Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Methanotrophic Community Detected by DNA-SIP at Bertioga's Mangrove Area, Southeast Brazil.

Methanotrophic bacteria can use methane as sole carbon and energy source. Its importance in the environment is related to the mitigation of methane emissions from soil and water to the atmosphere. Brazilian mangroves are highly productive, have potential to methane production, and it is inferred that methanotrophic community is of great importance for this ecosystem. The scope of this study was to investigate the functional and taxonomic diversity of methanotrophic bacteria present in the anthropogenic impacted sediments from Bertioga´s mangrove (SP, Brazil). Sediment sample was cultivated with methane and the microbiota actively involved in methane oxidation was identified by DNA-based stable isotope probing (DNA-SIP) using methane as a labeled substrate. After 4 days (96 h) of incubation and consumption of 0.7 mmol of methane, the most active microorganisms were related to methanotrophs Methylomonas and Methylobacter as well as to methylotrophic Methylotenera, indicating a possible association of these bacterial groups within a methane-derived food chain in the Bertioga mangrove. The abundance of genera Methylomonas, able to couple methane oxidation to nitrate reduction, may indicate that under low dissolved oxygen tensions, some aerobic methanotrophs could shift to intraerobic methane oxidation to avoid oxygen starvation.

Impact of Inoculation with Pseudomonas aestus CMAA 1215T on the Non-target Resident Bacterial Community in a Saline Rhizosphere Soil.

Plant growth reduction caused by osmotic stress, pathogens, and nutrient scarcity can be overcome by inoculation with plant growth-promoting rhizobacteria (PGPR). Knowing the effects of PGPR on the microbial community beyond those on plant growth can bring new options of soil microbiota management. The present study aimed to investigate the effect of inoculation with the newly described Pseudomonas aestus CMAA 1215T [a 1-aminocyclopropane-1-carboxylate (ACC) deaminase and glycine-betaine producer] on the rhizosphere bacterial community of Zea mays in natural (non-salinized) and saline soil. The bacterial community structure was assessed by sequencing the V6-V7 16S ribosomal RNA using the Ion Personal Genome Machine™. The non-metric multidimensional scaling (NMDS) of the OTU profile (ANOSIM P < 0.01) distinguishes all the treatments (with and without inoculation under saline and natural soils). Inoculated samples shared 1234 OTUs with non-inoculated soil. The most abundant classes in all samples were Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Acidobacteriia, Bacteroidia, Thermoleophilia, Verrucomicrobiae, Ktenodobacteria, and Bacilli. The inoculation, on the other hand, caused an increase in the abundance of the genera Bacillus, Bryobacter, Bradyrhizobium, "Candidatus Xiphinematobacter", and "Candidatus Udaeobacter" independent of soil salinization. "Candidatus Udaeobacter" has the largest Mean Decrease in Gini Values with higher abundance on inoculated salted soil. In addition, Pseudomonas inoculation reduced the abundance of Gammaproteobacteria and Phycisphaerae. Understanding how inoculation modifies the bacterial community is essential to manage the rhizospheric microbiome to create a multi-inoculant approach and to understand its effects on ecological function.

Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity.

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Williamsia aurantiacus sp. nov. a novel actinobacterium producer of antimicrobial compounds isolated from the marine sponge.

An antibiotic-producing actinobacterium, designated isolate B375T, was isolated from marine sponge Glodia corticostylifera collected from Praia Guaecá, São Paulo, Brazil (23°49S; 45°25W), and its taxonomic position established using data from a polyphasic study. The organism showed a combination of morphological, physiological, biochemical and chemotaxonomic characteristics consistent with its classification in the genus Williamsia. Comparative 16S rRNA gene sequence analysis indicated that the strain B375T was most closely related to Williamsia serinedens DSM 45037T and Williamsia spongiae DSM 46676T and having 99.43% and 98.65% similarities, respectively, but was distinguished from these strains by a low level of DNA-DNA relatedness (53.2-63.2%) and discriminatory phenotypic properties. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and N-glycolated muramic acid residues present in the wall cells. The cells contained C16:0 (23.3%), C18:0 10-methyl (23.2%) and C18:1 ω9c (21.6%) as the major cellular fatty acids. The strain B375T inhibited growing of Staphylococcus aureus and Colletotrichum gloeosporioides strains and was considered a producer of antimicrobial compounds. Based on the data obtained, the isolate B375T (= CBMAI 1090T = DSM 46677T) should, therefore, be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia aurantiacus sp. nov. is proposed.

