	+	turbid	2	+	turbid	1
S. bobili 33103	+ +	turbid	2	-	-	-
S. capoamus M31232	+ + +	clear	3	+ + +	clear	3
S. clavuligerus 270643	-	-	-	-	-	-
S. coelicolor A3(2)4	+ +	turbid	2	-	-	-
S. coelicolor 15014	+ +	turbid	1	-	-	-
S. felleus M30792	+ + +	clear	3	+ + +	clear	1
S. felleus M31822	+ + +	turbid	1	+ +	turbid	1
S. griseus M30592	-	-	-	-	-	-
S. lavenduluae M30032	-	-	-	-	-	-
S. lydicus 54611	+ + +	turbid	1	+ + +	turbid	2
S. lividans TK244	+	clear	3	+ +	turbid	1
S. lividans TK544	+	turbid	1	+	turbid	2
S. melanochromogenes31823	-	-	-	-	-	-
S. nigellus 54901	-	-	-	-	-	-
S. novaecasareae 30743	+	clear	3	+ + +	clear	4
S. olindensis M56222	+ + +	clear	3	+ + +	clear	3
S. parvulus M30422	+ + +	turbid	3	+ + +	turbid	4
S. plicatus M31662	-	-	-	-	-	-
S. recifenci M30642	+	clear	2	-	-	-
S. rimosus M31982	+	clear	1	+	turbid	1
Streptomyces sp. WL65	+ + +	clear	1	+ + +	turbid	2

Table I - Host range of the phages øBP1 and øBP2.

*p.f.u./ml: + less than 30; ++ between 30 to 70; +++ more than 70; - none; growth temperature 30^oC.

(1) ISP - International Streptomyces Project.

(2) DAUFPE - Department of Antibiotics, University of Pernambuco, Brazil.

(3) ATCC - American Type Culture Collection.

(4) JI - John Innes Institute, Norwich, England.

(5) CDBB - Biotecnology Department, University of Mexico.

Streptomyces strains and phages were manipuled as described by Chater and Carter (1979), but using modified R5 medium (R5M = R2YE medium without saccharose and with 3 g/l Tris instead of TES) (Hopwood et al., 1985). In order to detect phages, 2 g of soil samples were diluted in 4 ml of R5M medium, and enriched by incubating with 50 ml (10⁷ c.f.u.) of Streptomyces sp. WL6 spores for 60 h at 30^oC (adapted from Dowding, 1973). Phages were detected from supernatant solutions by their ability to form plaques on a bacterial lawn after overnight incubation, and distinguished by variations in morphology, size and turbidity of the plaques. High titer phage suspensions were obtained according to Anné et al., 1984, and phage DNA was extracted from pellets as described by Chater and Carter (1979). Growth step and adsorption experiments were carried out with phage suspensions added to mycelial fragments after 8-h incubation at 30^oC, according to Lomovskaya et al. (1980), Haket et al. (1990), and Long and Amphlett (1996). Electron microscopy observations of negative stained phage samples were done as described by Suarez and Chater (1980). Temperate phages could be distinguished by the appearance of immune colonies at the center of the plaques that were resistant to superinfection by the same phage. Host range was determined by using 100-ml phage suspension (10¹⁰ p.f.u. ml^-1) on lawns of different strains. If plaques appeared, the strain was considered to be sensitive to the specific phage.

In order to study the influence of the temperature in the morphology of the lysis plaques and infection efficiency, spore suspensions (10⁷ c.f.u ml^-1) were mixed with 50 ml (10¹⁰ p.f.u. ml^-1) of phage suspensions and incubated at 30^oC, 37^oC or 42^oC for 20 h. The stability of phage particles at high temperatures was tested by incubation for 10 min at 65^oC before plating at 30^oC. The G + C content of phage DNAs was determined by high-performance liquid chromatography (Ko et al., 1977). Restriction enzyme analysis of phage DNA was carried out with 23 different restriction enzymes, using assay conditions described by the manufacturers.

