Tabrizicola algicola sp. nov. isolated from culture of microalga Ettlia sp.

A novel Gram-stain-negative, aerobic, non-spore-forming, non-motile, and rod-shaped bacterium, strain ETT8T was isolated from a chemostat culture of microalga Ettlia sp. YC001. Optimal growth was with 0-2% NaCl and at 25-37 °C on R2A medium. Phylogenetic analysis based on the 16S rRNA gene and genome sequence showed that strain ETT8T belongs to the genus Tabrizicola, with the close neighbours being T. sediminis DRYC-M-16T (98.1 %), T. alkalilacus DJCT (97.6 %), T. fusiformis SY72T (96.9 %), T. piscis K13M18T (96.8 %), and T. aquatica RCRI19T (96.5 %). The genomic comparison of strain ETT8T with type species in the genus Tabrizicola was analysed using the genome-to-genome distance calculator (GGDC), average nucleotide identity (ANI), and average amino acid identity (AAI) (values indicated ≤17.7, ≤75.4 and ≤71.9 %, respectively). The genomic DNA G+C content of strain ETT8T was 64.4 %, plus C18 : 1 ω6c and C18 : 0-iso were the major fatty acids and Q-10 the major respiratory quinone. Strain ETT8T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine aminolipid, and four unidentified lipids as the major polar lipids. Based on the chemotaxonomic, genotypic, and phenotype results, strain ETT8T was recognized as a novel species of the genus Tabrizicola for which the name Tabrizicola algicola sp. nov. is proposed. The type strain is ETT8T (=KCTC 72206T=JCM 31893T=MCC 4339T).

Roseomonas algicola sp. nov., isolated from a green alga, Pediastrum duplex.

A Gram-stain-negative, strictly aerobic bacterium, designated strain PeD5T, was isolated from a green alga Pediastrum duplex from the Nakdong river of the Republic of Korea. Cells were non-motile cocci, catalase-negative and oxidase-positive. Growth of PeD5T was observed at 25-40 °C (optimum, 35 °C) and pH 5.0-10.0 (optimum, pH 7-8), and in the presence of 0-0.25% (w/v) NaCl (optimum, 0%). PeD5T contained C16:0, C18:1ω7c 11-methyl, summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) and summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) as major cellular fatty acids (>5%) and ubiquinone-10 as the sole isoprenoid quinone. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid and an unidentified aminolipid were detected as major polar lipids. The genomic DNA G+C content of PeD5T was 71.0 mol%. PeD5T was most closely related to Roseomonas stagni HS-69T with a 97.6% 16S rRNA sequence similarity and shared less than 96.3% 16S rRNA sequence similarities with type strains of other species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that PeD5T formed a phyletic lineage with Roseomonas stagni HS-69T within the genus Roseomonas. On the basis of results of phenotypic, chemotaxonomic and molecular analysis, strain PeD5T clearly represents a novel species of the genus Roseomonas, for which the name Roseomonas algicola sp. nov. is proposed. The type strain is PeD5T (=KACC 19925T=JCM 33309T).

Isolation and genome analysis of Winogradskyella algicola sp. nov., the dominant bacterial species associated with the green alga Dunaliella tertiolecta.

Microalgae and bacteria are known to be closely associated in diverse environments. To isolate dominant bacterial species associated with a green alga, Dunaliella tertiolecta, a photoreactor culture of the microalga was investigated using culture-based and culture-independent approaches. The bacterial community structure of the algal culture showed that the most abundant bacterial species under the culture conditions was related to the genus Winogradskyella. The closely related amplicon sequences, showing ≥ 99.5% 16S rRNA gene sequence similarity to one of the isolates, designated IMCC-33238T, constituted > 49% of the bacterial community and was therefore regarded as the most dominant species in the algal culture. Strain IMCC33238T was characterized by Gramstaining-negative and orange-colored rods. Phylogenetic analyses of the 16S rRNA genes as well as whole genome sequences revealed that strain IMCC33238T belonged to Winogradskyella and shared more than 97.2% 16S rRNA gene sequence similarity with Winogradskyella species. The strain contained iso-C15:1 G, iso-C15:0, iso-C15:0 3-OH, and summed feature 3 (C16:1ω6c and/or C16:1ω7c) as major fatty acids and MK-6 as the predominant quinone. The polar lipids found in strain IMCC33238T were phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. The genome of strain IMCC33238T was 3.37 Mbp in size with 33.9 mol% G + C content and proteorhodopsin. Many genes encoding folate and vitamin production are considered to play an important role in the bacteria-algae interaction. On the basis of phylogenetic and phenotypic characteristics, strain IMCC33238T represents a novel species in the genus Winogradskyella, for which the name Winogradskyella algicola sp. nov. is proposed. The type strain is IMCC33238T (= KACC 21192T = NBRC 113704T).

