Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.     

Ectomycorrhizal inoculum potential of northeastern US forest soils for American chestnut restoration: results from field and laboratory bioassays

American chestnut (Castanea dentata) was once a dominant overstory tree in eastern USA but was decimated by chestnut blight (Cryphonectria parasitica). Blight-resistant chestnut is being developed as part of a concerted restoration effort to bring this heritage tree back. Here, we evaluate the potential of field soils in the northern portion of the chestnut's former range to provide ectomycorrhizal (EM) fungus inoculum for American chestnut. In our first study, chestnut seedlings were grown in a growth chamber using soil collected from three sites dominated by red oak (Quercus rubra) as inoculum and harvested after 5 months. Of the 14 EM fungi recovered on these seedlings, four species dominated in soils from all three sites: Laccaria laccata, a Tuber sp., Cenococcum geophilum, and a thelephoroid type. Seedlings grown in the nonsterilized soils were smaller than those growing in sterilized soils. In the second study, chestnut seedlings were grown from seed planted directly into soils at the same three sites. Seedlings with intermingling roots of established trees of various species were harvested after 5 months. Seventy-one EM fungi were found on the root tips of the hosts, with 38 occurring on chestnut seedlings. Multiple versus single host EM fungi were significantly more abundant and frequently encountered. The fungi observed dominating on seedlings in the laboratory bioassay were not frequently encountered in the field bioassay, suggesting that they may not have been active in mycelial networks in the field setting but were in the soils as resistant propagules that became active in the bioassay. These results show that soil from red oak stands can be used to inoculate American chestnut with locally adapted ectomycorrhizal fungi prior to outplanting, a relatively cost effective approach for restoration efforts.     

Physical mapping of rDNA sites in possible diploid progenitors of polyploid Festuca species

Festuca species form a polyploid series but only two of the diploid species have been firmly proposed as progenitors of any polyploid. The number and distribution of rDNA sites on the chromosomes of F. scariosa (section Scariosae) and the four diploid species that comprise section Montanae are presented with their relative DNA amounts and key morphological features. Comparisons of the results with those of some polyploid Festuca species from section Bovinae published previously indicate that F. scariosa and F. altissima could be diploid progenitors of the polyploids. It is unlikely that any one of the other three Montanae species is a progenitor of these polyploids.     

Taxonomy and ecology of <Emphasis Type="Italic">Castanea sativa</Emphasis> Mill. forests in Greece

Castanea sativa Mill. is an important species of the Balkan Peninsula with high ecological and economic value. This article contributes to a phytosociological synsystematic approach of the C. sativa plant communities in Greece that covers the southern part of the Balkan Peninsula and describes the ecological parameters involved in their distribution and taxonomy. Phytosociological data have been collected from 14 mountainous areas across continental Greece, which are considered representative of the main forest types of C. sativa in the area. The 14 mountains demonstrate a variety of environmental conditions. Five communities (associations) and seven sub-communities (sub-associations) have been identified and described using TWINSPAN, Corespondence Analysis and the Braun-Blanquet classification scheme. Castanea sativa (chestnut) forests of Greece can be distinguished as three broad groups, defined primarily by their geographical distribution. In northern and central Greece chestnut stands are characterized by their high floristic homogeneity, which is reflected in the identification of only one community per region. Chestnut forests in northern Greece, in particular, exhibit strong floristic similarities with those of the rest of the Balkans. In southern Greece, on the other hand, the southern distribution limit of chestnut in the Balkans, there is a greater floristic diversity between the sampled mountains which is reflected in the presence of three confirmed communities and two sub-associations. Several factors have been identified as important in determining the structure and floristic composition of chestnut forests in Greece. Whilst climate and the grazing regime are influential, the degree of silvicultural management appears to be the most important factor determining the floristic composition of chestnut forests and their long-term sustainability.     