Streptomyces rhizosphaericola sp. nov., an actinobacterium isolated from the wheat rhizosphere.

A novel actinobacterium, designated strain 1AS2cT, was isolated from the wheat rhizosphere collected from the Brazilian Cerrado biome. A polyphasic approach, including phenotypic characterization and phylogenetic multilocus sequence analysis (MLSA), was used to determine the taxonomic position of this strain. Analysis of the 16S rRNA gene sequence indicated that the novel strain is closely related to Streptomyces cavourensis NBRC 13026T, Streptomyces albolongus NBRC 13465T and Streptomyces araujoniae ASBV-1T, sharing a similarity value of 98.2, 98.1 and 97.9 %, respectively. Additionally, MLSA of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) showed evolutionary distances beyond the 0.007 threshold, as well as low DNA-DNA relatedness between strain 1AS2cT and its closest phylogenetic neighbours (S. cavourensis NBRC 13026T, S. albolongus NBRC 13465T and S. araujoniae ASBV-1T: 56, 62.5 and 63.0 %, respectively), indicating a new phylogenetic lineage. The phylogenetic, chemotaxonomic and phenotypic characteristics support the assignment of strain 1AS2cT to the genus Streptomyces, representing a novel species. It is concluded that strain 1AS2cT (=CMAA 1679T=NRRL B-65479T=DSM 105299T) can be classified as the type strain of a novel species of the genus Streptomyces, for which the name Streptomyces rhizosphaericola sp. nov. is proposed.

Amazoninema gen. nov., (Synechococcales, Pseudanabaenaceae) a novel cyanobacteria genus from Brazilian Amazonian rivers.

The genus Leptolyngbya includes morphotypes with thin cells and simple morphology, and is one of the most common cyanobacterial genera found in a wide range of environments. In many cases, however, the morphotypes assigned to this genus do not share a common ancestor based on 16S rRNA gene phylogeny, which has led to the description of novel genera, such as Nodosilinea, Oculatella, Pantanalinema, Alkalinema, Thermoleptolyngbya, Onodrimia, Timaviella and Toxifilum. Thus, four novel isolates, with a comparable morphology to Leptolyngbya, were recovered from the Amazon and Solimões rivers. The novel 16S rRNA gene sequences obtained from these strains were placed together as a new and distinct phylogenetic lineage that is more closely related to the clusters embracing the genera Nodosilinea, Haloleptolyngbya and Halomicronema than to the genus Leptolyngbya. Additionally, these novel 16S rRNA gene sequences showed similarity values lower than 95 % compared with those from the most phylogenetic related groups and/or established genera. Altogether, these results supported the erection of a novel genus, named Amazoninema, to accommodate the novel isolates. Likewise, a comparison of their 16S rRNA gene sequences revealed similarities higher than 99.8 %, indicating that they belong to a single species, which was corroborated by analysing their 16S-23S internal transcribed spacer regions and unique Box-B helix pattern. Few studies have been undertaken to uncover the cultured diversity of cyanobacteria from Amazonia, and to our knowledge, this is the first cyanobacteria genus erected, considering morphotypes isolated exclusively from Brazilian Amazonian rivers.

Bioguided isolation, characterization and media optimization for production of Lysolipins by actinomycete as antimicrobial compound against Xanthomonas citri subsp. citri.

Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8 µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.

Higher phylogenetic diversity prevents loss of functional diversity caused by successive drying and rewetting cycles.

Microbial communities regulate nutrient cycling in soil, thus the impact of climate change on the structure and function of these communities can cause an imbalance of nutrients in the environment. Structural and functional changes of soil bacterial communities in two contrasting biomes in Brazil, the Atlantic Forest and the Tropical Dry Forest (Caatinga), were studied by simulating, in microcosms, rainfall and drought events. Soil samples were collected in three Brazilian states: Bahia, Pernambuco and São Paulo, in a total of four sampling sites. Analysis of 16S rRNA amplicon libraries revealed changes in microbial communities after three drying-rewetting cycles (60-30% water holding capacity). Alpha diversity indexes were obtained for bacterial communities, as well as the functional diversity index (Shannon) based on the activity of the following enzymes: acid and alkaline phosphatase, arylsulfatase, dehydrogenase, cellulase, amylase, urease and phytase. In general, the soils of Caatinga showed a decrease in the diversity indexes studied, conversely, however, the soils of Atlantic Forest were found to be more resistant during the drying-rewetting cycles. Functional diversity was significantly different for the two biomes, with a decrease in Caatinga soils, while Atlantic Forest samples demonstrated a greater stability of enzymatic activity. Further, the Atlantic Forest samples showed more resistance when compared to samples from Caatinga. The results found in this study have confirmed the hypothesis that biomes, independent of climate, when subjected to successive events of drought and rewetting exhibit structural and metabolic changes.

