Identification and genomic analysis of Pseudosulfitobacter koreense sp. nov. isolated from toxin-producing dinoflagellate Alexandrium pacificum.

The bacterial strain AP-MA-4T isolated from the marine dinoflagellate Alexandrium pacificum (KCTC AG60911), was subjected to a taxonomic analysis. Cells of strain AP-MA-4T were Gram-stain-negative, aerobic, rod-shaped, optimum growth at 20 °C, pH 7.0, in the presence of 5% (w/v) NaCl. Strain AP-MA-4T shared the highest 16S rRNA gene sequence similarity to Pseudosulfitobacter pseudonitzschiae DSM 26824T (98.5%), followed by Ascidiaceihabitans donghaensis RSS1-M3T (96.3%), Pseudoseohaeicola caenipelagi BS-W13T (95.7%), and Sulfitobacter pontiacus CHLG 10T (95.3%). Based on 16S rRNA phylogeny, strain AP-MA-4T is phylogenetically closely related to Pseudosulfitobacter pseudonitzschiae (type species of Pseudosulfitobacter) and could be distinguished from the type species based on their phenotypic properties. The genome length of strain AP-MA-4T was 3.48 Mbp with a 62.9% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain AP-MA-4 T and its closely related type strains were 72.2-83.3 and 18.2-27.6%, respectively. Summed feature 8 (C18:1ω7c and/or C18:1ω6c) was identified the major fatty acids (> 10%). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phospholipid (PL) were demonstrated as the major polar lipids. The major respiratory quinone is ubiquinone-10 (Q-10). Based on genotypic and phenotypic features, strain AP-MA-4T (= KCTC 92289T = GDMCC 1.3585T) represents a new Pseudosulfitobacter species, in which the name Pseudosulfitobacter koreense sp. nov. is proposed.

Gymnodinialimonas phycosphaerae sp. nov., a phycosphere bacterium isolated from Karlodinium veneficum.

A facultative anaerobic, Gram-negative, non-motile, rod-shaped bacterial strain, designated N5T, was obtained from the phycosphere microbiota of the marine planktonic dinoflagellate, Karlodinium veneficum. Strain N5T showed growth on marine agar at 25 °C, pH 7 and 1 % (w/v) NaCl and produced a yellow colour. According to a phylogenetic study based on 16S rRNA gene sequences, strain N5T has a lineage within the genus Gymnodinialimonas. The G+C content in the genome of strain N5T is 62.9 mol% with a total length of 4 324 088 bp. The NCBI Prokaryotic Genome Annotation Pipeline revealed that the N5T genome contained 4230 protein-coding genes and 48 RNA genes, including a 5S rRNA, 16S rRNA, 23S rRNA, 42 tRNA, and three ncRNAs. Genome-based calculations (genome-to-genome distance, average nucleotide identity and DNA G+C content) clearly indicated that the isolate represents a novel species within the genus Gymnodinialimonas. The predominant fatty acids were C19 : 0 cyclo ω8c and feature 8 (comprising C18 : 1 ω6c and/or C18 : 1 ω7c). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The main respiratory quinone was Q-10. Based on its phenotypic, phylogenetic, genomic and chemotaxonomic features, strain N5T represents a novel species of the genus Gymnodinialimonas, for which the name Gymnodinialimonas phycosphaerae sp. nov. is proposed. The type strain is N5T (=KCTC 82362T=NBRC 114899T).

Dinoflagellate hosts determine the community structure of marine Chytridiomycota: Demonstration of their prominent interactions.

The interactions of parasitic fungi with their phytoplankton hosts in the marine environment are mostly unknown. In this study, we evaluated the diversity of Chytridiomycota in phytoplankton communities dominated by dinoflagellates at several coastal locations in the NW Mediterranean Sea and demonstrated the most prominent interactions of these parasites with their hosts. The protist community in seawater differed from that in sediment, with the latter characterized by a greater heterogeneity of putative hosts, such as dinoflagellates and diatoms, as well as a chytrid community more diverse in its composition and with a higher relative abundance. Chytrids accounted for 77 amplicon sequence variants, of which 70 were found exclusively among different blooming host species. The relative abundance of chytrids was highest in samples dominated by the dinoflagellate genera Ostreopsis and Alexandrium, clearly indicating the presence of specific chytrid communities. The establishment of parasitoid-host co-cultures of chytrids and dinoflagellates allowed the morphological identification and molecular characterization of three species of Chytridiomycota, including Dinomyces arenysensis, as one of the most abundant environmental sequences, and the discovery of two other species not yet described.