RESULTS AND DISCUSSION

Nine different phages, named øBP1 to øBP9, were isolated from tropical soils and characterized according to morphology, genome size and adsorption curve. The incubation time during phage isolation was relatively short (in average 20 h) to avoid problems such as readsorption, death and the presence of older phages. Previous reports have demonstrated the isolation of actinophages after 1 week of incubation such as phage SAt1, phages of Micromonospora purpurea and Brevibacterium lactofermentum (Klaus et al., 1981). Due to their ability to undergo lytic and lysogenic growth in Streptomyces sp. WL6, phages øBP1 and øBP2 were selected for more detailed characterization. øBP1 infected 18 out of 24 Streptomyces strains tested, developing a lysogenic response in 9 strains (Table I). Lysogeny was demonstrated by immunity of putative lysogens to super infection and by release of phage particles from the lysogenic host strain. The phages could be adsorbed but yielded no progeny. øBP2 infected 14 Streptomyces strains and evoked the lysogenic response in 9 strains. Both phages did not infect S. clavuligerus, S. griseus, S. lavenduluae, S. melanochromogenes, S. nigellus and S. plicatus, probably because these species have strong restriction systems (Baltz and Cox, 1984 and Diaz et al., 1989). The host range of both phages is wider than related actinophages, such as øC31, R4 and øA7 (Lomovskaya et al., 1980; Baltz and Cox, 1984; Diaz et al., 1991). This feature could be useful for taxonomic applications and also for the elimination of Streptomyces strains most frequently found in soil samples, in the screening of novel organisms (Kurtboke et al., 1992; Long et al., 1993). The optimum temperature for plaque formation was 30^oC in S. felleus, S. parvulus, S. olidensis, S. capoamus, S. lydicus and Streptomyces sp. WL6. Plaque formation required the presence of 10 mM Ca²⁺. Electron microscopy of purified øBP1 and øBP2 virions revealed that both phages have icosahedral heads of 70 nm and 60 nm in diameter, and tails of 300 nm and 290 nm in length, respectively (Figure 1). The phages belong to Type B according to Bradley’s classification system (Bradley, 1967).

Growth step and adsorption experiments showed that øBP1 replicated more efficiently in both host strains than øBP2, inducing total bacterial lysis and liberating more particles to the culture medium in a shorter period of time. The most efficient adsorption rates were obtained for mycelium germinated from spores after 8-h incubation. The latent period (biosynthesis and maduration) was 60 min for øBP1 in both hosts; øBP2 took 60 min in S. lydicus and 120 min in Streptomyces sp. WL6 (Figure 2), whereas the reference phage øC31 took only 40 min in S. coelicolor (Lomovskaya et al., 1980). The long rise period of both phages indicated that phage release from single infected cells was asynchronous.

In order to study the influence of the temperature on the replication of phages øBP1 and øBP2, the host strains were incubated at 30^oC, 37^oC and 42^oC (Table II). When the incubation temperature increased from 30^oC to 37^oC, phage øBP1 developed lysogenic and lytic responses, respectively. In Streptomyces sp. WL6, the plaque and burst sizes were greater at 37^oC. At 30^oC, øBP2 replicated by lysogenic growth in both hosts (Table II); temperature shift to 37^oC and 42^oC induced lytic response and also increased the plaque and burst sizes (Table II). Because øBP2 was more resistant to higher temperatures than øBP1, it was subjected to heat-shock treatment (65^oC for 15 min) before normal incubation at 30^oC with Streptomyces sp. WL6. Low infection efficiency and irregular forms of the lytic plaques were observed as product of the temperature shifts. These kinds of alterations have been observed for other actinophages, but at lower intensity (Williams et al., 1987). Tolerance to high temperatures (max. 45^oC) has been described only for phages of thermophilic microorganisms such as Bacillus, Micromonospora and Thermoactinomyces (Haket et al., 1990).

