Biologically Active Endophytic Streptomycetes from <Emphasis Type="Italic">Nothofagus</Emphasis> spp. and Other Plants in Patagonia

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.     

The evolution of development in Streptomyces analysed by genome comparisons

There is considerable information about the genetic control of the processes by which mycelial Streptomyces bacteria form spore-bearing aerial hyphae. The recent acquisition of genome sequences for 16 species of actinobacteria, including two streptomycetes, makes it possible to try to reconstruct the evolution of Streptomyces differentiation by a comparative genomic approach, and to place the results in the context of current views on the evolution of bacteria. Most of the developmental genes evaluated are found only in actinobacteria that form sporulating aerial hyphae, with several being peculiar to streptomycetes. Only four (whiA, whiB, whiD, crgA) are generally present in nondifferentiating actinobacteria, and only two (whiA, whiG) are found in other bacteria, where they are widespread. Thus, the evolution of Streptomyces development has probably involved the stepwise acquisition of laterally transferred DNA, each successive acquisition giving rise either to regulatory changes that affect the conditions under which development is initiated, or to changes in cellular structure or morphology.     

Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo).

A variety of isolation procedures were carried out to study the involvement of bacteria in the colonisation and biodeterioration of Spanish caves with paleolithic rock art (Altamira and Tito Bustillo). The applied techniques mainly aimed to isolate heterotrophic bacteria such as streptomycetes, nocardioform and coryneform actinomycetes, and other gram-positive and gram-negative bacteria. The results demonstrated that actinomycetes were the most abundant gram-positive bacteria in the caves. Actinomycetes revealed a great taxonomic diversity with the predominant isolates belonging to the genus Streptomyces. Members of the genera Nocardia, Rhodococcus, Nocardioides, Amycolatopsis, Saccharothrix, Brevibacterium, Microbacterium, and coccoid actinomycetes (family Micrococcaceae) were also found.     

PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

Nitrate-nonutilizing mutants ofGibberella zeae (Fusarium graminearum) and their use in determining vegetative compatibility

Twenty-four single-spore isolates ofFusarium graminearum were obtained from scabby wheat seeds or glumes collected from 23 locations in Kansas in 1990. All isolates were sexually fertile and homothallic. Nitrate-nonutilizing (nit) mutants of each isolate were generated on a medium amended with 1.5% KCIO₃. Of 378 mutants, 161 were able to utilize nitrite and hypoxanthine (nit1), 165 utilized hypoxanthine but not nitrite (nit3), 47 utilized nitrite but not hypoxanthine (NitM), and 5 appeared to be global nitrogen regulatory mutants similar to the previously describednnu mutant. Complementation was tested by pairingnit1 mutants of each isolate with either a NitM or anit3 mutant from each isolate on media containing nitrate as the sole nitrogen source. Complementation was more pronounced whennit1 and NitM mutants were paired. Mutants were only able to complement with other mutants from the same wild-type isolate. Therefore, each wild-type isolate belonged to a genetically distinct vegetative compatibility group. The genetic diversity suggests that sexual genetic recombination may be important in the field.     

Selective isolation of bioactive soil actinomycetes belonging to the Streptomyces violaceusniger phenotypic cluster

AIMS: To devise and evaluate a strategy for isolating members of the Streptomyces violaceusniger phenotypic cluster, which are known to be a promising source of bioactive metabolites. METHODS AND RESULTS: The treatment of four soil samples with 1.5% phenol (30 degrees C, 30 min) prior to inoculation on humic acid-vitamin (HV) agar eliminated most of the streptomycetes and other bacterial populations. The surviving streptomycetes on the HV isolation plates were subcultured, and species-group identification was made according to the probabilistic identification system of Williams et al. (1989). Of the 133 streptomycetes subcultured, 102 (77%), were assigned to the S. violaceusniger cluster. A test with an overlay technique revealed that all of these S. violaceusniger-cluster isolates had broad antimicrobial spectra, as they inhibited the growth of all test Gram-positive bacteria, yeasts and filamentous fungi. Antitumour activity against colon carcinoma cells was found among 68 or 67%, of these S. violaceusniger-cluster isolates, following growth in submerged culture. CONCLUSIONS: Chemical pretreatment of soil samples with phenol reduces the growth of ubiquitous Streptomyces species, thereby facilitating the recovery of less-abundant S. violaceusniger-cluster strains that are characterized by high antimicrobial and antitumour activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to selectively isolate rare, bioactive streptomycete groups is important for discovering novel secondary metabolites with bioactive properties.     

Diet, morphology, and interspecific killing in carnivora

Interspecific killing is a key determinant of the abundances and distributions of carnivores, their prey, and nonprey community members. Similarity of body size has been proposed to lead competitors to seek similar prey, which increases the likelihood of interference encounters, including lethal ones. We explored the influence of body size, diet, predatory habits, and taxonomic relatedness on interspecific killing. The frequency of attacks depends on differences in body size: at small and large differences, attacks are less likely to occur; at intermediate differences, killing interactions are frequent and related to diet overlap. Further, the importance of interspecific killing as a mortality factor in the victim population increases with an increase in body size differences between killers and victims. Carnivores highly adapted to kill vertebrate prey are more prone to killing interactions, usually with animals of similar predatory habits. Family-level taxonomy influences killing interactions; carnivores tend to interact more with species in the same family than with species in different families. We conclude that although resource exploitation (diet), predatory habits, and taxonomy are influential in predisposing carnivores to attack each other, relative body size of the participants is overwhelmingly important. We discuss the implications of interspecific killing for body size and the dynamics of geographic ranges.     