Investigating the Relationship between Nitrate, Total Dissolved Nitrogen, and Phosphate with Abundance of Pathogenic Vibrios and Harmful Algal Blooms in Rehoboth Bay, Delaware.

Vibrio spp. and phytoplankton are naturally abundant in marine environments. Recent studies have suggested that the co-occurrence of phytoplankton and the pathogenic bacterium Vibrio parahaemolyticus is due to shared ecological factors, such as nutrient requirements. We compared these communities at two locations in the Delaware Inland Bays, representing a site with high anthropogenic inputs (Torquay Canal) and a less developed area (Sloan Cove). In 2017 to 2018, using light microscopy, we were able to identify the presence of many bloom-forming algal species, such as Karlodinium veneficum, Dinophysis acuminata, Heterosigma akashiwo, and Chattonella subsalsa. Dinoflagellate biomass was higher at Torquay Canal than that at Sloan Cove. D. acuminata and Chloromorum toxicum were found only at Torquay Canal and were not observed in Sloan Cove. Most probable number real-time PCR revealed V. parahaemolyticus and Vibrio vulnificus in environmental samples. The abundance of vibrios and their virulence genes varied between sites, with a significant association between total dissolved nitrogen (TDN), PO4-, total dissolved phosphorus (TDP), and pathogenic markers. A generalized linear model revealed that principal component 1 of environmental factors (temperature, dissolved oxygen, salinity, TDN, PO4-, TDP, NO3:NO2, NO2-, and NH4+) was the best at detecting total (tlh+) V. parahaemolyticus, suggesting that they are the prime drivers for the growth and distribution of pathogenic Vibrio spp. IMPORTANCE Vibrio-associated illnesses have been expanding globally over the past several decades (A. Newton, M. Kendall, D. J. Vugia, O. L. Henao, and B. E. Mahon, Clin Infect Dis 54:S391-S395, 2012, https://doi.org/10.1093/cid/cis243). Many studies have linked this expansion with an increase in global temperature (J. Martinez-Urtaza, B. C. John, J. Trinanes, and A. DePaola, Food Res Int 43:10, 2010, https://doi.org/10.1016/j.foodres.2010.04.001; L. Vezzulli, R. R. Colwell, and C. Pruzzo, Microb Ecol 65:817-825, 2013, https://doi.org/10.1007/s00248-012-0163-2; R. N. Paranjpye, W. B. Nilsson, M. Liermann, and E. D. Hilborn, FEMS Microbiol Ecol 91:fiv121, 2015, https://doi.org/10.1093/femsec/fiv121). Temperature and salinity are the two major factors affecting the distribution of Vibrio spp. (D. Ceccarelli and R. R. Colwell, Front Microbiol 5:256, 2014, https://doi.org/10.3389/fmicb.2014.00256). However, Vibrio sp. abundance can also be affected by nutrient load and marine plankton blooms (V. J. McKenzie and A. R. Townsend, EcoHealth 4:384-396, 2007; L. Vezzulli, C. Pruzzo, A. Huq, and R. R. Colwell, Environ Microbiol Rep 2:27-33, 2010, https://doi.org/10.1111/j.1758-2229.2009.00128.x; S. Liu, Z. Jiang, Y. Deng, Y. Wu, J. Zhang, et al. Microbiologyopen 7:e00600, 2018, https://doi.org/10.1002/mbo3.600). The expansion of Vibrio spp. in marine environments calls for a deeper understanding of the biotic and abiotic factors that play a role in their abundance. We observed that pathogenic Vibrio spp. were most abundant in areas that favor the proliferation of harmful algal bloom (HAB) species. These results can inform managers, researchers, and oyster growers on factors that can influence the growth and distribution of pathogenic Vibrio spp. in the Delaware Inland Bays.

Gymnodinialimonas ceratoperidinii gen. nov., sp. nov., isolated from rare marine dinoflagellate Ceratoperidinium margalefii.

