Mary P. Lechevalier’s research while affiliated with Rutgers, The State University of New Jersey and other places

Strain NRRL 12292T, which produces the bleomycin-like antibiotics LL-BO1208 alpha and LL-BO1208 beta, forms umbels consisting of chains of smooth-surface ovoid spores that are borne on verticils on the serial mycelia, indicating that it is a member of the verticillate group of the genus Streptomyces formerly classified in the genus Streptoverticillium. This strain was compared morphologically and physiologically to 54 other verticillate Streptomyces strains. The levels of DNA relatedness between strain NRRL 12292T and 34 other verticillate Streptomyces strains, including strains representing at least 19 genetic species clusters, were also determined. Strain NRRL 12292T is morphologically and physiologically distinct from the other verticillate strains studied, particularly because of the straw yellow color of its aerial mycelia and spore mass. DNA hybridization data support the uniqueness of this strain, since the levels of DNA relatedness between NRRL 12292T and the other verticillate strains used in this study were low. Our data support designation of a new species, Streptomyces stramineus, whose type strain is NRRL 12292.

Members of the genus Frankia have been classified in the order Actinomycetales on the basis of morphology, cell chemistry, and 16S rRNA sequences and catalogs. This genus, which is presently defined by morphology, cell chemistry, the ability to fix nitrogen, and infectivity for and ability to enter into symbiotic relationships with certain plant hosts, may be heterogeneous. Frankia species groups have been difficult to delineate by classical phenotypic methods. The recent use of DNA-DNA pairing and low- frequency restriction fragment analysis, as well as probes composed of certain sequences from the nif (nitrogen fixation) genes or the variable regions of 16S rRNA, has contributed to substantial progress in developing species concepts. In this review I trace the taxonomic history of the genus and outline some of the problems to be resolved in the future.

Using a method known as low-frequency restriction fragment analysis (LFRFA) (M. L. Beyazova and M. P. Lechevalier, Int. J. Syst. Bacteriol. 42:422-433, 1992), we determined the molecular weights of AseI restriction fragments of Streptomyces DNAs by pulsed-field gel electrophoresis. The levels of similarity of fragment patterns among strains were determined by using the simple matching coefficient, and clustering was performed by using the unweighted pair group with mathematical average algorithm. A total of 59 strains representing eight species and the numerically classified taxon Streptomyces cyaneus group A18 (S. T. Williams, M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A. Sneath, and M. J. Sackin, J. Gen. Microbiol. 129:1743-1813, 1983) were studied. Forty-two strains (six species) formed eight clusters at levels of similarity of more than 80%; 17 strains (including the entire S. cyaneus group) were unclustered. Cluster 1 contained all of the Streptomyces albus strains studied plus two strains of Streptomyces somaliensis and two strains of Streptomyces lavendulae. Cluster 2 contained 8 of the 12 Streptomyces fradiae strains examined plus one strain each of S. somaliensis and S. lavendulae. Cluster 3 was heterogeneous in terms of species. Cluster 4 contained two S. somaliensis strains; cluster 5 contained three S. fradiae strains; cluster 6 contained three Streptomyces rimosus strains; and clusters 7 and 8 contained seven and three Streptomyces ipomoea strains, respectively. The S. cyaneus group strains exhibited no clustering among themselves or with the other species examined. Some Streptomyces species which exhibited high levels of similarity (85 to 95%) in physiological tests (e.g., S. albus and the S. fradiae strains in cluster 2) exhibited high levels of similarity in the LFRFA (84 and 81%, respectively). Other taxa (S. cyaneus group) which exhibited equally high levels of physiological similarity (90%) appeared to be unrelated as determined by the LFRFA. Species with lower levels of physiological similarity (e.g., S. somaliensis [75%], S. lavendulae [63%], and S. rimosus [68%]) exhibited low levels of LFRFA similarity (75, 64, and 54%, respectively). High levels of DNA-DNA relatedness (>90%) (S. ipomoea) were reflected in high levels of similarity as determined by the LFRFA (75 to 100%); lower levels of DNA-DNA relatedness (ca. 70%) (S. cyaneus group) were reflected in low levels of LFRFA similarity (strains not clustered). We concluded that the presently used physiological tests reflect too small a portion of the genome to be universally useful in streptomycete species characterization. In contrast, high levels of DNA-DNA relatedness (>90%) and high LFRFA similarity values will probably both be valuable in species delineation in actinomycetes.

