Saonia flava gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from coastal seawater.

A Gram-stain-negative, aerobic, yellow-pigmented, straight rod-shaped bacterium, strain MOLA117T, was isolated from a coastal water sample from the north-western Mediterranean Sea, near Banyuls-sur-Mer, France. On the basis of phylogenetic analysis of the 16S rRNA gene sequence, strain MOLA117T was placed within the family Flavobacteriaceae, but showed less than 93 % 16S rRNA gene sequence similarity to other recognized species within the family. The most closely related genera included Arenibacter, Cellulophaga, Maribacter and Zobellia. The only isoprenoid quinone was menaquinone MK-6 and the predominant fatty acid was iso-C17 : 0 3-OH, representing over 33 % of the total fatty acids. The DNA G+C content was 36.9 mol%. Strain MOLA117T required NaCl for growth, and did not exhibit gliding motility or produce flexirubin. Based on the phenotypic and phylogenetic data, strain MOLA117T should be considered to represent a novel species of a new genus, for which the name Saonia flava gen. nov., sp. nov. is proposed. The type strain of Saonia flava is MOLA117T (=CIP 110873T=DSM 29762T).

Epiphytic Planctomycetes communities associated with three main groups of macroalgae.

Planctomycetes, a unique group of widespread and understudied bacteria, are known to be associated with macroalgae. The temporal dynamics and the host-specific association of planctomycetal communities on Fucus spiralis, Ulva sp. and Chondrus crispus from two locations in the North Coast of Portugal were assessed both by denaturing gradient gel electrophoresis with group-specific primers and 16S rDNA amplicon libraries. The epiphytic planctomycetal communities showed a significant association with the host macroalgal species independently of the geographical location and the season. This pattern was confirmed by clone libraries of winter and summer samples: we obtained 720 16S rRNA gene sequences that represented 44 operational taxonomic units (OTUs) within the phylum Planctomycetes. Most of the OTUs belonged to Blastopirellula, followed by Rhodopirellula, Planctomyces, the Pir4 lineage and the uncultured class OM190 (this last one nearly 30% of the OTUs). Ulva sp. and C. crispus had more diverse planctomycetal communities than F. spiralis. Analysis of beta diversity showed that the planctomycetal microbiome was host specific. We hypothesize that the specific association of Planctomycetes and their macroalgal hosts is likely determined by nutritional molecules provided by the algae and the set of sulfatases inherent to each Planctomycetes species.

The antimicrobial activity of heterotrophic bacteria isolated from the marine sponge Erylus deficiens (Astrophorida, Geodiidae).

Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds.

Roseimaritima ulvae gen. nov., sp. nov. and Rubripirellula obstinata gen. nov., sp. nov. two novel planctomycetes isolated from the epiphytic community of macroalgae.

Four isolates, belonging to the deep-branching phylum Planctomycetes, were recovered from the biofilm of two marine macroalgae, Ulva sp. and Laminaria sp., from the Northern coast of Portugal. These strains were light pink- or red-pigmented; the cells were variable in shape and usually organized in rosettes. They had a dimorphic cell cycle with budding reproduction. The organisms were chemoheterotrophic, strictly aerobic and mesophilic. The 16S rRNA gene sequence analysis showed that the strains belong to the family Planctomycetaceae with Rhodopirellula as the closest genus. The isolates form two separate branches (strain LF1(T) forms one branch and the strains UC8(T), UF3 and UF42 form a second branch) clearly separated from Rhodopirellula baltica with 94.2% and 93.8% 16S rRNA gene sequence similarity, respectively. Based on differential characteristics that distinguish the novel genera from R. baltica, such as cell size and shape, ultrastructure, enzymatic activities, substrate utilization pattern, fatty acid composition, phospholipid profiles and phylogeny we propose that the isolates represent two novel genera of the order Planctomycetales, Roseimaritima ulvae gen. nov., sp. nov. (type strain is UC8(T)=DSM 25454(T)=LMG 27778(T)) and Rubripirellula obstinata gen. nov., sp. nov. (type strain is LF1(T)=LMG 27779(T)=CECT 8602(T)).

Planctomycetes and macroalgae, a striking association.

Planctomycetes are part of the complex microbial biofilm community of a wide range of macroalgae. Recently, some studies began to unveil the great diversity of Planctomycetes present in this microenvironment and the interactions between the two organisms. Culture dependent and independent methods revealed the existence of a great number of species but, so far, only less than 10 species have been isolated. Planctomycetes comprise the genera Rhodopirellula, Blastopirellula, and Planctomyces, Phycisphaera and the uncultured class OM190 and some other taxa have only been found in this association. Several factors favor the colonization of macroalgal surfaces by planctomycetes. Many species possess holdfasts for attachment. The macroalgae secrete various sulfated polysaccharides that are the substrate for the abundant sulfatases produced by planctomycetes. Specificity between planctomycetes and macroalgae seem to exist which may be related to the chemical nature of the polysaccharides produced by each macroalga. Furthermore, the peptidoglycan-free cell wall of planctomycetes allows them to resist the action of several antimicrobial compounds produced by the macroalgae or other bacteria in the biofilm community that are effective against biofouling by other microorganisms. Despite the increase in our knowledge on the successful planctomycetes-macroalgae association, a great effort to fully understand this interaction is needed.

