Stable and Enriched Cenarchaeum symbiosum and Uncultured Betaproteobacteria HF1 in the Microbiome of the Mediterranean Sponge Haliclona fulva (Demospongiae: Haplosclerida).

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

Culture-independent characterization of a novel magnetotactic member affiliated to the Beta class of the Proteobacteria phylum from an acidic lagoon.

Magnetotactic bacteria (MTB) comprise a group of motile microorganisms common in most mesothermal aquatic habitats with pH values around neutrality. However, during the last two decades, a number of MTB from extreme environments have been characterized including: cultured alkaliphilic strains belonging to the Deltaproteobacteria class of the Proteobacteria phylum; uncultured moderately thermophilic strains belonging to the Nitrospirae phylum; cultured and uncultured moderately halophilic or strongly halotolerant bacteria affiliated with the Deltaproteobacteria and Gammaproteobacteria classes and an uncultured psychrophilic species belonging to the Alphaproteobacteria class. Here, we used culture-independent techniques to characterize MTB from an acidic freshwater lagoon in Brazil (pH ∼ 4.4). MTB morphotypes found in this acidic lagoon included cocci, rods, spirilla and vibrioid cells. Magnetite (Fe3 O4 ) was the only mineral identified in magnetosomes of these MTB while magnetite magnetosome crystal morphologies within the different MTB cells included cuboctahedral (present in spirilla), elongated prismatic (present in cocci and vibrios) and bullet-shaped (present in rod-shaped cells). Intracellular pH measurements using fluorescent dyes showed that the cytoplasmic pH was close to neutral in most MTB cells and acidic in some intracellular granules. Based on 16S rRNA gene phylogenetic analyses, some of the retrieved gene sequences belonged to the genus Herbaspirillum within the Betaproteobacteria class of the Proteobacteria phylum. Fluorescent in situ hybridization using a Herbaspirillum-specific probe hybridized with vibrioid MTB in magnetically-enriched samples. Transmission electron microscopy of the Herbaspirillum-like MTB revealed the presence of many intracellular granules and a single chain of elongated prismatic magnetite magnetosomes. Diverse populations of MTB have not seemed to have been described in detail in an acid environment. In addition, this is the first report of an MTB phylogenetically affiliated with Betaproteobacteria class.

Planktonic microbial profiling in water samples from a Brazilian Amazonian reservoir.

Our comprehension of the dynamics and diversity of freshwater planktonic bacterial communities is far from complete concerning the Brazilian Amazonian region. Therefore, reference studies are urgently needed. We mapped bacterial communities present in the planktonic communities of a freshwater artificial reservoir located in the western Amazonian basin. Two samples were obtained from rainy and dry seasons, the periods during which water quality and plankton diversity undergo the most significant changes. Hypervariable 16S rRNA and shotgun sequencing were performed to describe the first reference of a microbial community in an Amazonian lentic system. Microbial composition consisted mainly of Betaproteobacteria, Cyanobacteria, Alphaproteobacteria, and Actinobacteria in the dry period. The bacteria distribution in the rainy period was notably absent of Cyanobacteria. Microcystis was observed in the dry period in which the gene cluster for cyanotoxins was found. Iron acquisition gene group was higher in the sample from the rainy season. This work mapped the first inventory of the planktonic microbial community of a large water reservoir in the Amazon, providing a reference for future functional studies and determining other communities and how they interact.

A glimpse of the endophytic bacterial diversity in roots of blackberry plants (Rubus fruticosus).

The aim of this study was to explore the diversity of culturable bacterial communities residing in blackberry plants (Rubus fruticosus). Bacterial endophytes were isolated from plant roots, and their 16S rDNA sequences were amplified and sequenced. Our results show that the roots of R. fruticosus exhibit low colony forming units of bacterial endophytes per gram of fresh tissue (6 x 102 ± 0.5 x 102). We identified 41 endophytic bacterial species in R. fruticosus by BLAST homology search and a subsequent phylogenetic analysis, belonging to the classes Actinobacteria, Bacilli, Alfaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Predominantly, genera belonging the Proteobacteria (Burkholderia, 29.4%; Herbaspirillum, 10.7%; Pseudomonas, 4.9%; and Dyella, 3.9%), Firmicutes (Bacillus, 42.1%), and Actinobacteria (two isolates showing high identity with the Streptomyces genus, 1.9%) divisions were identified. Fifty percent of the bacterial endophytes produced the phytohormone indole-acetic acid (IAA), eleven of which exhibited higher IAA production (>5.8 mg/mL) compared to the plant growth-promoting strain, Pseudomonas fluorescens UM270. Additionally, the endophytic isolates exhibited protease activity (22%), produced siderophores (26.4%), and demonstrated antagonistic action (>50% inhibition of mycelial growth) against the grey mold phytopathogen Botrytis cinerea (3.9%). These results suggested that field-grown R. fruticosus plants contain bacterial endophytes within their tissues with the potential to promote plant growth and display antagonism towards plant pathogens.

