Distribution and diversity of actinopages in soils populated with streptomycetes

Abstract

Distribution and diversity of actinophages from soil samples collected from several parts of Thailand were investigated. At first, streptomycetes were isolated and used as hosts for isolation of phages. By enrichment procedure, 24 phages were obtained and their hosts were further studied for antimicrobial producing activity. The phages were partially purified by single plaque isolation and then distinguished by plaque morphology and morphology examination under transmission electron microscope. All phages gave differences in size and shape but they all had a hexagonal head and long non-contractile tails. Thus, they were belonged to group B of Bradleyʼs classification and type B1, in Family Siphoviridae of Ackermann and Eisenstarkʼs taxonomy. Determination of host range of phage with 164 streptomycetes strains, it revealed that all phages displayed the different host range pattern by forming plaques on Streptomyces strains and varied from narrow to broad host range. Phage Roi-1 and Yok-15 each gave very broad host range. The phages giving broad host range were brought to further studied. They showed dissimilar patterns in one step growth experiment. Most of them were able to give maximum replication at neutral pH except phage Yok-15, which was at basidic pH. Requirement of cations, Ca 2+and Mg 2+, they were also different. Some phages did not require at all, some required small amounts except Yok-15, Sin-27, Nsaw-28 and Nsaw-30 that required high amounts of Ca2+. Antigenic relationship between the 9 phages and anti-phage Roi-1 serum revealed that Nsaw-30 and Sing-27 were highly inactivated, thus they were closely serological relatedness. SDS-PAGE showed that the phages displayed a different size of protein bands. The number and size of fragments of all phage DNA, which obtained after digestion with restriction enzymes, showed a unique and different molecular weights. The homology of phage DNA was also employed by plaque hybridization. When using DNA of Surat-11, Ray-7, Yok-15 and Roi-1 DNA as probes, it resulted that the homology of phage DNA toward these probes were 5, 4, 4 and 0, respectively. The fate of actinophage Yok-15, and its host bacteria, Streptomyces sp. 15 ( isolated from Thai soil as well as phage Yok-15), S. coelicolor, S. griseus and S. viridochromogenes in two kinds of Thai and Japanese sterile soils using batch microcosms, was also investigated. Phage Yok-15 had a very wide host range, and could infect 39 important strains of Streptomyces species from 48 strains tested. In two kinds of Thai soils, phage Yok-15 multiplied in host strain 15 at a maximal titer level, then decreased rapidly in titer level during incubation. However, no multiplication of phage YoK-15 was observed during the incubation with other three streptomycetes in Thai soils, and also with all used streptomycetes in Japanese soil. During incubation with these streptomycetes, the phages decreased in their titer more rapidly than free phages in both Thai and Japanese soils. These results indicated that natures or conditions of soil and strains of host streptomycetes may affect on the survival and multiplication of phages in soil. On the other hand, the survival of host streptomycetes seemed to be strongly affected by the conditions of soil rather than phage infection. Streptomyces sp. 15 was identified by partial 16S rDNA sequences covering the variable region. It resulted that Streptomyces sp. 15 was closely resembled to Streptomyces sp. NRRL 5183 (accession number AJ391815) which has published in year 2000 and has no any report supported since then. Thus, this strain was designated as a new strain (accession number AY128706). To further characterization of phages Yok-15, the phage was sensitive to chloroform, it stabilized at temperature of 4 0C, at pH 9 for 5 h, and rapidly inactivated by detergents: sodium pyrophosphate and EDTA. In addition, it could multiply in liquid culture with maximum yields at 200 rpm, with initial host and phage concentration at 1.41x 108 cfu/ml and 106 pfu/ml, respectively, and could survive at pH 5.