Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium . Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, β-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.     

Oxyanthraquinones and flavonoids from garland chrysanthenum

The stems of the vegetable plant garland chrysanthemum (Chrysanthemum coronarium L.) were shown to contain emodin (in its aglycon and glycoside forms) and chrysophanol. Chrysophanol and chrysazin were isolated from the roots of the plant. Because the pigments identified are derivatives of 1,8-dihydroxyanthroquinone, garland chrysanthemum may be a medicinal plant and have utility as a component of laxative species. The leaves of C. coronarium were shown to be rich in quercetin and its glycosides, rutin and isoquercetin. Taken together, this observation and the known high content of ascorbic acid and carotenoids in the plant suggest that C. coronarium may be useful in preventing cardiac and vascular diseases.     

Exiguobacterium: an overview of a versatile genus with potential in industry and agriculture

The bacterial genus Exiguobacterium accommodates many versatile species isolated from diverse environments. Exiguobacterium was described as a genus approximately three decades ago, and now, 17 species, growing over a broad range of temperatures and pH, have been recognized. Various isolates from different niches have been explored for biotechnological and industrial purposes, including enzyme production, bioremediation and degradation of toxic substances released into the environment. Some isolates possess plant growth promoting capabilities, and they are currently being explored for increasing agricultural production. The genome sequences of various strains of this genus have shown the presence of many genes encoding products of importance to agriculture and the environment. In addition, many strains possess stress-responsive genes helping them to colonize and thrive in diverse ecological niches. This review provides a broad view of the versatile genus Exiguobacterium and its potential for applications in agriculture, the environment and industry, as well as the underlying genomic determinants that drive its diversity and adaptability to various extreme environments.     

Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity

AIMS: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media. METHODS AND RESULTS: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates). CONCLUSIONS: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.     

Phylogenetic characterization of culturable actinomycetes associated with the mucus of the coral Acropora digitifera from Gulf of Mannar

The marine environment is a virtually untapped source of novel actinomycete diversity and its metabolites. Investigating the diversity of actinomycetes in other marine macroorganisms, like seaweeds and sponges, have resulted in isolation of novel bioactive metabolites. Actinomycetes diversity associated with corals and their produced metabolites have not yet been explored. Hence, in this study we attempted to characterize the culturable actinomycetes population associated with the coral Acropora digitifera. Actinomycetes were isolated from the mucus of the coral wherein the actinomycetes count was much higher when compared with the surrounding seawater and sediment. Actinobacteria-specific 16S rRNA gene primers were used for identifying the isolates at the molecular level in addition to biochemical tests. Amplified ribosomal DNA restriction analysis using three restriction enzymes revealed several polymorphic groups within the isolates. Sequencing and blast analysis of the isolates revealed that some isolates had only 96.7% similarity with its nearest match in GenBank indicating that they may be novel isolates at the species level. The isolated actinomycetes exhibited good antibacterial activity against various human pathogens. This study offers for the first time a prelude about the unexplored culturable actinomycetes diversity associated with a scleractinian coral and their bioactive capabilities.     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.     

Symbiotic incompatibility between two forage species of Hedysarum, grown in Morocco, and their homologous rhizobia

Abstract Symbiotic experiments in the glasshouse demonstrated that two species of sulla, Hedysarum coronarium and Hedysarum flexuosum , grown in Morocco were mutually incompatible in their requirements for effectively nitrogen-fixing strains of rhizobia: nitrogen-fixing Rhizobium strains isolated from H. coronarium nodulated H. flexuosum but did not fix nitrogen, conversely strains from H. flexuosum were ineffective for H. coronarium . The agronic implications are discussed.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.     

A study of the polymorphism of mec dna in methycillin-resistant strains of Staphylococcus aureus isolated at permanent stations in different regions of Russia

Polymorphism of the chromosome staphylococcus cassette mec (SCCmec), a mobile and heterological genetic element providing resistance to beta-lactam antibiotics was studied in methycillin-resistant strains of Staphylococcus aureus (MRSA) isolated at permanent stations situated in different regions of Russia. Type SCCmec was identified using the PCR method by determining allotypes of 3 different structural genetic complexes incorporated in the cassettes mec. It was found that the isolates studied in this work contained 3 different types of SCCmec: I, III, and IVb. Both isolates containing 2 different copies of SCCmec and isolates containing defective copies of SCCmec were identified. It was demonstrated that determination of the SCC-mec type provided an opportunity to differentiate the isolates studied in this work from one another. The isolates attributed to the same genotype variant (identified by polymorphism of coagulase gene) but isolated at different hospitals located in different regions of Russia were found to contain the same type of the chromosome staphylococcus cassette mec, whereas the isolates of different coagulase groups (i.e., different genotype variants) contained different types of SCCmec. It was found that at least 2 epidemic strains circulated in the permanent hospitals of Russia. The strains differ from one another by the polymorphism of the coagulase gene and the mec DNA polymorphism. According to results of studies of several molecular markers (including mec DNA), these strains proved to be identical to the international strains EMRSA-1 and EMRSA-2. Possible mechanisms of MRSA formation and circulation in Russia and CIS countries are discussed.     