Decomposition of plant-sourced carbon compounds by heterotrophic betaproteobacteria isolated from a tropical Costa Rican bromeliad.

Betaproteobacteria were the most common isolates from the water-filled tank of a Costa Rican bromeliad. Isolates included eight species from the orders Neisseriales and Burkholderiales, with close relatives recovered previously from tropical soils, wetlands, freshwater, or in association with plants. Compared to close relatives, the isolates displayed high temperature and comparatively low pH optima, reflecting the tropical, acidic nature of the bromeliad tank. Bromeliad-associated bacteria most closely related to Chromobacterium, Herbaspirillum, and Aquitalea were all isolated exclusively at pH 6, while Ralstonia, Cupriavidus, and three species of Burkholderia were isolated mostly at pH 4. Activity profiles for the isolates suggest pervasive capabilities for the breakdown of plant-sourced organics, including d-galacturonic acid, mannitol, d-xylose, and l-phenylalanine, also reflecting a niche dominated by decomposition of leaves from the overlying canopy, which become entrained in the tanks. Metabolic activity profiles were overlapping between the Burkholderiales, isolated at pH 4, and the Neisseriales, isolated at pH 6, suggesting that plant material decomposition, which is presumably the underlying process sustaining the tank community and possibly the plant itself, occurs in the tanks at both pH extremes. These results suggest that bromeliad-associated betaproteobacteria may play an important role in the cycling of carbon in this unusual aquatic habitat.

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species.

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts.

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.

Microbial activity and diversity during extreme freeze-thaw cycles in periglacial soils, 5400 m elevation, Cordillera Vilcanota, Perú.

High-elevation periglacial soils are among the most extreme soil systems on Earth and may be good analogs for the polar regions of Mars where oligotrophic mineral soils abut with polar ice caps. Here we report on preliminary studies carried out during an expedition to an area where recent glacial retreat has exposed porous mineral soils to extreme, daily freeze-thaw cycles and high UV fluxes. We used in situ methods to show that inorganic nitrogen (NO(3) (-) and NH(4) (+)) was being actively cycled even during a period when diurnal soil temperatures (5 cm depth) ranged from -12 to 27 degrees C and when sub-zero, soil cooling rates reached 1.8 degrees C h(-1) (the most rapid soil cooling rates recorded to date). Furthermore, phylogenetic analyses of microbial phylotypes present at our highest sites (5410 m above sea level) showed the presence of nitrifying bacteria of the genus Nitrospira and newly discovered nitrite-oxidizing Betaproteobacteria. These soils were overwhelmingly dominated (>70% of phylotypes) by photosynthetic bacteria that were related to novel cyanobacteria previously found almost exclusively in other plant-free, high-elevation soils. We also demonstrated that soils from our highest sites had higher potential for mineralizing glutamate and higher microbial biomass than lower elevation soils that had been more recently covered by ice. Overall, our findings indicate that a diverse and robustly functioning microbial ecosystem is present in these previously unstudied high-elevation soils.

Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions.

Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.

Mircobial community composition in petroleum-contaminated and uncontaminated soil from Francisco de Orellana, in the northern Ecuadorian Amazon.

The microbial compositions of two soils from the northern Ecuadorian Amazon (Francisco de Orellana province), one contaminated with petroleum and the other uncontaminated, were compared. Classical culture and molecular techniques were used to analyze microbial diversity. The cultivable Bacteria from contaminated soil belonged to betaproteobacteria (16.6%), gammaproteobacteria (66.6%), and Firmicutes (16,6%), whereas in uncontaminated soil, cultivable Bacteria were identified as gammaproteobacteria (80%) and Firmicutes (20%). Analysis of the 16S rRNA showed that in the contaminated soil proteobacterial populations (alpha-, beta- and deltaproteobacteria) were more abundant than acidobacterial populations. The Shannon index (H cent ) was used to estimate diversity in the contaminated and uncontaminated soil. Diversity was higher in the uncontaminated (H cent = 2.16) than in the contaminated (H cent = 1.72) soil sample. Further studies are needed to determine whether the differences between contaminated and non-contaminated soil samples were due to spontaneous bioremediation microbial activity.