Rhodococcus psychrotolerans sp. nov., isolated from rhizosphere of Deschampsia antarctica.

A novel actinobacterium, designated strain CMAA 1533T, was isolated from the rhizosphere of Deschampsia antarctica collected at King George Island, Antarctic Peninsula. Strain CMAA 1533T was found to grow over a wide range of temperatures (4-28 °C) and pH (4-10). Macroscopically, the colonies were observed to be circular shaped, smooth, brittle and opaque-cream on most of the culture media tested. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CMAA 1533T belongs to the family Nocardiaceae and forms a distinct phyletic line within the genus Rhodococcus. Sequence similarity calculations indicated that the novel strain is closely related to Rhodococcus degradans CCM 4446T, Rhodococcus erythropolis NBRC 15567T and Rhodococcus triatomae DSM 44892T (≤ 96.9%). The organism was found to contain meso-diaminopimelic acid, galactose and arabinose in whole cell hydrolysates. Its predominant isoprenologue was identified as MK-8(H2) and the polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The major fatty acids were identified as Summed feature (C16:1 ω6c and/or C16:1 ω7c), C16:0, C18:1 ω9c and 10-methyl C18:0. The G+C content of genomic DNA was determined to be 65.5 mol%. Unlike the closely related type strains, CMAA 1533T can grow at 4 °C but not at 37 °C and was able to utilise adonitol and galactose as sole carbon sources. Based on phylogenetic, chemotaxonomic and physiological data, it is concluded that strain CMAA 1533T (= NRRL B-65465T = DSM 104532T) represents a new species of the genus Rhodococcus, for which the name Rhodococcus psychrotolerans sp. nov. is proposed.

Phylogenetic insights into the diversity of homocytous cyanobacteria from Amazonian rivers.

The Amazon Rainforest holds great tropical biodiversity, mainly because of its favourable climatic conditions. The high temperatures, luminosity and humidity coupled with the nutritional simplicity of cyanobacteria allow undiscovered diversity to flourish within this group of microorganisms. Some efforts to reveal this diversity have been attempted; however, most were focused on the microscopic observation of environmental samples without any genetic information. Very few studies focusing on morphological, ecological and molecular criteria have been conducted, and none have been devoted to homocytous cyanobacteria forms in Amazonia region. Therefore, the genetic relationships amongst strains retrieved from this ecosystem with regard to other environments from Brazil and the world have not been tested and, consequently, the Amazonian strains would naturally be assumed as novel to science. To examine these relationships, cultured homocytous cyanobacteria isolated from two Amazonian rivers (Amazonas and Solimões) were evaluated using a phylogenetic perspective, considering the 16S rRNA gene sequence. A total of eleven homocytous cyanobacterial strains were isolated. Morphologically, they were identified as Pseudanabaena, Leptolyngbya, Planktothrix and Phormidium, but genetically they were included in the typical clusters of Planktothrix, Pseudanabaena, Cephalothrix, Pantanalinema and Alkalinema. These three latter genera have been detected in other Brazilian ecosystems only (Pantanal, Atlantic Rainforest and Pampa), while those remaining have been extensively found in many parts of the world. The data provided here indicate that Amazonian rivers support a homocytous cyanobacterial diversity previously reported from other geographical and ecological environments.

Pseudomonas aestus sp. nov., a plant growth-promoting bacterium isolated from mangrove sediments.