A bacterial strain, designated J12C1-MA-4T, was isolated from liquid culture of the dinoflagellate Ceratoperidinium margalefii. The bacterium was Gram-negative, aerobic, and rod-shaped. Oxidase and catalase were positive. Optimal growth was observed at 30 °C, pH 7.0, in the presence of 1% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene and a 92 core gene set suggested that the strain J12C1-MA-4T belongs to the family Rhodobacteraceae in the class Alphaproteobacteria and represents a taxon separated from other genera. 16S rRNA gene sequence of the strain J12C1-MA-4T showed high similarities to Loktanella ponticola KCTC 42133T (95.7%), Pseudooctadecabacter jejudonensis KCTC 32525T (95.5%) and Jannaschia helgolandensis KCTC 12191T (95.3%). The genome length of strain J12C1-MA-4T was 3,621,968 bp with a DNA G + C content of 64.48 mol%. The major cellular fatty acids of strain J12C1-MA-4T were summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) (> 10%). Phosphatidylglycerol (PG), phosphatidylcholine (PC), phospholipids (PL), lipids 1 (L1) and aminolipid (AL) were shown to be the major polar lipids. The sole predominant isoprenoid quinone was Q-10. Based on phylogenetic, phenotypic, chemotaxonomic and genomic features, we propose that strain J12C1-MA-4T represent a novel species in the novel genus of the family Rhodobacteraceae, with the proposed name Gymnodinialimonas ceratoperidinii gen. nov., sp. nov.. The type strain is J12C1-MA-4T (=KCTC 82770T =GDMCC 1.2729T).

Single-cell genomics unveiled a cryptic cyanobacterial lineage with a worldwide distribution hidden by a dinoflagellate host.

Cyanobacteria are one of the most important contributors to oceanic primary production and survive in a wide range of marine habitats. Much effort has been made to understand their ecological features, diversity, and evolution, based mainly on data from free-living cyanobacterial species. In addition, symbiosis has emerged as an important lifestyle of oceanic microbes and increasing knowledge of cyanobacteria in symbiotic relationships with unicellular eukaryotes suggests their significance in understanding the global oceanic ecosystem. However, detailed characteristics of these cyanobacteria remain poorly described. To gain better insight into marine cyanobacteria in symbiosis, we sequenced the genome of cyanobacteria collected from a cell of a pelagic dinoflagellate that is known to host cyanobacterial symbionts within a specialized chamber. Phylogenetic analyses using the genome sequence revealed that the cyanobacterium represents an underdescribed lineage within an extensively studied, ecologically important group of marine cyanobacteria. Metagenomic analyses demonstrated that this cyanobacterial lineage is globally distributed and strictly coexists with its host dinoflagellates, suggesting that the intimate symbiotic association allowed the cyanobacteria to escape from previous metagenomic studies. Furthermore, a comparative analysis of the protein repertoire with related species indicated that the lineage has independently undergone reductive genome evolution to a similar extent as Prochlorococcus, which has the most reduced genomes among free-living cyanobacteria. Discovery of this cyanobacterial lineage, hidden by its symbiotic lifestyle, provides crucial insights into the diversity, ecology, and evolution of marine cyanobacteria and suggests the existence of other undiscovered cryptic cyanobacterial lineages.

Flagellatimonas centrodinii gen. nov., sp. nov., a novel member of the family Nevskiaceae isolated from toxin-producing dinoflagellate Centrodinium punctatum.

A Gram-stain-negative, aerobic, rod-shaped strain (R2A-3T) was isolated from the toxin-producing dinoflagellate Centrodinium punctatum and identified as a novel genus and new species based on a polyphasic taxonomic approach. The optimum conditions for growth of the strain were at 25 °C, pH 8.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA genes and 92 core genes sets revealed that strain R2A-3T belongs to the family Nevskiaceae in the class Gammaproteobacteria and represented an independent taxon separated from other genera. The 16S rRNA gene of strain R2A-3T showed the highest sequence similarity to Polycyclovorans algicola TG408T (95.2%), Fontimonas thermophila HA-01T (94.1%) and Sinimarinibacterium flocculans NH6-24T (93.2%), and less than 92.8 % similarity to other genera in the family Nevskiaceae. The genome length of strain R2A-3T was 3608892 bp with 65.2 mol% G+C content. Summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c) was the major fatty acid (>10 %). Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were detected as the major polar lipids. The major respiratory quinone was ubiquinone-8. According to its phylogenetic, phenotypic, chemotaxonomic and genomic features, strain R2A-3T represents a new species in the new genus of the family Nevskiaceae. It is recommended to name it Flagellatimonas centrodinii gen. nov., sp. nov. The type strain is R2A-3T (=KCTC 82469T=GDMCC 1.2523T).