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry)

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.     

Phylogenetic and phenotypic diversity of 4-chlorobenzoate-degrading bacteria isolated from soils

Twenty numerically dominant 4-chlorobenzoate (4-CBA)-degrading bacteria were isolated from agricultural soils. The isolates were able to utilize 4-CBA as a sole source of carbon and energy. A total of 65% of the isolates was identified to the species level by fatty acid methyl ester (FAME) analysis, and the isolates were strains of Micrococcus, Pseudomonas, Oerskovia, Cellulomonas, and Arthrobacter species. The chromosomal DNA patterns of the isolates obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from each other. Most of the isolates grew rapidly in 4-CBA medium, but their substrate utilization capabilities were generally restricted. Plasmid DNAs were detected from 55% of the isolates, and one strain, HR7, was shown to have self-transmissible, 4-CBA degradative plasmids. 4-CBA degradative enzymes were inducible by the presence of 4-CBA and most of the isolates appeared to mineralize it through 4-hydroxybenzoate rather than 4-chlorocatechol.     

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry).

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.     

A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents

Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clusters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. thermoleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus. The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T.     

A phylogenetic analysis of micro-organisms isolated from subsurface environments

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis— a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria— including the pseudomonads and Agrobacterium tumefaciens— or the low-G+C Gram-positive bacteria.     

Phylogenetic diversity of aerobic saprotrophic bacteria isolated from the Daqing oil field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G + C content of DNA and gram-negative bacteria of the gamma-subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Microbial diversity in ikaite tufa columns: an alkaline,cold ecological niche in Greenland

Ikaite tufa columns from the Ikka Fjord in south-western Greenland constitute a natural, stable environment at low temperature and with a pH ranging from neutral at the exterior to very alkaline (pH 10.4) at the interior of the column. Phylogenetic analysis of culturable organisms revealed ten different isolates representing three of the major bacterial divisions. Nine of the isolates showed 94-99% similarity to known sequences, whereas one isolate displayed a low degree of similarity (less than 90%) to a Cyclobacterium species. Seven of the isolates were shown to be cold active alkaliphiles, whereas three isolates showed optimal growth at neutral pH. Phylogenetic analysis of DNA isolated directly from the ikaite material demonstrated the presence of a microbial flora more diverse than the culturable isolates. Whereas approximately half of the phylotypes showed 90-99% similarity to known meso- or thermophilic alkaliphiles, the rest of the sequences displayed less than 90% similarity when compared to known 16S rRNA gene sequences in databases. Thus, in the present paper, we demonstrate that ikaite columns that host a specialized macroscopic flora and fauna also contain a unique, cold active, alkaliphilic microflora.     

Rep-PCR应用于快速鉴定啤酒污染菌的研究

为评价rep-PCR在快速鉴定啤酒污染菌中的应用,首先比较了DNA提取方法,确定CTAB法作为制备rep-PCR的DNA模板的方法。并通过PCR产物直接测序的方法,从分离菌中鉴定得到11种常见的啤酒污染菌。用BOXA1R和(GTG)_5引物扩增分离菌,采用Gel ComparⅡ软件处理电泳图,构建污染菌的标准指纹图库。经过聚类分析表明,BOXA1R和(GTG)5对Lactobacillusbrevis、L.buchneri、L.casei/paracasei、L.plantarum和L.fermentum的聚类效果具有互补性,并首次提出指纹比对快速鉴定的相似系数阈值的概念。对来自三个不同来源的9株乳酸菌的快速鉴定结果表明,rep-PCR鉴定技术简单、快速、可靠,在快速鉴定方面将具有重要的应用价值。     

Alkalitolerant micromycetes in acidic and neutral soils of the temperate zone

A wide diversity of micromycetes from various taxonomic groups in acidic and neutral soils is known from the literature data. In the present work, the fungi isolated from these soils and capable of growth at high pH are analyzed. The fungi were isolated from acidic sod-podzol and neutral cultivated soils by plating on alkaline agar (pH 10.0–10.5). Their identification was carried out using morphological, cultural, and molecular genetic criteria. Phylogenetic analysis was performed and the rates of linear growth within a broad pH range (4.0–10.4) were determined. The isolates represented a polyphyletic group of ascomycetes (Sordariomycetes), which included members of Plectosphaerellaceae (5 species) and various families of Hypocreales (4 species). The most common species were Gibellulopsis nigrescens, Acrostalagmus luteoalbus, Chordomyces antarcticum, and Plectosphaerella spp. Investigation of fungal growth at different pH values revealed all isolates to be alkalitolerant, with no alkaliphilic fungi isolated from acidic sod-podzol and neutral cultivated soils. Although the group of isolates was polyphyletic and its members originated from different ecological and trophic niches, most alkalitolerant isolates exhibited common morphological traits with acremonium- and verticillium-like conidial spore formation, abundant slime formation, and a tendency for aggregation of their mycelium in bundles. Our research confirmed the presence of fungi with alkalitolerant adaptation to external pH in the sod-podzolic and cultivated soils of the Moscow region.