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor

Cyclic dimeric GMP(c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase(DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.     

Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L)

The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X(L)) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X(L) using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X(L) prevented all BAX-mediated responses. We also assessed the function of BCL-X(L) and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic beta subunit or the delta subunit of the F(0)F(1)-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho(0)] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the beta subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X(L) protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X(L): cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.     

链霉菌次级代谢调控相关的双组分系统研究进展

链霉菌具有强大的次级代谢能力, 能够产生众多具有生物活性的次级代谢产物, 如目前广泛应用的抗生素、抗肿瘤药物以及免疫抑制剂等。在链霉菌中, 次级代谢产物的生物合成受到包括途径特异性、多效性以及全局性调控基因在内的多层次严格调控。关键调控基因的缺失或过表达可以显著影响次级代谢产物的生物合成, 提示对于链霉菌次级代谢重要调控基因的功能及其作用机制的研究具有巨大的潜在应用价值。其中, 作为细菌信号传导系统的双组分系统(Two-component system, TCS)一直是大家研究的关注点。越来越多的研究表明TCS在链霉菌次级代谢过程中发挥着全局性的调控功能。本文重点介绍链霉菌模式菌株——天蓝色链霉菌中TCS(包括典型TCS)、孤立的组氨酸蛋白激酶(HK)以及应答调控蛋白(RR)参与次级代谢调控的研究进展。这些TCS的功能鉴定及机制解析为工业链霉菌的定向遗传改造以提高重要次级代谢产物的含量提供了理论依据。     

Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants.

The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane. Bacteria were chosen to include penicillin and cephalosporin beta-lactam producers and nonproducers. No significant amount of glutathione was found in any of the streptomycetes. Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17. Those streptomycetes that make beta-lactam antibiotics also produce significant amounts of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis. In Streptomyces clavuligerus, a potent producer of beta-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production. These and other observations indicate that ACV does not function as a protective thiol in streptomycetes. U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV. Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids. This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus. A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes.     

Genetic and phenotypic traits of streptomycetes used to characterize antibiotic activities of field-collected microbes

Although antibiotic production may contribute significantly to microbial fitness, there is limited information on the ecology of antibiotic-producing microbial populations in soil. Indeed, quantitative information on the variation in frequency and intensity of specific antibiotic inhibitory and resistance abilities within soil microbial communities is lacking. Among the streptomycetes, antibiotic production is highly variable and resistance to antibiotics is highly specific to individual microbial strains. The objective of this work was to genetically and phenotypically characterize a reference collection of streptomycetes for use in distinguishing inhibition and resistance phenotypes of field-collected microbes. Specifically, we examined inhibition and resistance abilities of all isolates in all possible pairwise combinations, genetic relatedness using BOX-PCR and 16S rDNA sequence analyses, nutrient utilization profiles, and antibiotic induction among all possible three-way combinations of isolates. Each streptomycete isolate possessed a unique set of phenotypic and genetic characteristics. However, there was little correspondence between phenotypic and genetic traits. This collection of reference isolates provides the potential for distinguishing 1024 inhibition and resistance phenotypes in field-collected microbes. Relationships between the genetic and phenotypic characteristics examined may provide preliminary insight into the distinct strategies that microbes use in optimizing their fitness in natural environments.     

Genetic and biological comparisons of four nucleopolyhedrovirus isolates that are infectious to Adoxophyes honmai (Lepidoptera: Tortricidae)

The smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae), is one of the most important pests of tea plants in Japan. Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) isolates from Tsukuba (AdhoNPV-Ts) and Tokyo (AdhoNPV-To), Japan, and Adoxophyes orana nucleopolyhedrovirus (AdorNPV) isolates from England (AdorNPV-En) and the Netherlands (AdorNPV-Ne) were subjected to genetic and biological comparisons to select a candidate NPV isolate to control A. honmai. Restriction endonuclease (REN) analysis demonstrated that AdhoNPV-Ts and AdhoNPV-To had similar REN patterns, whereas AdorNPV-En and AdorNPV-Ne exhibited different REN patterns from each other as well as those of AdhoNPV-Ts and AdhoNPV-To. Bioassays with fourth-instar A. honmai larvae showed that AdorNPV-En was most pathogenic, with the lowest LD₅₀ of 37 occlusion bodies (OBs) per larva. When A. honmai neonates were inoculated with each isolate, most larvae infected with AdhoNPV-Ts and AdhoNPV-To were killed in the final (fifth)-instar, whereas larvae infected with AdorNPV-Ne were killed at every instar and larvae infected with AdorNPV-En were killed at the first- to third-instar. AdorNPV-En or AdhoNPV-Ts fed to neonates had the shortest or longest killing times, respectively, with ST₅₀ values of 6 and 19 days. AdhoNPV-To and AdorNPV-Ne had intermediate killing times. The OB yield per larva of AdhoNPV-Ts and AdhoNPV-To was significantly higher than that of AdorNPV-En and AdorNPV-Ne. Our results suggest that AdorNPV-En is suitable as an inundative agent because it is a quick-killing, highly virulent NPV, and AdhoNPV-Ts and AdhoNPV-To are more appropriately used as inoculative agents because of their high OB production.     