Table II
- Influence of the temperature on the development of the life cycles of phages øBP1 and øBP2 after 20 h of incubation.

Phage host	Temperature of incubation Plaques morphology
	30oC	37oC	42oC
øBP1/ Streptomyces sp. WL6	46 p.f.u.* 1 mm clear	36 p.f.u. 2-3 mm turbid	- - -
øBP1/ S. lydicus	81 p.f.u. 1 mm turbid	206 p.f.u. 2 mm clear	- - -
øBP2/ Streptomyces sp. WL6	45 p.f.u. 2 mm turbid	124 p.f.u. 4 mm clear	23 p.f.u. 7 mm clear
øBP2/ S. lydicus	25 p.f.u. 2 mm turbid	28 p.f.u. 2-3 mm turbid	27 p.f.u. 2 mm turbid

*Average per plate; - = no growth.

The G + C content of the phages (69.6% for øBP1 and 67.2% for øBP2) was in agreement with the G + C range of Streptomyces spp., which is about 60 to 76 mol%. The genome size of øBP1 (43.5 kb) and øBP2 (35.5 kb) was determined by restriction analysis. The most informative restriction profiles were obtained using ClaI (7 fragments) and EcoRI (4 fragments) for øBP1, and ClaI (10 fragments), EcoRV (2 fragments) and by BamHI (3 fragments) for øBP2 (Figure 3A). Restriction profiles of phage genomes digested with restriction enzymes that cut preferentially G/G-rich sites are shown in Figure 3B. The remaining enzymes had either multiple sites or did not cut the phage genomes (Table III). The comparison of øBP1 and øBP2 restriction profiles with those of other actinophages did not show any obvious similarities between them (Lomovskaya et al., 1980; Anné et al., 1984; Diaz et al., 1989). These results lead to the conclusion that øBP1 and øBP2 are two novel Streptomyces phage isolated from tropical soils. They present interesting characteristics, particularly a broad host range and replication cycle inducible by high temperatures, that could be used as molecular tools.

Table III
- Physical properties of DNA of phages øBP1 and øBP2.

Phage	G + C content (mol%)	DNA size (Kb)	Restriction endonucleases cleavage sites	Enzymes with no cleavage sites
øBP1	69.6	43.5	ApaI ( 15); AvaI ( 11);	AccI; AluI; BclI;
			BalI ( 9); BglII ( 13)	BamHI; BglI;
			ClaI (7); EcoRI (4);	DraI; EcoRV; HincII;
			HaeIII ( 6); KpnI ( 13);	HpaI; PstI; PvuI
			MluI ( 11); NcoI ( 11);
			ScaI ( 5); XhoI ( 12)
øBP2	67.2	35.5	AvaI ( 20); ApaI ( 4);	AccI; AluI; BclI; BglII;
			BalI ( 8); BamHI (3);	DraI; EcoRI; HincII;
			BglI ( 15); ClaI (10);	HpaI; KpnI; PstI;
			EcoRV (2); HaeIII ( 14)	PvuI
			MluI ( 15); NcoI ( 11);
			ScaI ( 12); XhoI ( 8)

ACKNOWLEDGMENTS

The authors wish to thank Drs. S. Newton, Ch. Thompson, G.P. Manfio and M. Marins for valuable advice and suggestions; Dr. V.P Canhos for G + C determination, and to the Electron Microscopy Unit of Institut Pasteur, Paris, France. A.B. was the recipient of a FAPESP fellowship (Proc. No. 92/1517-5). Publication supported by FAPESP.