Evolutionary trend in feeding habits of <Emphasis Type="Italic">Girella</Emphasis> (Perciformes: Girellidae)

Although some Girella species are herbivorous, having basically tricuspid teeth, some are omnivorous. To determine the evolutionary trends in feeding habits of Girella, the phylogenetic relationships of several species of Girella were estimated by partially sequencing the mitochondrially encoded NADH dehydrogenase subunit 2 gene, and the dentition and adductor mandibulae complex of each species were examined. The cladogram determined from the mitochondrial DNA analysis indicated that multiple tooth-rows containing incisor-like teeth existed in adults of the ancestral species of Girella, species with a single tooth-row of tricuspid teeth in the adult stage having diverged subsequently on several occasions. The tendinous connections between each section of the adductor mandibulae complex are believed to have been simple in the ancestral species, more complicated connections also having diverged later on several occasions. Multiple tooth-rows containing incisor-like teeth and the simple adductor mandibulae complex are deduced as adaptations to herbivory; on the other hand, a single tooth-row of tricuspid teeth and the complicated adductor mandibulae complex are deduced as adaptations to omnivory. Therefore, the ancestral species of Girella is suggested as having been adapted to herbivory, with species adapted to omnivory having diverged on several subsequent occasions.     

The biogeography of Streptomyces in New Zealand enabled by high-throughput sequencing of genus-specific rpoB amplicons

We evaluated Streptomyces biogeography in soils along a 1200 km latitudinal transect across New Zealand (NZ). Streptomyces diversity was examined using high-throughput sequencing of rpoB amplicons generated with a Streptomyces specific primer set. We detected 1287 Streptomyces rpoB operational taxonomic units (OTUs) with 159 ± 92 (average ± SD) rpoB OTUs per site. Only 12% (n = 149) of these OTUs matched rpoB sequences from cultured specimens (99% nucleotide identity cutoff). Streptomyces phylogenetic diversity (Faith's PD) was correlated with soil pH, mean annual temperature and plant community richness (Spearman's r: 0.77, 0.64 and −0.79, respectively; P < 0.05), but not with latitude. In addition, soil pH and plant community richness both explained significant variation in Streptomyces beta diversity. Streptomyces communities exhibited both high dissimilarity and strong dominance of one or a few species at each site. Taken together, these results suggest that dispersal limitation due to competitive interactions limits the colonization success of spores that relocate to new sites. Cultivated Streptomyces isolates represent a major source of clinically useful antibiotics, but only a small fraction of extant diversity within the genus have been identified and most species of Streptomyces have yet to be described.     

The activity of streptomyces phage-virus in fresh water

Streptomyces (Actinomycetales) host strains and their virulent phages were obtained from a restricted locality, both fresh waters and soils being sampled. There was some evidence that one category of host strains was confined to fresh water and was susceptible to phage there. There were several instances where a particular host strain elicited phages both from fresh water and from soil and the phages from these two environments subsequently exhibited differing polyvalency ranges in the laboratory. The possible significance of this finding is that it hints at the existence of phage ecotypes, with properties and polyvalency ranges adapted to the immediate environment.     

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.     

Studies on the microbial populations of the rhizosphere of big sagebrush (<Emphasis Type="Italic">Artemisia tridentata</Emphasis>)

Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture. New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents. Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist. One such habitat is the rhizosphere of desert shrubs. Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush (Artemisia tridentata) is a source of actinomycetes capable of producing an extensive array of antifungal metabolites. Culturable microbial populations from both the sagebrush rhizosphere and nearby bulk soils from three different sites were enumerated and compared, using traditional plate-count techniques and antibiotic activity bioassays. There were no statistical differences between the relative numbers of culturable non-actinomycete eubacteria, actinomycetes and fungi in the rhizosphere versus bulk soils, but PCR amplification of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soils. A high percentage of actinomycetes produced antimicrobials; and the percentage of active producers was significantly higher among the rhizosphere isolates, as compared with the bulk soil isolates. Also, the rhizosphere strains were more active in the production of antifungal compounds than antibacterial compounds. 16S rRNA gene sequence analysis showed that sagebrush rhizospheres contained a variety of Streptomyces species possessing broad spectrum antifungal activity. Scanning electron microscopy studies of sagebrush root colonization by one of the novel sagebrush rhizosphere isolates, Streptomyces sp. strain RG, showed that it aggressively colonized young sagebrush roots, whereas another plant rhizosphere-colonizing strain, S. lydicus WYEC108, not originally isolated from sagebrush, was a poor colonizer of the roots of this plant, as were two other Streptomyces isolates from forest soil. These results support the hypothesis that the rhizosphere of desert big sagebrush is a promising source of habitat-adapted actinomycetes, producing antifungal antibiotics.     