Friends and foes: streptomycetes as modulators of plant disease and symbiosis

The ecological role of soil streptomycetes within the plant root environment is currently gaining increased attention. This review describes our recent advances in elucidating the complex interactions between streptomycetes, plants, pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-associated microbial communities. Some act as biocontrol agents, inhibiting plant interactions with pathogenic organisms. Owing to the antagonistic properties of streptomycetes, they exert a selective pressure on soil microbes, which may not always be for plant benefit. Others promote the formation of symbioses between plant roots and microbes, and this is in part due to their direct positive influence on the symbiotic partner, expressed as, e.g., promotion of hyphal elongation of symbiotic fungi. Recently, streptomycetes have been identified as modulators of plant defence. By repressing plant responses to pathogens they facilitate root colonisation with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or enhance plant growth. In conclusion, while streptomycetes have a clear potential of acting as biocontrol agents, care has to be taken to avoid strains that select for virulent pathogens or enhance disease development. We argue towards the use of an integrated screening approach in the search for efficient biocontrol agents, including assays on in vitro antagonism, plant growth, and disease suppression.     

Biotypic diversity in Colorado Russian wheat aphid (Hemiptera: Aphididae) populations

The biotypic diversity of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), was assessed in five isolates collected in Colorado. Three isolates, RWA 1, RWA 2, and an isolate from Montezuma County, CO, designated RWA 6, were originally collected from cultivated wheat, Triticum aestivum L., and obtained from established colonies at Colorado State University. The fourth isolate, designated RWA 7, was collected from Canada wildrye, Elymus canadensis L., in Baca County, CO. The fifth isolate, designated RWA 8, was collected from crested wheatgrass, Agropyron cristatum (L.) Gaertn., in Montezuma County, CO. The four isolates were characterized in a standard seedling assay, by using 24 plant differentials, 22 wheat lines and two barley, Hordeum vulgare L., lines. RWA 1 was the least virulent of the isolates, killing only the four susceptible entries. RWA 8 also killed only the four susceptible entries, but it expressed intermediate virulence on seven wheat lines. RWA 6, killing nine entries, and RWA 7, killing 11 entries, both expressed an intermediate level of virulence overall, but differed in their level of virulence to 'CO03797' (Dn1), 'Yumar' (Dn4), and 'CO960293-2'. RWA 2 was the most virulent isolate, killing 14 entries, including Dn4- and Dny-containing wheat. Four wheat lines, '94M370' (Dn7), 'STARS 02RWA2414-11', CO03797, and 'CI2401', were resistant to the five isolates. The results of this screening confirm the presence of five unique Russian wheat aphid biotypes in Colorado.     

不同生境放线菌的卤化酶基因分析及其对卤代产物筛选的意义

摘要：【目的】在基因水平上分析并比较陆地来源与海洋来源的放线菌产生卤化代谢产物的潜力。【方法】基于依赖黄素腺嘌呤二核苷酸的卤化酶基因筛选,从经过表型去重复的70株陆地来源和71株海洋来源的放线菌中,通过PCR筛选获得卤化酶基因片段,并进行测序鉴定;通过卤化酶氨基酸序列的系统发育分析,比较不同来源放线菌的卤化酶序列,以及海洋链霉菌和小单孢菌的卤化酶序列。另外,对卤化酶阳性菌株进行了聚酮合酶和非核糖体多肽合成酶基因的检测。【结果】本研究中36.6%的海洋放线菌具有卤化酶基因,其阳性率远高于本研究所涉及的陆地放     

Alkaliphilic Soil Actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

Screening and Production of Arylsulfatases for Target Therapy with Etoposide 4′-Sulfate,an Antitumor Prodrug

Two arylsulfatase-producing streptomycetes that desulfated etoposide 4′-sulfate were isolated from soil samples. Taxonomical study identified one soil isolate as Streptomyces griseorubiginosus S980-14 (Es-1 arylsulfatase producer), while the other was considered new and tentatively designated Streptomyces sp. T109-3 (Es-2 arylsulfatase producer). Both strains produced extracellular arylsulfatase activities, provided that cultivation media were prepared with distilled water. Unlike the two known types of arylsulfatases, which had significant activity on p-nitrophenyl sulfate but none on etoposide 4′-sulfate, the crude streptomycete arylsulfatases efficiently desulfated etoposide 4′-sulfate and p-nitrophenyl sulfate, which supports the establishment of a new type of arylsulfatases.     

The SsgA-like proteins in actinomycetes: small proteins up to a big task

Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been successfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family.