Diversity of aquatic prokaryotic communities in the Cuatro Cienegas basin.

The Cuatro Cienegas basin (Coahuila, México) is a composite of different water systems in the middle of the desert with unusually high levels of endemism and diversity in different taxa. Although the diversity of macrobiota has been well described, little is known about the diversity and distribution of microorganisms in the oligotrophic ponds. Here we describe the extent and distribution of diversity found in aquatic prokaryotic communities by analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes and phylogenetic analysis of cloned genes. Twelve locations within the basin were sampled. Among all the samples, we found a total of 117 operational taxonomic units (OTUs) using T-RFLPs, which ranged in any single sample from four to 49. OTU richness and Shannon diversity indices for different sites varied, but none were particularly high. 16S rRNA gene sequence data showed 68 different phylotypes among 198 clones. The most abundant phylotypes were Gamma- and Betaproteobacteria, and extreme halophiles. The differences among sites were significant; 45 TRFs were found only once, and 37% of the total diversity was represented by differences between sites, suggesting high beta-diversity. Further studies are needed to test whether this is a direct consequence of environmental heterogeneity in the basin.

Phylogenetic analysis of bacterial populations in waters of the former Texcoco Lake, Mexico.

Molecular techniques were used to compare the compositions of the bacterial communities of the 2 following lagoons from the former soda Texcoco Lake, Mexico: the restored Facultativa lagoon and the Nabor Carrillo lagoon. Ribosomal intergenic spacer analysis (RISA) revealed that bacterial communities of the 2 lagoons were different and presented a relatively low diversity. Clone libraries of 16S rDNA genes were constructed, and significant phylotypes were distinguished by restriction fragment length polymorphism (RFLP). A representative clone from each phylotype was partially sequenced. Molecular identification and phylogenetic analyses based on ribosomal sequences revealed that the Facultativa lagoon harbored mainly gamma- and beta-Proteobacteria, low G+C Gram-positive bacteria, and several members of the Halobacteriaceae family of archaea. The Nabor Carrillo lagoon mainly included typical halophilic and alkaliphilic low G+C Gram-positive bacteria, gamma-Proteobacteria, and beta-Proteobacteria similar to those found in other soda lakes. Several probably noncultured new bacterial species were detected. Three strains were isolated from the Nabor Carrillo lagoon, their partial 16S rDNA sequences were obtained. On this basis, they were identified as Halomonas magadiensis (H1), Halomonas eurihalina (H2), and Staphylococcus sciuri (H3). This is the first study that uses molecular techniques to investigate potential genetic diversity in the Texcoco lakes. In this preliminary evaluation, we infer the presence of alkalophilic, halophilic, or haloalkaliphilic bacteria potentially useful for biotechnology.

Sterolibacterium denitrificans gen. nov., sp. nov., a novel cholesterol-oxidizing, denitrifying member of the beta-Proteobacteria.

A bacterial strain (Chol-1S(T)) that is able to oxidize cholesterol to CO2 and reduce nitrate to dinitrogen was enriched and isolated from an upflow sludge bed (USB) anoxic reactor that treats sanitary landfill leachate from the city of Montevideo, Uruguay. Cells of strain Chol-1S(T) were gram-negative, rod-shaped to slightly curved, measured 0.5-0.6 x 1.0-1.3 microm and were motile by a single polar flagellum. Strain Chol-1S(T) grew optimally at 30-32 degrees C and pH 7.0, with a doubling time of 44-46 h when cholesterol was used as the sole carbon and energy source. The metabolism of strain Chol-1S(T) was strictly respiratory, with oxygen or nitrate as the terminal electron acceptor. The presence of ubiquinone Q-8 as the sole respiratory lipoquinone indicated that strain Chol-1S(T) belonged to the beta-subclass of the Proteobacteria. Phosphatidylethanolamine was the predominant polar lipid and the G + C content of the DNA was 65.3 mol%. The fatty acid profile of strain Chol-1S(T), cultivated under denitrifying conditions by using a defined mineral medium supplemented with cholesterol, was characterized by the following major components: summed feature 4 (C16:1 omega7c and/or iso C15:0 2-OH), C16:0, C18:1 omega7c and hydroxy acid C10:0 3-OH. Minor components included C10:0, C11:0, C12:0, C14:0, C15:0, C19:0, C19:0 10-methyl and hydroxylated acids C8:0 3-OH and C16:0 3-OH. Analysis of the 16S rDNA sequence showed that strain Chol-1S(T) represents a separate lineage within the Thauera, Azoarcus, Zoogloea and Rhodocyclus assemblage of the beta-Proteobacteria. Strain Chol-1S(T) had highest sequence similarity (96.5%) with strain 72Chol, a denitrifying beta-Proteobacterium. On the basis of polyphasic evidence, strain Chol-1S(T) (=DSM 13999T=ATCC BAA-354T) is proposed as the type strain of Sterolibacterium denitrificans gen. nov., sp. nov.