RESUMO

Dois novos fagos de Streptomyces, denominados øBP1 e øBP2, foram isolados de amostras de solos tropicais. Estes fagos apresentaram uma grande faixa de hospedeiros e foram capazes de desenvolver os ciclos lítico e lisogênico em diferentes espécies de Streptomyces testadas. A temperatura de incubação mostrou-se importante para o desenvolvimento do ciclo de replicação. O aumento desta temperatura de 30^oC para 37^oC ou 42^oC induziu o ciclo lítico de øBP2 e lisogênico de øBP1 na linhagem hospedeira Streptomyces sp. WL6. Ambos os fagos apresentaram cabeça icosaédrica com talos longos e foram caracterizados em relação à morfologia, tamanho do genoma, conteúdo de G + C e curva de adsorção.

REFERENCES

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Diaz, L.A., Hardisson, C. and Rodicio, M.R. (1989). Isolation and characterization of actinophages infecting Streptomyces species and their interaction with host restriction-modification systems. J. Gen. Microbiol. 135: 1847-1856.

Diaz, L.A., Hardisson, C. and Rodicio, M.R. (1991). Characterization of the temperate actinophage fA7 and its deletion derivatives. J. Gen. Microbiol. 137: 293-298.

Dowding, J.E. (1973). Characterization of a bacteriophage virulent for Streptomyces coelicolor A3(2). J. Gen. Microbiol. 76: 163-176.

Haket, J., Desmarais, D., Mehindate, K. and Dery, C.V. (1990). Saccharopolyspora hirsuta strain 367 releases JHJ-1, a bacteriophage capable of propagation on old mycelium. J. Gen. Microbiol. 136: 573-579.

Hopwood, D.A., Bibb., M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, J.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schrempf, H. (1985). Genetic Manipulation of Streptomyces. A Laboratory Manual. The John Innes Institute, Norwich.

Klaus, S., Krugel, H., Suss, F., Nergenfind, M., Zimmerman, I. and Tauibeneck, U. (1981). Properties of the temperate actinophage SH10. J. Gen. Microbiol. 123: 269-279.

Ko, C.Y., Johnson, J.L., Branett, L.B., McNair, H.M. and Vercelloti, J.R. (1977). Sensitive estimation of the percentage of guanine plus cytosine in deoxyribonucleic acid by high performance liquid cromatography. Anal. Biochem. 80: 183-192.

Kobler, L., Schwertfirm, G., Schmieger, H., Bolotin, A. and Sladkova, I. (1991). Construction and transduction of a shuttle vector bearing the cos site of Streptomyces phage øC31 and determination of its cohesive ends. FEMS Microbiol. Lett. 78: 347-354.

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Kurtboke, D.I., Chen, C.F. and Williams, S.T. (1992). Use of polyvalent phage reduction of streptomycetes on soil dilution plates. J. Appl. Bacteriol. 72: 103-111.

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(Received March 3, 1997)

Figure 1 -
Electron micrographs of phages øBP1 (A) and øBP2 (B) negatively stained with 2% uranil acetate. Magnification: 45000X. Dimensions are indicated in nanometers.

Figure 2 - One-step growth curve of phages øBP1 and øBP2 in the hosts Streptomyces sp. WL6 and S. lydicus. Growing mycelial cultures (8 h) were inoculated with phage suspensions and incubated at 30^oC for 16 h

Figure 3
- Restriction analysis of the phages øBP1 and øBP2. (A): 1, øBP1/ClaI; 2, øBP1/EcoRI; 3, øBP1/BamHI; 4, lHindIII (23.1, 9.4, 6.5, 4.3, 2.3, 2.0, 0.5 kb); 5, øBP2/ClaI; 6, øBP2/EcoRV; 7, øBP2/BamHI. (B): 1, øBP1/HaeIII; 2, øBP1/AvaI; 3, øBP1/ApaI; 4, øBP1/XhoI; 5, lHindIII (23.1, 9.4, 6.5, 4.3, 2.3, 2.0, 0.5 kb); 6, øBP2/HaeIII; 7, øBP2/AvaI; 8, øBP2/ApaI; 9, øBP2/XhoI.

Publication Dates

• Publication in this collection
  06 Oct 1998
• Date of issue
  Dec 1997

History

• Received
  03 Mar 1997