Sexual identification of<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>

The absence of sexual dimorphism in Pyrenean desmanGalemys pyrenaicus (E. Geoffroy, 1811) makes it difficult to sex live animals. This paper presents a method of sexing desmans observing their external genitals. Based in a sampling of 49 animals from the north of Spain, it was possible to identify the penis of males, independently of the age or season.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Effectiveness of<Emphasis Type="Italic">Cyanothece</Emphasis> spp. and<Emphasis Type="Italic">Cyanospira capsulata</Emphasis> exocellular polysaccharides as antiadhesive agents for blocking attachment of<Emphasis Type="Italic">Helicobacter pylori</Emphasis> to human gastric cells

The effect of cyanobacterial polysaccharides (from Cyanothece spp. and Cyanospira capsulata) on the binding of Helicobacter pylori to gastric epithelial cells was evaluated. The antiadhesive action on Kato III and HeLa S3 human gastric cell lines was established.     

Isolation and characterization of<Emphasis Type="Italic"> Erythrobacter</Emphasis> sp. strains from the upper ocean

Seven strains of marine aerobic anoxygenic phototrophs belonging to the genus Erythrobacter were isolated. The strains were characterized regarding their physiological and biochemical properties, 16S rDNA and pufM gene sequences, morphological features, substrate preference, as well as pigment and lipid composition. All strains had functional type-2 reaction centers containing bacteriochlorophyll, served by small, light-harvesting complex 1, and were photosynthetically competent. In addition, large pools of carotenoids were found, but only some of the accessory pigments transfer energy to the reaction centers. All of the isolates were facultative photoheterotrophs. They required an organic carbon substrate for growth; however, they are able to supplement a significant fraction of their metabolic requirements with photosynthetically derived energy.Abbreviations BChl Bacteriochlorophyll - Chl Chlorophyll - Erb. Erythrobacter - Erm. Erythromicrobium - FAMEs Fatty acid methyl esters - IRFRR Infrared fast repetition rate - LH1, LH2 Light-harvesting complex 1 and 2, respectively - Por. Porphyrobacter - PUFAs Polyunsaturated fatty acids - Rsb. Roseobacter - RubisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase - µ Growth rate - ₄₇₀ Functional cross-section of the photosynthetic unit at 470 nm     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Selective inhibition of the cyanobacterium, <Emphasis Type="Italic">Microcystis</Emphasis>, by a <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

A Streptomyces strain, NT0401, was isolated from soil that selectively inhibited Microcystis strains but did not affect other microorganisms. Based on its morphology, physiology and 16S rDNA sequence, it was identified as Streptomyces grisovariabilis. The active substance produced by NT0401 was a water-soluble compound with a Mr <1 kDa that was stable over a broad pH range and at 100°C for 20 min. This organism should be a potential environment-friendly strain for control of Microcystis blooms.     

Mutation of<Emphasis Type="Italic">gyrA</Emphasis> and<Emphasis Type="Italic">parC</Emphasis> in clinical isolates of<Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> and its relationship with antimicrobial drugs resistance in Taiwan

Acinetobacter baumannii is a species of non fermentative Gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Herein we have determined the mutation frequency of two hot spot residue 83 in gyrA of gyrase and residue 80 in parC of topoisomerase IV and performed a comparative screen the drug resistance ability in 128 clinical isolates ofAcinetobacter baumannii in Taiwan region. Low frequency of mutation was found (11.7%, 11.7%, and 10.2% ingyrA, parC, or both, respectively). Mutation of these sites was not correlated with drug resistance. Our study suggested that mutation ofgyrA andparC may play a minor role in quinolone resistance and other mechanisms may contribute to the drug resistance ofA. Baumannii.     

Arbuscular mycorrhizal fungi colonize decomposing leaves of<Emphasis Type="Italic"> Myrica parvifolia</Emphasis>,<Emphasis Type="Italic"> M. pubescens</Emphasis> and<Emphasis Type="Italic"> Paepalanthus</Emphasis> sp.

Hyphae and vesicles of arbuscular mycorrhizal fungi (AMF) were found within the decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. at three montane sites in Colombia. Hyphae, vesicles, and arbuscule-like structures were also found within scale-like leaves of the rhizomes of Paepalanthus sp. The litter found in the vicinity of the roots was divided into three decomposition layers. The highest AMF colonization occurred in the most decomposed leaves, which were in close association with roots. In contrast, there were no differences in AMF colonization of roots present in the different decomposition layers. Colonization of decomposing leaves by AMF did not differ between the two closely related species M. parvifolia and M. pubescens, nor between two sites (Guatavita and Zipacón, Colombia) differing in soil fertility. Occurrence of vesicles in decomposing leaves was correlated with abundant AMF extraradical hyphae among the leaves. We propose that AMF enter decomposing leaves mechanically through vascular tissue. As a consequence, AMF are well positioned to obtain and efficiently recycle mineral nutrients released by decomposer microorganisms before their loss by leaching or immobilization in soil.     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Biosynthesis of silver nanoparticles by a new strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. compared with <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">fumigatus</Emphasis>

Locally isolated strains of a thermoalkalotolerant Streptomyces sp. and Aspergillus fumigatus were used for the in vitro biosynthesis of silver nanoparticles from AgNO₃ solutions. An autolysed cell-free culture filtrate from each strain was used, indicating that the formation mechanism depends on intra-cellular components for both organisms, since culture broths had no significant nanoparticle formation potential. Nanoparticle formation was indicated by a change of the solution from colourless or light brown to dark brown after 24 h or more, and UV–visible spectroscopy and x-ray diffraction analysis confirmed the formation by both organisms. The initial formation kinetics were faster with Aspergillus, but formation continued for a longer period with Streptomyces, resulting in higher concentrations after 48 h. Transmission electron microscope images revealed well dispersed nanoparticles with diameters ranging from 15 to 45 nm from A. fumigatus, while those from Streptomyces sp. had a narrower size distribution of 15–25 nm. The higher productivity and preferred narrower size distribution of Streptomyces, together with its well established industrial use, may make it the preferred choice for further optimization studies.     