Low-frequency restriction fragment analysis of more than 100 strains of the genus Frankia showed that restriction enzyme DraI (recognition site, TTT'AAA) gave rise to large DNA fragments (200 to 1,500 kb), which, when they were subjected to cluster analysis, reflected the host plants from which the strains were isolated. Our results support the conclusions of Lalonde and his colleagues (M. Lalonde, L. Simon, J. Bousquet, and A. Seguin, p. 671-680, in H. Bothe, F. J. de Bruijn, and W. E. Newton, ed., Nitrogen Fixation: Hundred Years After, 1988; P. Normand, P. Simonet, and R. Bardin, Mol. Gen. Genet. 213:238-246, 1988) and Fernandez et al. (M. P. Fernandez, H. Meugnier, P. A. D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989) that various biochemical and genomic analyses can give rise to groupings of Frankia strains that are consistent with the host plants from which the strains are isolated and that the resulting groups may form a basis for defining Frankia species.

Whole-cell amino acid and whole-cell sugar patterns using thin-layer chromatography of cell hyclrolysates of 40 strains of the actinomycete Frankia from North America, Western Europe and Asia were determined. All strains contained meso-diaminopimelic acid. Of the strains studied, 35 contained xylose as the major diagnostic sugar residue (whole cell sugar type D); 3 strains contained glucose without xylose (whole cell sugar type C); and 2 contained fucose (whole cell sugar type E). Madurose was not observed as a sugar residue in any of the strains tested in this study. The thin-layer chromatographic method combined with the sensitive spray N-naphthylethylenediamine dihydrochloride were determined to be convenient for analysis of the slow-growing frankiae.

A previously undescribed cryptic phage was found associated withMicromonospora purpurea ATCC 15835.

A Type II restriction endonuclease, designated Fsel, has been partially purified from a Frankia species (NRRL 18528). This enzyme cleaves Adenovirus 2 DNA at three sites, but does not cleave the DNAs from bacteriophages lambda, T7, and øX174, the animal virus SV40, pUC18 and pBR322. Fsel recognizes the octanucleotide sequence 5′ GGCCGGICC 3′ and cleaves as indicated by the arrow. The frequency of occurrence of Fsel sites within sequenced regions of the human genome is similar to that for Notl sites.

A temperate actinophage was isolated from soil using the gentamicin-producing microorganism, Micromonospora purpurea ATCC 15835 as host. The characterization of the phage represents the initial step in its development as a cloning vector. The phage isolated, MPphiWR-1, formed red- to purple-pigmented turbid plaques. Cells isolated from these plaques were resistant to superinfection with lytic mutants of MPphiWR-1. Southern blots of genomic DNA from a resistant culture showed that MPphiWR-1 integrated into the host genome. The phage was UV- or Mitomycin C-inducible. The integration, resistance to superinfection and inducibility indicated a lysogenic relationship with the host. Using MPphiE-RCPM, a lytic derivative, the phage host range was demonstrated to include members of three genera: one species each of Ampullariella and Catellatospora, and 12 species of Micromonospora. The phage belonged to Ackerman's B1 morphotype having an isometric head and a flexible noncontractile tail. The density of the phage was 1.525 g/cc. Restriction site mapping demonstrated that the phage DNA was 57.9 kb long and had cohesive ends. Using EDTA enrichment, viable mutants with deletions of at least 3.5 kb were isolated and mapped. Phage adsorption, sensitivities and plating efficiency were investigated. Non-liposome PEG-mediated transfection was demonstrated.

A temperate actinophage was isolated from soil using the gentamicin-producing microorganism,Micromonospora purpurea ATCC 15835 as host. The characterization of the phage represents the initial step in its development as a cloning vector. The phage isolated, MPphiWR-1, formed red- to purple-pigmented turbid plaques. Cells isolated from these plaques were resistant to superinfection with lytic mutants of MPphiWR-1. Southern blots of genomic DNA from a resistant culture showed that MPphiWR-1 integrated into the host genome. The phage was UV- or Mitomycin C-inducible. The integration, resistance to superinfection and inducibility indicated a lysogenic relationship with the host. Using MPphiE-RCPM, a lytic derivative, the phage host range was demonstrated to include members of three genera: one species each ofAmpullariella andCatellatospora, and 12 species ofMicromonospora. The phage belonged to Ackerman's B1 morphotype having an isometric head and a flexible noncontractile tail. The density of the phage was 1.525 g/cc. Restriction site mapping demonstrated that the phage DNA was 57.9 kb long and had cohesive ends. Using EDTA enrichment, viable mutants with deletions of at least 3.5 kb were isolated and mapped. Phage adsorption, sensitivities and plating efficiency were investigated. Non-liposome PEG-mediated transfection was demonstrated.