Rhodopirellula lusitana sp. nov. and Rhodopirellula rubra sp. nov., isolated from the surface of macroalgae.

Twenty two strains of Rhodopirellula were isolated from the epiphytic community of several marine macroalgae and separated into two groups, designated as group B and group C. In this study, we characterized these groups as two novel species belonging to the genus Rhodopirellula. These strains were represented by pleomorphic cells that were arranged in rosettes and formed pink- or red-pigmented colonies. The organisms were chemoorganotrophic and required vitamin B12 for growth. Their optimal temperature for growth was around 25°C. Major fatty acids were C18:1 ω9c, C16:0 and C16:1 ω7c/C16:1 ω6c. Phosphatidylcholine and phosphatidylglycerol were the major polar lipids. Unidentified phospholipids were also present. The 16S rDNA sequence analysis confirmed the affiliation of these organisms to the order Planctomycetales, genus Rhodopirellula, with R. baltica as the closest phylogenetic relative. The analysis of a partial sequence of the gene encoding the ß-subunit of RNA polymerase (rpoB) confirmed the phylogenetic separation of the isolates into two different species of the genus Rhodopirellula. The 16S rRNA sequences from strains of group B revealed their widespread occurrence across the world, whereas strains of group C were not observed before. On the basis of physiological, biochemical, chemotaxonomic and genetic characteristics we propose that our isolates represent two new species of Rhodopirellula, Rhodopirellula rubra sp. nov. (type strain is LF2(T)=DSM 25,459=CECT 8075) and Rhodopirellula lusitana sp. nov. (type strain is UC17(T)=DSM 25,457=LMG 27,777).

Community composition of the Planctomycetes associated with different macroalgae.

Insights into the diversity of marine natural microbial biofilms, as for example those developing at the surface of marine macroalgae, can be obtained by using molecular techniques based on 16S rRNA genes. We applied denaturing gradient gel electrophoresis (DGGE) with 16S rRNA genes-specific primers for Planctomycetes to compare the communities of these organisms developing on six different macroalgae (Chondrus crispus, Fucus spiralis, Mastocarpus stellatus, Porphyra dioica, Sargassum muticum, and Ulva sp.) sampled in spring 2012 in two rocky beaches in the north of Portugal. Planctomycetes can be one of the dominant organisms found in the epibacterial community of macroalgae, and we wanted to determine the degree of specificity and the spatial variation of these group. Shannon diversity indexes obtained from the comparison of DGGE profiles were similar in all the macroalgae, and in both sites, F. spiralis was the algae presenting lower Planctomycetes diversity, while M. stellatus and P. dioica from Porto showed the highest diversity. The analysis of DGGE profiles, including anosim statistics, indicate the existence of a specific Planctomycetes community associated with the algal host, likely independent of geographical variation. Sequencing of DGGE bands indicated that Planctomycetes communities were highly diverse, and some Operational Taxonomic Units seemed to be specifically associated with each macroalgae.

Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus (Astrophorida, Geodiidae).

Heterotrophic bacteria associated with two specimens of the marine sponge Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.

rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales.

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA-DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95-96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.

Insights into the ultrastructural morphology of novel Planctomycetes.

Knowledge of the interesting phylum of Planctomycetes has increased in the last decades both due to cultural and molecular methods. Although a restricted number of species have been described to date, this group presents a much larger diversity that has been mainly revealed by molecular ecology studies. Isolation experiments allowed us to get a number of new Planctomycetes taxa that extend the already described ones. In this work we present the ultrastructural morphological characterization of these new taxa as well as we give new details of Aquisphaera giovannonii ultrastructure. Furthermore, our interpretation on Planctomycetes cell envelope is provided.

Determination of zeta potential in Planctomycetes and its application in heavy metals toxicity assessment.