<Emphasis Type="Italic">Streptomyces tunisialbus</Emphasis> sp. nov., a novel <Emphasis Type="Italic">Streptomyces</Emphasis> species with antimicrobial activity

A novel actinomycete strain designated S2^T was isolated from Tunisian rhizosphere soil of Lavandula officinalis. This isolate exhibited broad spectrum antibacterial activity against several Gram-positive and Gram-negative bacteria and also antifungal activity against yeast and filamentous fungi. The isolate S2^T presents morphological and chemotaxonomic characteristics typical of the members of the genus Streptomyces. Whole cell hydrolysates of S2^T were found to contain LL-diaminopimelic acid. The major fatty acids were identified as C16:0, anteiso-C15:0 and iso-C16:0 whereas the predominant menaquinones were found to be MK-9(H6) and MK-9(H8). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and three unidentified compounds. The G+C content of the genomic DNA was determined to be 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2^T belongs to the genus Streptomyces and is closely related to Streptomyces netropsis DSM 40259^T with 99.86% sequence similarity. Multi-locus sequence analysis (MLSA) based on four house-keeping gene alleles (gyrB, recA, trpB, rpoB) showed that isolate S2^T is closely related to S. netropsis, with an MLSA distance greater than 0.007. The DNA–DNA relatedness between strain S2^T and its near phylogenetic neighbour was 63.6 ± 2.3%, which is lower than the 70% threshold value for delineation of genomic prokaryotic species. This isolate was also distinguished from the type strain S. netropsis DSM 40259^T, using a combination of morphological and physiological features. Based on its phenotypic and molecular properties, strain S2^T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces tunisialbus sp. nov. is proposed. The type strain is S2^T (= JCM 32165^T = DSM 105760^T).     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

New isolates and physiological properties of the<Emphasis Type="Italic"> Aquificales</Emphasis> and description of<Emphasis Type="Italic"> Thermocrinis albus</Emphasis> sp. nov.

The ecology of the Aquificales was studied using a combination of phylogenetic and cultivation approaches. Enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the United States (Yellowstone National Park), Russia (Kamchatka), Italy, Germany, Djibouti, Iceland, and Africa (Lake Tanganyika). Isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. Comparisons of their 16S rRNA gene sequences showed that most of the isolates were new representatives of the major lineages of the Aquificaceae, represented by the genera Aquifex, Thermocrinis, Hydrogenobaculum, and Hydrogenobacter. Isolate HI 11/12, which was obtained from whitish streamers in the Hveragerthi area of Iceland, represents a separate branch within the Aquificaceae. The organism grew at salinities up to 0.7% NaCl and at temperatures up to 89 degrees C. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed whitish streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, sulfur, or thiosulfate as the electron donor under microaerophilic conditions. It represents a second species within the order Thermocrinis, which we name Thermocrinis albus HI 11/12 (DSM 14484, JCM 11386).     

Molecular cloning of the phospholipase D gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. YU100 and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.     

Antibacterial metabolites from the Actinomycete <Emphasis Type="Italic">Streptomyces</Emphasis> sp. P294

The Actinomycete strain P294 was isolated from soil and identified as Streptomyces sp. based upon the results of 16S rRNA sequence analysis. Three compounds obtained from the solid fermentation products of this strain have been determined by 1D, 2D NMR and HRMS experiments. These compounds include two new compounds angumycinones C (1) and D (2), and the known compound X-14881 E (3). All compounds were assayed for antibacterial and nematicidal activity. The results showed the three compounds had different degrees of inhibitory activity against several target bacteria but no significant toxicity against the nematode Caenorhabditis elegans.     

Bioactive compounds from marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. VITPSA as therapeutics

Background

Marine actinomycetes are efficient producers of new secondary metabolites that show different biological activities, including antibacterial, antifungal, anticancer, insecticidal, and enzyme inhibition activities.

Methods

The morphological, physiological, and biochemical properties of the strain Streptomyces sp. VITPSA were confirmed by conventional methods. Antibacterial, anti-oxidant, anti-inflammatory, anti-diabetic, and cytotoxic activities of Streptomyces sp. VITPSA extract were determined. The media were optimized for the production of secondary metabolites. Characterization and identification of secondary metabolites were conducted by high-performance liquid chromatography, gas chromatography-mass spectroscopy, and Fourier transform infrared spectroscopy analysis.

Results

The strain showed significant antibacterial, anti-oxidant, and cytotoxic activities, moderate anti-inflammatory activity, and no satisfactory anti-diabetic activity. The ethyl acetate extract of Streptomyces sp. VITPSA showed maximum antibacterial activity against two gram-positive and gram-negative bacteria at 0.5 mg/mL. The antioxidant potential of the crude extract exhibited strong reducing power activity at 0.5 mg/mL with 95.1% inhibition. The cytotoxic effect was found to be an IC₅₀ of 50 μg/mL on MCF-7 cell lines. Experimental design of optimization by one-factor analysis revealed the most favorable sucrose, yeast extract, pH (7.25), and temperature (28°C) conditions for the effective production of secondary metabolites.

Conclusion

This study revealed that Streptomyces sp. VITPSA is an excellent source of secondary metabolites with various bioactivities.

     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.