An initial comparison of growth rates in the rare grass Achnatherum hendersonii and its common associate Poa secunda

The rare grass, Achnatherum hendersonii (Vasey) Barkworth, and the more widely distributed Poa secunda J. S. Presl grow together on shallow soils derived from basaltic rock that are subject to cryogenic disturbance. It has been proposed that the solifluction allows A. hendersonii to persist by inhibiting the competitive ability of P. secunda. A correlate of this proposal is that P. secunda will have a superior growth rate in undisturbed soils. This idea was tested by growing both species in potting soil, both singly and in combination. Under these conditions, P. secunda grew faster and attained a larger above-ground size than A. hendersonii. Thus, one factor allowing the survival of the rare A. hendersonii seems to be its occurrence on sites where the competitive ability of a wide-ranging species is restricted. The survival of A. hendersonii on disturbed sites may also be partly explained by its roots that are apparently adapted to growing where solifluction occurs.     

Repressed multidrug resistance genes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment. Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump. In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans. The resistance genes of three of these systems were cloned and sequenced. Two of them are accompanied by a repressor gene. These MDR gene sequences are found in most other Streptomyces species investigated. Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes.Abbreviations MDR Multidrug resistance     

地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定

药用植物内生放线菌具有合成天然活性化合物的潜力,放线菌新种是寻找新型抗生素先导化合物的一个重要来源。【目的】挖掘药用植物地黄内生放线菌资源,并对地黄轮纹病拮抗菌株leaf-16进行新种鉴定。【方法】本研究采用五步消毒法分离河南道地药材地黄的内生放线菌,以地黄轮纹病原真菌草茎点霉(Phoma herbarum)为指示菌,采用平板对峙法筛选对该病菌有抑制作用的菌株,16S rRNA基因测序发现一株抗地黄轮纹病的放线菌新种leaf-16。通过形态、生理生化、细胞壁化学组分和分子生物学等特征对菌株leaf-16进行多相分类学鉴定。【结果】经平板对峙实验得到8株抗地黄轮纹病的放线菌,其中菌株leaf-16经16S rRNA基因测序、形态比较、生理生化、化学组分和分子生物学以及DNA-DNA杂交分析,确定菌株leaf-16为1株链霉菌新种,并命名为Streptomyces folium。【结论】菌株leaf-16为1株链霉菌新种,具有抑制地黄轮纹病原真菌的活性,为进一步分离新型抗地黄轮纹病的生物制剂奠定物质基础。     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine+cytosine (G+C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

A genetic contribution to the taxonomy ofOenothera sect.Oenothera (including subsectionsEuoenothera,Emersonia, Raimannia andMunzia)

Based on an analysis of results from experimental hybridization, the plants assigned byMunz toOenothera subg.Oenothera and subg.Raimannia, now divided into approximately 76 species, are referred to a single section,Oenothera. This section is in turn divided into five subsections:Euoenothera, Munzia, Raimannia, Emersonia, and an undescribed group of three species related toOenothera pubescens. Euoenothera is maintained in the traditional sense, and includes about 14 species of North America, widely naturalized elsewhere.Munzia consists of 45 species, comprising three series, and native to South America.Raimannia is restricted to a group of approximately 11 North American species.Emersonia comprises four rather heterogenous species of northern Mexico and southern New Mexico, of whichOenothera macrosceles, O. maysillesii, andO. organensis have been described. Within these four subsections, interspecific hybrids can be made in general, although plastid differentiation often leads to incompatibilities. With varying degrees of difficulty, hybrids were produced in all intersectional combinations involvingEuoenothera, Emersonia, Munzia, andRaimannia, the most difficult being those betweenEmersonia andRaimannia. Based on their habit and distribution,Emersonia species, and especiallyOenothera maysillesii, appear to resemble most closely the common ancestor of the section,Euoenothera andMunzia to have been derived from it or its common ancestor, andRaimannia perhaps to be more closely related to the phylogenetic branch that leads toEuoenothera.     