Molecular diversity of mesophilic and thermophilic bacteria in a membrane bioreactor determined by fluorescent in situ hybridization with mxaF- and rRNA-targeted probes.

An evaluation of the efficiency of treatment of kraft mill foul condensates in a membrane bioreactor was carried out in the laboratory. Efficiency and rate of methanol removal were quantified at operating temperatures of 35, 45 and 55 degrees C. The structure of the bacterial community present in the reactor biomass at the different operating temperatures was evaluated by in situ hybridization of the biomass samples with fluorescently-labelled probes (FISH) targeting the Eubacteria, the alpha, beta and gamma subclasses of the Proteobacteria, the low G + C content Gram-positive bacteria (Bacillus spp.), while community function was evaluated by in situ hybridization with a methanol dehydrogenase gene (mxaF) probe. Methanol removal efficiency decreased from 99.4 to 92%, and removal rate from 2.69 mg MeOH/l x min to 2.49 mg MeOH/l x min when the operating temperature was increased from 35 to 55 degrees C. This decrease in methanol removal was accompanied by a decrease (from 58% to 42%) in the relative proportion of cells that hybridized with the mxaF probe. The relative proportion of Bacillus spp. increased from 5 to 20% while the proportion of members of the alpha subclass of Proteobacteria decreased from 16% to 6% when the bioreactor operating temperature was raised from 35 to 55 degrees C. The relative proportions of bacteria belonging to the beta (22-25%) and gamma (18-20%) subclasses of the Proteobacteria remained relatively constant regardless of operating temperature. Proteobacteria (alpha, beta and gamma subclasses) and Bacillus spp. represented 61, 67 and 71% of the Eubacteria in the biomass sampled at 35, 45 and 55 degrees C, respectively. The FISH technique was shown to be an efficient method for detection of both structural and functional changes in the bacterial communities that could be related to efficiency of methanol removal in a membrane bioreactor operating at different temperatures.

16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril).

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.

Acidovorax anthurii sp. nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium

The bacterial leaf-spot of anthurium emerged during the 1980s, in the French West Indies and Trinidad. This new bacterial disease is presently wide spread and constitutes a serious limiting factor for commercial anthurium production. Twenty-nine strains isolated from leaf-spots of naturally infected anthurium were characterized and compared with reference strains belonging to the Comamonadaceae family, the genera Ralstonia and Burkholderia, and representative fluorescent pseudomonads. From artificial inoculations 25 out of 29 strains were pathogenic on anthurium. Biochemical and physiological tests, fatty acid analysis, DNA-DNA hybridization, 16S rRNA gene sequence analysis, DNA-16S RNA hybridization were performed. The 25 pathogenic strains on anthurium were clustered in one phenon closely related to phytopathogenic strains of the genus Acidovorax. Anthurium strains were 79-99% (deltaTm range 0.2-1.6) related to the strain CFBP 3232 and constituted a discrete DNA homology group indicating that they belong to the same species. DNA-rRNA hybridization, 16S rRNA sequence and fatty acid analysis confirmed that this new species belongs to the beta-subclass of Proteobacteria and to rRNA superfamily III, to the family of Comamonadaceae and to the genus Acidovorax. The name Acidovorax anthurii is proposed for this new phytopathogenic bacterium. The type strain has been deposited in the Collection Fran‡aise des Bact‚ries PhytopathogŠnes as CFBP 3232T.