Zeta potential of Planctomycetes was evaluated under different environmental conditions and correlated to cell viability. Phylogenetically distinct strains of the Planctomycetes presented different negative zeta potential values. More negative values were associated with Rhodopirellula spp. and related to the great amount of fimbriae in these species. Milli-Q water was chosen as the best dispersion media to perform the measurements. Zeta potential increased with ionic strength and varied with pH. In the physiological range of pH 5.0-9, zeta potential remained low and Rhodopirellula sp. strain LF2 cells were viable. Out of this range, zeta potential increased significantly and viability decreased. The effect on zeta potential of arsenic, cadmium, chromium, copper, lead, nickel, and zinc was assessed in Rhodopirellula sp. strain LF2. Zeta potential increased with increasing toxicity of the heavy metals in a dose-response way. This result was confirmed by the results observed for Rhodopirellula baltica strain SH1 under copper toxicity. Lead was the most toxic metal and zinc was the least toxic as observed by zeta potential and viability. The results support a correlation between zeta potential and cell viability which seem to indicate the possibility to use it as a viability predictor for the effects of heavy metals toxicity.

Isolation and characterization of Planctomycetes from the sediments of a fish farm wastewater treatment tank.

The increasing ecological significance of Planctomycetes and the still limited knowledge of this group prompted us to obtain cultured isolates from the sediment of a treatment water recycling tank of a marine fish farm. Presence of strains from this group was assessed in the sediments and water column of the tank. Eleven isolates were obtained from the sediment sample by exploiting Planctomycetes natural resistance to several antibiotics and their capacity to degrade organic matter. Based on morphological characteristics and resistance to antibiotics, Planctomycetes were identified. Their phylogenetic affiliation was confirmed by the sequence analysis of the 16S rRNA gene that revealed the presence of a group of 6 isolates closely related to Rhodopirellula baltica and a cluster of 5 isolates with 97.7-97.9 % of similarity to this species, which probably are a different species of Rhodopirellula. ERIC-PCR profiles showed a higher discrimination within the two groups and allowed the identification of nine different genotypes within the isolated strains. This work corroborates the association of Rhodopirellula spp. with fish farm environments.

Bringing Planctomycetes into pure culture.

Planctomycetes have been known since the description of Planctomyces bekefii by Gimesi at the beginning of the twentieth century (1924), although the first axenic cultures were only obtained in the 1970s. Since then, 11 genera with 14 species have been validly named and five candidatus genera belonging to the anaerobic ammonium oxidation, anammox bacteria have also been discovered. However, Planctomycetes diversity is much broader than these numbers indicate, as shown by environmental molecular studies. In recent years, the authors have attempted to isolate and cultivate additional strains of Planctomycetes. This paper provides a summary of the isolation work that was carried out to obtain in pure culture Planctomycetes from several environmental sources. The following strains of planctomycetes have been successfully isolated: two freshwater strains from the sediments of an aquarium, which were described as a new genus and species, Aquisphaera giovannonii; several Rhodopirellula strains from the sediments of a water treatment recycling tank of a marine fish farm; and more than 140 planctomycetes from the biofilm community of macroalgae. This collection comprises several novel taxa that are being characterized and described. Improvements in the isolation methodology were made in order to optimize and enlarge the number of Planctomycetes isolated from the macroalgae. The existence of an intimate and an important relationship between planctomycetes and macroalgae reported before by molecular studies is therefore supported by culture-dependent methods.

Planctomycetes diversity associated with macroalgae.

Planctomycetes associated with 12 macroalgae from the north coast of Portugal were isolated, using an improved method. A total of 138 isolates were found to comprise 10 operational taxonomic units (OTUs), with 65% of the strains being closely related to the species Rhodopirellula baltica. The other strains are probably new species or genera related to Rhodopirellula, Blastopirellula and Planctomyces. Some of the OTUs isolated are unique and have never been found before in previous studies. Catalyzed reporter deposition-FISH confirmed the presence of Planctomycetes on macroalgal surfaces. This study provides the first report of the cultured diversity of Planctomycetes on the epiphytic macroalgae community and presents clear evidence of their nutritional and intimate relationship.

Aquisphaera giovannonii gen. nov., sp. nov., a planctomycete isolated from a freshwater aquarium.

As part of a study of the diversity of planctomycetes, two novel strains, designated OJF2(T) and OJF8, were isolated from the sediments of a freshwater aquarium. The organisms were chemoheterotrophic, spherical and pink-pigmented, had an optimum growth temperature of about 30-35 °C and an optimum pH for growth of around 7.5-8.5. The predominant fatty acids were C(18:1)ω9c and C(16:0). The two strains were able to assimilate several sugars and organic acids. 16S rRNA gene sequence analysis confirmed the affiliation of these organisms to the phylum 'Planctomycetes'; they showed highest similarity to the type strains of Singulisphaera acidiphila (92.4%) and Isosphaera pallida (91.9%). On the basis of physiological, biochemical and chemotaxonomic characteristics, strains OJF2(T) and OJF8 are considered to represent a novel species of a new genus of the order Planctomycetales, for which the name Aquisphaera giovannonii gen. nov., sp. nov. is proposed. The type strain of Aquisphaera giovannonii is OJF2(T) (=CECT 7510(T) =DSM 22561(T)).