Resource Amendments Influence Density and Competitive Phenotypes of <Emphasis Type="Italic">Streptomyces</Emphasis> in Soil

Carbon from plant rhizospheres is a source of energy for soil microbial communities in native habitats. Soil amendments have been used as a means for deliberately altering soil community composition in agricultural soils to enhance plant health. However, little information is available in agricultural or natural soils on how specific carbon compounds or quantities influence soil microbial communities. Streptomyces are important soil saprophytes noted for their ability to produce antibiotics and influence plant health. To explore how specific types and amounts of carbon compounds influence Streptomyces in soil, glucose, cellulose, and lignin were added alone and in combination with six other carbon substrates of varying complexity to mesocosms of native prairie soil for 9 months at amounts equivalent to natural inputs from plants. Estimated culturable population densities, antibiotic inhibitory phenotypes, and resource utilization profiles were examined for Streptomyces communities from each treatment. The type and quantity of carbon compounds influenced densities, proportions, antibiotic phenotypes, and substrate utilization profiles of Streptomyces. Cellulose and lignin inputs produced the largest Streptomyces densities. Also, Streptomyces communities receiving high-resource inputs were more inhibitory whereas those receiving low-resource inputs used substrates more efficiently. Knowledge of how the availability and quantity of particular carbon compounds influences Streptomyces communities and their function, specifically resource use and inhibitory phenotypes, may be helpful in understanding the roles of resource availability in Streptomyces community dynamics and the potential of Streptomyces to suppress pathogens and enhance plant fitness in native and agricultural soils.

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羊毛硫肽SapB类多肽促进链霉菌形态分化作用的研究

【目的】链霉菌属于革兰氏阳性菌,以复杂的形态分化过程和强大的次级代谢产物合成能力为主要特征。链霉菌的形态分化与次级代谢产物的产生密切相关。Ⅲ型羊毛硫肽SapB能够促进天蓝色链霉菌气生菌丝体形成,暗示这类多肽可以作为靶标用于形态分化改造工程开发。本研究表征了SapB类多肽对多种链霉菌形态分化的影响,为该类多肽的工程化应用提供理论基础。【方法】生物信息学分析多个链霉菌基因组中SapB类多肽的生物合成基因簇,构建SapB类多肽的异源表达载体,利用接合转移方法导入不同链霉菌中进行异源表达,探究SapB类多肽对链霉菌形态分化的影响。【结果】SapB类多肽在不同程度上促进了多个链霉菌由营养菌丝向气生菌丝分化,表现为气生菌丝体数量的增多和分化速度的加快,缩短了链霉菌形态分化周期。【结论】SapB类多肽的过表达有助于缩短链霉菌形态分化周期,可用于针对链霉菌形态分化的工程改造。     

Forest Management and Plant Species Diversity in Chestnut Stands of Three Mediterranean Areas

Over many centuries, chestnut fruits had an important role as food, while chestnut wood was used for local purposes. Today sweet chestnut stands are very common around the western Mediterranean Basin, and it is necessary to analyze the dynamic of plant species diversity in different chestnut stand types (groves and coppices) to guide management strategies that will allow the conservation of biodiversity. Our objective was to analyze consequences on plant species diversity of various management strategies in chestnut stands of three Mediterranean areas, Salamanca (Spain), the Cévennes (France), and Etna volcano (Italy). We found that plant species diversity is different according to management types; it is higher in groves than in coppice stands. We also demonstrated that Castanea sativa cultivated groves were characterized by small heliophillous therophytes. C. sativa abandoned groves, mixed C. sativa–Quercus pyrenaica coppice stands, Q. pyrenaica coppice stands, and young C. sativa coppice stands were characterized by hemicryptophytes with anemochorous dispersal mode and chamaephytes. Medium and old C. sativa coppice stands (that differ by the shoot age) were characterized by phanerophytes with zoochorous dispersal mode. Human perturbations maintain a quite high level of species diversity. In contrast, the abandonment of chestnut stands leads to homogeneous vegetation with decreasing diversity. One solution could be to maintain a landscape mosaic constituted of diverse chestnut stands modified by human activities (groves, cultivated or abandoned, and coppice stands). This could enhance regional plant diversity.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep mutagenesis-selection procedure allowed a genetically stable strain,Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.