Streptomycin sensitivity of ribosomes isolated from a streptomycin-producing Streptomyces griseus.

The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.     

DNA-DNA hybridization in the systematics of Streptomyces.

The DNA relatedness among strains in several different phenotypically defined Streptomyces species clusters was evaluated. It was found that the data from DNA-relatedness studies do not necessarily agree with the clustering generated using numerical taxonomic techniques. A study of the morphologically heterogeneous 'S. cyaneus' cluster showed that morphological criteria traditionally used to classify and identify Streptomyces species still have value, since strains in DNA-relatedness cluster groups were also similar morphologically (i.e., they had similar spore color, surface properties, and sporophore morphology). An evaluation of DNA relatedness among strains in the S. violaceusniger and S. lavendulae clusters indicated that, if anything, the genus is underspeciated, based on the number of single-member clusters observed. A study of strains of the sweet potato pathogen, S. ipomoea, collected in various locations in the United States and Japan indicated, not surprisingly, that all of the strains belong to the same species.     

Real-time PCR技术在弧菌DNA-DNA同源性测定中的应用

目前在微生物学领域, 研究各级分类单元的最有效手段是多相分类, 该法能较客观地反映生物间自然系统进化的关系1。DNA-DNA同源性分析在多相分类中扮演重要的角色, 常用于亲缘关系密切的微生物种内相似度描述。它在检测错分菌株以及新描述的菌株划归已有分类单元时非常有用。       

Genetic relatedness among species in Aspergillus section Clavati as measured by electrophoretic comparison of enzymes,DNA base composition,and DNA-DNA hybridization

Aspergillus subgenus Clavati has four recognized species: A. clavatus (the type species), A. clavatonanicus, A. giganteus, and A. longivesica. These species are strictly anamorphic (mitotic) and defined by the morphological species concept. However, their genealogical relationships remain uncertain. In this study, we examined the genetic relatedness among the four species in this section, using electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. In a dendrogram based on the calculated similarity values of four enzymes, 10 strains in section Clavati, 3 strains in the xerophilic species, a strain in section Ornati, and a strain in section Cremei were separated into nine major clusters at a 60% similarity level. A. longivesica JCM 10186(T) had Q-10 in our analysis, but Kuraishi et al. (1990) reported A. longivesica JCM 1720(T) had Q-9 (49%) and Q-10 (46%). The G+C contents of the four species of section Clavati ranged from 48 to 50 mol%. The degree of the intraspecific reassociation among the DNAs from the strains of these species ranged from 77 to 99%, whereas the degrees of interspecific relative binding among strains of the four species ranged from 30 to 59%. Our data from enzyme patterns and DNA relatedness support the validity of the three species in section Clavati, except for A. longivesica.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison

The pragmatic species concept for Bacteria and Archaea is ultimately based on DNA-DNA hybridization (DDH). While enabling the taxonomist, in principle, to obtain an estimate of the overall similarity between the genomes of two strains, this technique is tedious and error-prone and cannot be used to incrementally build up a comparative database. Recent technological progress in the area of genome sequencing calls for bioinformatics methods to replace the wet-lab DDH by in-silico genome-to-genome comparison. Here we investigate state-of-the-art methods for inferring whole-genome distances in their ability to mimic DDH. Algorithms to efficiently determine high-scoring segment pairs or maximally unique matches perform well as a basis of inferring intergenomic distances. The examined distance functions, which are able to cope with heavily reduced genomes and repetitive sequence regions, outperform previously described ones regarding the correlation with and error ratios in emulating DDH. Simulation of incompletely sequenced genomes indicates that some distance formulas are very robust against missing fractions of genomic information. Digitally derived genome-to-genome distances show a better correlation with 16S rRNA gene sequence distances than DDH values. The future perspectives of genome-informed taxonomy are discussed, and the investigated methods are made available as a web service for genome-based species delineation.     

Biotransformations of 1',2'-dihydrorotenone by Streptomyces griseus.

Dihydrorotenone yields three major products when incubated with growing cultures of Streptomyces griseus. These were isolated by solvent extraction and characterized by spectral methods as 1',2'-dihydro-6abeta-hydroxyrotenone, 1',2'-dihydro-2',6abeta-dihydroxyrotenone, and 1',2'-dihydro-1',6abeta-dihydroxyrotenone.     

Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.Abbreviation PBP penicillin-binding protein     

Hybridization of A-factor deficient mutants of Streptomyces griseus by means of fusion of protoplasts

Characteristics of 6 A-factor deficient mutants of S. griseus are presented. The common feature of the mutants was impairment of sporulation, formation of aerial mycelium and streptomycin synthesis. Pair-by-pair hybridization of the mutants was performed with protoplast fusion followed by regeneration. 9 pair couplings of the mutants were performed. In 3 of them sporulating recombinants were detected. The antibiotic production level in 70 hybrids was different and ranged from 0 to 1700 micrograms/ml. The morphological features of the colonies and the number of the spores formed were also different. The common feature of all the 70 sporulating hybrid strains was recovery of synthesis of A-factor, an endogenic regulator of S. griseus development. Therefore, in the A-factor deficient mutants impairment of A-factor synthesis was induced not by the plasmid elimination, as was suggested, but by mutation of separate genes.     

Mismatching base-pair dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNAs consisting of complementary base pairs except for one mismatching base pair (MM1) can form double-stranded DNA by molecular recognition. This type of duplex is not as stable as that formed by MM0. In order to add to a better understanding of the physical mechanism of the hybridization and dissociation processes at sensor (chip) surfaces, we studied the kinetics of the MM1 hybridization by surface plasmon fluorescence spectroscopy. Target DNA strands labelled with a fluorescent molecule Cy5 at the 5′ end and hybridizing with the surface-attached probe DNA can be excited by the strong optical field of a surface plasmon resonance mode. The emitted fluorescence can be detected with high sensitivity. The affinity of a duplex was found to depend on the chemical nature, i.e. G–G, G–T etc., and on the position of the mismatching base pair along the 15mer duplex.     

Biosynthesis of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus.

Biosynthesis of N-methyl-L-glucosamine moiety of streptomycin from D-glucose by Streptomyces griseus was studied. A mixture of D-[1-(14) C] glucose and D-[6(-3)H]glucose was given to the culture of S. griseus. The 3H/14C ratio found in N-methyl-L-glucosamine further supports a mechanism that the conversion of D-glucose to L-hexose is carried out without scission of carbon skeleton. When D-[1-14C]glucose and D-[3-3H]glucose were used, the fall of 3H/14C ratio in N-metyl-L-glucosamine showed that the hydrogen atom at C-3 plays a r?le in such a transformation.     

Intergeneric hybridization between Erucastrum canariense and Brassica rapa. Genetic relatedness between E(C) and A genomes

An intergeneric hybrid between a wild species, Erucastrum canariense (2n = 18; E^CE^C), and a cultivated oilseed brassica species, Brassica rapa (2n = 20; AA), was synthesized through ovary culture in White's basal medium supplemented with casein hydrolysate. Morphological, cytological and DNA-based analysis helped to establish the hybrid nature of the derived plants. Hybrid plants were morphologically intermediate between the two parents and were completely male, as well as female sterile. Cytological analysis revealed the occurrence of 19 I in about 38% of the PMCs investigated. However 1-8 bivalents/PMC were also observed, indicating a significant level of homology between the two genomes. Normal chromosome pairing and pollen fertility was restored following colchiploidy. The intergeneric amphiploid developed during the investigation can be used as a bridging species for the transfer of desirable genes from E^C to cultivated genomes (especially A and C), and for resistance to Alternaria blight and mustard aphid. Under field conditions, the E. canariense intergeneric hybrid and the amphiploid appeared to be moderately resistant to Alternaria blight and also harboured a significantly lower population of mustard aphid than the cultivated B. rapa.     

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.     

Detection and changes of nucleotide pyrophosphotransferase activity of Streptomyces griseus strains in submerged culture.

Nucleotide pyrophosphotransferase (NPT) activity of two Streptomyces griseus strains was studied in submerged culture during their life cycle. NPT activity could be detected only in the culture filtrate but not in the membrane fraction or in cell extract of the sporulating (streptomycin-non-producing) S. griseus No. 45-H. No enzyme could be detected in the non-sporulating (streptomycin-producing) S. griseus No 52--1 cultures.     

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.