Interspecies interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus SAG 86.81 in a co-cultivation system at various growth phases.

In lakes, cyanobacterial blooms are frequently associated with green algae and dominate the phytoplankton community in successive waves. In the present study, the interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus were studied to clarify the probable ecological significance of algal secondary metabolites; focusing on the role of cyanotoxin 'microcystin-LR' (MC-LR). A dialysis co-cultivation technique was applied where M. aeruginosa was grown inside and D. subspicatus was cultured outside of the dialysis tubing. The concentration of the intra- and extracellular MC-LR and the growth of two species were measured at different time points over a period of one month. Additionally, the growth of the two species in the culture filtrate of one another and the effect of the purified MC-LR on the growth of the green alga were studied. The results indicated that the co-existing species could affect each other depending on the growth phases. Despite the early dominance of D. subspicatus during the logarithmic phase, M. aeruginosa suppressed the growth of the green alga at the stationary phase, which coincided with increased MC production and release. However, the inhibitory effects of Microcystis might be related to its other extracellular metabolites rather than, or possibly in addition to, MC.

Cellulomonas algicola sp. nov., an actinobacterium isolated from a freshwater alga.

An actinomycete strain, TKZ-21T, was isolated from a freshwater alga (Chetophoraceae) collected from the Takizawa River, Yamanashi, Japan, and examined using a polyphasic taxonomic approach. Cells were Gram-stain positive, aerobic, non-sporulating, motile, and coccoid or short rod-shaped. The strain grew in the presence of 0-4 % (w/v) NaCl, between pH 6-9.4, and over a temperature range of 15-40 °C, with optimum growth at 30 °C. The peptidoglycan type of strain TKZ-21T was A4ß, containing l-ornithine as diagnostic diamino acid and d-glutamic acid as the interpeptide bridge. The predominant menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, ninhydrin-positive glycolipid, and unidentified phospholipids. The major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, and the DNA G+C content was 75.6 mol%. On the basis of 16S rRNA gene sequence analysis, strain TKZ-21T was closely related to Cellulomonas fimi (98.5 % sequence similarity) and Cellulomonas biazotea (98.3 %). The genome orthoANI value between strain TKZ-21T and C. biazotea and C. fimi were 84.7 and 84.2 %, respectively. On the basis of fatty acid and MALDI-TOF MS profile analysis, phylogenetic analyses, genomic analysis, and phenotypic data, it is proposed that the isolate be classified as a representative of a novel species of the genus Cellulomonas, with the name Cellulomonas algicola sp. nov. The type strain is TKZ-21T (=NBRC 112905T=TBRC 8129T).

RNA Interference by Ingested dsRNA-Expressing Bacteria to Study Shell Biosynthesis and Pigmentation in Crassostrea gigas.

RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Although the Pacific oyster Crassostrea gigas is an economically important species with fully sequenced genome, very few mechanistic studies have been carried out due to the lack of molecular techniques to alter gene expression without inducing stress. In this present study, we used unicellular alga Platymonas subcordiformis and Nitzschia closterium f. minutissima as a vector to feed oysters with Escherichia coli strain HT115 engineered to express double-stranded RNAs (dsRNAs) targeting specific genes involved in shell pigmentation. A C. gigas strain with black shell was used to target tyrosinase or peroxidase gene expression by RNAi using the above-mentioned approach. The results showed that feeding oyster with dsRNA of tyrosinase could knock down the expression of corresponding tyrosinase and hinder the developed shell growth. Feeding oyster with dsRNA of peroxidase could knock down the expression of the corresponding peroxidase and result in reduced black pigmentation in the newly developed shell. This non-invasive RNAi study demonstrated that tyrosinase played a vital role in the assembly and maturation of shell matrices and peroxidase was essential for black pigmentation in the shell. Moreover, the RNA interference by ingested dsRNA-expressing bacteria is a relatively simple and effective method for knockdown of a gene expression in adult oysters, thus further advances the use of C. gigas as model organism in functional genomic studies.

Stimulation and Isolation of Paraphysoderma sedebokerense (Blastocladiomycota) Propagules and Their Infection Capacity Toward Their Host Under Different Physiological and Environmental Conditions.

Paraphysoderma sedebokerense (P. sedebokerense) (Blastocladiomycota) is a facultative pathogenic chytrid that causes irreversible damage to some green microalgae. Specific attacks leading to culture collapse under different conditions have only been described in the lucrative microalga Haematococcus pluvialis (H. pluvialis), while generating biomass for ketocarotenoid astaxanthin production, both indoors and outdoors. In order to manage the infection, parasite propagules (zoospores/amoeboid swarmers), the initiators of the disease, must be studied. Until now, no report on isolated P. sedebokerense propagules has been published. Here, we report on a reproducible method for the stimulation of P. sedebokerense propagule release and their isolation from fungal cultures in synthetic media and infected H. pluvialis cultures, and we further studied their development under different conditions. The isolated propagules featured different spore morphotypes, with coatless spherical spores and amoeboid swarmers being the most dominant in the first pulse of propagule release in both cultures. Inoculating the pure propagules with the host, in both the presence and absence of nitrogen, resulted in epidemic development in both green and red cells; however, in red cells, the epidemic developed more quickly in the presence of nitrogen. Biologically non-active autoclaved host cells were used to distinguish the initial stages of recognition from more progressive stages of the epidemics; on these cells, propagules encysted but did not develop further. These results prove the existence of heat-stable recognition sites on the host and an obligatory signal transduction from the host to support fungal cyst development. The propagule isolation method described herein is a breakthrough that will enable researchers to study the influence of different substances on the propagules, specifically as the initiators of the infection, and thus assist in the management of chytrid diseases. Moreover, it will be useful in studying host-parasite recognition and, therefore, will increase our understanding of the multiple chytrid infections found in nature.

Diversity and Hidden Host Specificity of Chytrids Infecting Colonial Volvocacean Algae.

Chytrids are zoosporic fungi that play an important, but yet understudied, ecological role in aquatic ecosystems. Many chytrid species have been morphologically described as parasites on phytoplankton. However, the majority of them have rarely been isolated and lack DNA sequence data. In this study we isolated and cultivated three parasitic chytrids, infecting a common volvocacean host species, Yamagishiella unicocca. To identify the chytrids, we characterized morphology and life cycle, and analyzed phylogenetic relationships based on 18S and 28S rDNA genes. Host range and specificity of the chytrids was determined by cross-infection assays with host strains, characterized by rbcL and ITS markers. We were able to confirm the identity of two chytrid strains as Endocoenobium eudorinae Ingold and Dangeardia mamillata Schröder and described the third chytrid strain as Algomyces stechlinensis gen. et sp. nov. The three chytrids were assigned to novel and phylogenetically distant clades within the phylum Chytridiomycota, each exhibiting different host specificities. By integrating morphological and molecular data of both the parasitic chytrids and their respective host species, we unveiled cryptic host-parasite associations. This study highlights that a high prevalence of (pseudo)cryptic diversity requires molecular characterization of both phytoplankton host and parasitic chytrid to accurately identify and compare host range and specificity, and to study phytoplankton-chytrid interactions in general.