Strain CMAA 1215T, a Gram-reaction-negative, aerobic, catalase positive, polarly flagellated, motile, rod-shaped (0.5-0.8 × 1.3-1.9 µm) bacterium, was isolated from mangrove sediments, Cananéia Island, Brazil. Analysis of the 16S rRNA gene sequences showed that strain CMAA 1215T forms a distinct phyletic line within the Pseudomonas putida subclade, being closely related to P. plecoglossicida ATCC 700383T, P. monteilii NBRC 103158T, and P. taiwanensis BCRC 17751T of sequence similarity of 98.86, 98.73, and 98.71%, respectively. Genomic comparisons of the strain CMAA 1215T with its closest phylogenetic type strains using average nucleotide index (ANI) and DNA:DNA relatedness approaches revealed 84.3-85.3% and 56.0-63.0%, respectively. A multilocus sequence analysis (MLSA) performed concatenating 16S rRNA, gyrB and rpoB gene sequences from the novel species was related with Pseudomonas putida subcluster and formed a new phylogenetic lineage. The phenotypic, physiological, biochemical, and genetic characteristics support the assignment of CMAA 1215T to the genus Pseudomonas, representing a novel species. The name Pseudomonas aestus sp.nov. is proposed, with CMAA 1215T (=NRRL B-653100T = CBMAI 1962T) as the type strain.

Bradyrhizobium sacchari sp. nov., a legume nodulating bacterium isolated from sugarcane roots.

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280T), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303T. Average nucleotide identity (ANI) analyses confirmed that BR 10280T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303T, but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

Saccharopolyspora spongiae sp. nov., a novel actinomycete isolated from the marine sponge Scopalina ruetzleri (Wiedenmayer, 1977).

A novel marine actinomycete, designated strain CMAA 1452T, was isolated from the sponge Scopalina ruetzleri collected from Saint Peter and Saint Paul Archipelago, in Brazil, and subjected to a polyphasic taxonomic investigation. The organism formed a distinct phyletic line in the Saccharopolyspora 16S rRNA gene tree and had chemotaxonomic and morphological properties consistent with its classification in this genus. It was found to be closely related to Saccharopolyspora dendranthemae KLBMP 1305T (99.5% 16S rRNA gene sequence similarity) and shared similarities of 99.3, 99.2 and 99.0 % with 'Saccharopolyspora endophytica' YIM 61095, Saccharopolyspora tripterygii YIM 65359T and 'Saccharopolyspora pathumthaniensis' S582, respectively. DNA-DNA relatedness values between the isolate and its closest phylogenetic neighbours, namely S. dendranthemae KLBMP 1305T, 'S. endophytica' YIM 61095 and S. tripterygii YIM 65359T, were 53.5, 25.8 and 53.2 %, respectively. Strain CMAA 1452T was also distinguished from the type strains of these species using a range of phenotypic features. On the basis of these results, it is proposed that strain CMAA 1452T (=DSM 103218T=NRRL B-65384T) merits recognition as the type strain of a novel Saccharopolyspora species, Saccharopolyspora spongiae sp. nov.

Williamsia spongiae sp. nov., an actinomycete isolated from the marine sponge Amphimedon viridis.

A novel actinobacterium, designated isolate B138T, was isolated from the marine sponge, Amphimedon viridis, which was collected from Praia Guaecá (São Paulo, Brazil), and its taxonomic position was established using data from a polyphasic study. The organism showed a combination of chemotaxonomic and morphological characteristics consistent with its classification in the genus Williamsia and it formed a distinct phyletic line in the Williamsia 16S rRNA gene tree. It was most closely related to Williamsia serinedens DSM 45037T and Williamsia deligens DSM 44902T (99.0 % 16S rRNA gene sequence similarity) and Williamsia maris DSM 44693T (97.5 % 16S rRNA gene sequence similarity), but was distinguished readily from these strains by the low DNA-DNA relatedness values (62.3-64.4 %) and by the discriminatory phenotypic properties. Based on the data obtained, the isolate B138T (=CBMAI 1094T=DSM 46676T) should be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia spongiae sp. nov. is proposed.

Dry Season Constrains Bacterial Phylogenetic Diversity in a Semi-Arid Rhizosphere System.

The rhizosphere is viewed as a deterministic environment led by the interaction between plants and microorganisms. In the case of semi-arid plants, this interaction is strengthened by the harshness of the environment. We tested the hypothesis that dry season represents a constraint on the bacterial diversity of the rhizosphere from semi-arid plants. To accomplish this, we sampled two leguminous species at five locations during the dry and rainy seasons in the Caatinga biome and characterised bacterial community structures using qPCR and 16S rRNA sequencing. We found that the main differences between seasons were due to reduced phylogenetic diversity caused by dryness. Variation partitioning indicated that environmental characteristics significant impacts in ß-diversity. Additionally, distance decay relationship and taxa area relationship indicate a higher spatial turnover at the rainy season. During the dry season, decreased bacterial abundance is likely due to the selection of resistant or resilient microorganisms; with the return of the rain, the sensitive populations start to colonise the rhizosphere by a process that is strongly influenced by environmental characteristics. Thus, we propose that the reduction of PD and strong influence of environmental parameters on the assemblage of these communities make them prone to functional losses caused by climatic disturbances.

Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest.

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.

Bradyrhizobium stylosanthis sp. nov., comprising nitrogen-fixing symbionts isolated from nodules of the tropical forage legume Stylosanthes spp.

The introduction of legumes and nitrogen-fixing bacteria in tropical areas under pasture is a key factor for improvement of soil fertility. However, there are still very few studies concerning the symbionts of tropical forage legumes. We performed a polyphasic study with three strains representing the genus Bradyrhizobium (BR 446T, BR 510 and BR 511) isolated from the tropical perennial forage legume of the genus Stylosanthes. On the basis of 16S rRNA gene sequences, the three strains showed highest similarity with B. huanghuaihaiense, and in the analysis of the intergenic transcribed spacer (ITS) they showed less than 93.4 % similarity to all described species of the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) with three, four or five (dnaK, glnII, gyrB, recA and rpoB) housekeeping genes confirmed that the BR strains belong to a distinct clade, with <96.5 % nucleotide identity with other members of the genus Bradyrhizobium. Average nucleotide identity (ANI) of genome sequences between strain BR 446T and B.huanghuaihaiense was below the threshold for species circumscription (90.7 %). DNA-DNA hybridization resulted in ΔTm values over 6.7 °C with the most closely related species. Similarities among the BR strains and differences from other species were confirmed by rep-PCR analysis. Interestingly, the BR strains were grouped in the analysis of nifH and nodC genes, but showed higher similarity with B. iriomotense and B. manausense than with B.huanghuaihaiense, indicating a different evolutionary history for nitrogen-fixation genes. Morpho-physiological, genotypic and genomic data supported that these BR strains represent a novel species for which the name Bradyrhizobium stylosanthis sp. nov. is suggested. The type strain is BR 446T (=CNPSo 2823T=HAMBI 3668T=H-8T), isolated from Stylosanthes guianensis.

Gordonia didemni sp. nov. an actinomycete isolated from the marine ascidium Didemnum sp.

A novel actinobacterium, designated isolate B204(T), was isolated from a marine ascidian Didemnum sp., collected from São Paulo, Brazil, and its taxonomic position established using data from a polyphasic study. The organism showed a combination of chemotaxonomic and morphological characteristics consistent with its classification in the genus Gordonia and formed a distinct phyletic line in the Gordonia 16S rRNA gene tree. It was closely related to Gordonia terrae DSM 43249(T) (99.9 % 16S rRNA gene sequence similarity) and Gordonia lacunae DSM 45085(T) (99.3 % 16S rRNA gene sequence similarity) but was distinguished from these strains by a moderate level of DNA-DNA relatedness (63.0 and 54.7 %) and discriminatory phenotypic properties. Based on the data obtained, the isolate B204(T) (=CBMAI 1069(T) = DSM 46679(T)) should therefore be classified as the type strain of a novel species of the genus Gordonia, for which the name Gordonia didemni sp. nov. is proposed.

Streptomyces atlanticus sp. nov., a novel actinomycete isolated from marine sponge Aplysina fulva (Pallas, 1766).

The taxonomic position of a novel marine actinomycete isolated from a marine sponge, Aplysina fulva, which had been collected in the Archipelago of Saint Peter and Saint Paul (Equatorial Atlantic Ocean), was determined by using a polyphasic approach. The organism showed a combination of morphological and chemotaxonomic characteristics consistent with its classification in the genus Streptomyces and forms a distinct branch within the Streptomyces somaliensis 16S rRNA gene tree subclade. It is closely related to Streptomyces violascens ISP 5183T (97.27 % 16S rRNA gene sequence similarity) and Streptomyces hydrogenans NBRC 13475T (97.15 % 16S rRNA gene sequence similarity). The 16S rRNA gene similarities between the isolate and the remaining members of the subclade are lower than 96.77 %. The organism can be distinguished readily from other members of the S. violacens subclade using a combination of phenotypic properties. On the basis of these results, it is proposed that isolate 103T (=NRRL B-65309T = CMAA 1378T) merits recognition as the type strain of a new Streptomyces species, namely Streptomyces atlanticus sp. nov.