Revealing changes in the microbiome of Symbiodiniaceae under thermal stress.

Symbiodiniaceae are a diverse family of marine dinoflagellates, well known as coral endosymbionts. Isolation and in vitro culture of Symbiodiniaceae strains for physiological studies is a widely adopted tool, especially in the context of understanding how environmental stress perturbs Symbiodiniaceae cell functioning. While the bacterial microbiomes of corals often correlate with coral health, the bacterial communities co-cultured with Symbiodiniaceae isolates have been largely overlooked, despite the potential of bacteria to significantly influence the emergent physiological properties of Symbiodiniaceae cultures. We examined the physiological response to heat stress by Symbiodiniaceae isolates (spanning three genera) with well-described thermal tolerances, and combined these observations with matched changes in bacterial composition and abundance through 16S rRNA metabarcoding. Under thermal stress, there were Symbiodiniaceae strain-specific changes in maximum quantum yield of photosystem II (proxy for health) and growth rates that were accompanied by changes in the relative abundance of multiple Symbiodiniaceae-specific bacteria. However, there were no Symbiodiniaceae-independent signatures of bacterial community reorganisation under heat stress. Notably, the thermally tolerant Durusdinium trenchii (ITS2 major profile D1a) had the most stable bacterial community under heat stress. Ultimately, this study highlights the complexity of Symbiodiniaceae-bacteria interactions and provides a first step towards uncoupling their relative contributions towards Symbiodiniaceae physiological functioning.

Metabolic relationships of uncultured bacteria associated with the microalgae Gambierdiscus.

Microbial communities inhabit algae cell surfaces and produce a variety of compounds that can impact the fitness of the host. These interactions have been studied via culturing, single-gene diversity and metagenomic read survey methods that are limited by culturing biases and fragmented genetic characterizations. Higher-resolution frameworks are needed to resolve the physiological interactions within these algal-bacterial communities. Here, we infer the encoded metabolic capabilities of four uncultured bacterial genomes (reconstructed using metagenomic assembly and binning) associated with the marine dinoflagellates Gambierdiscus carolinianus and G. caribaeus. Phylogenetic analyses revealed that two of the genomes belong to the commonly algae-associated families Rhodobacteraceae and Flavobacteriaceae. The other two genomes belong to the Phycisphaeraceae and include the first algae-associated representative within the uncultured SM1A02 group. Analyses of all four genomes suggest these bacteria are facultative aerobes, with some capable of metabolizing phytoplanktonic organosulfur compounds including dimethylsulfoniopropionate and sulfated polysaccharides. These communities may biosynthesize compounds beneficial to both the algal host and other bacteria, including iron chelators, B vitamins, methionine, lycopene, squalene and polyketides. These findings have implications for marine carbon and nutrient cycling and provide a greater depth of understanding regarding the genetic potential for complex physiological interactions between microalgae and their associated bacteria.

Symbiodiniaceae-bacteria interactions: rethinking metabolite exchange in reef-building corals as multi-partner metabolic networks.

The intimate relationship between scleractinian corals and their associated microorganisms is fundamental to healthy coral reef ecosystems. Coral-associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi and viruses) support coral health and resilience through metabolite transfer, inter-partner signalling, and genetic exchange. However, much of our understanding of the coral holobiont relationship has come from studies that have investigated either coral-Symbiodiniaceae or coral-bacteria interactions in isolation, while relatively little research has focused on other ecological and metabolic interactions potentially occurring within the coral multi-partner symbiotic network. Recent evidences of intimate coupling between phytoplankton and bacteria have demonstrated that obligate resource exchange between partners fundamentally drives their ecological success. Here, we posit that similar associations with bacterial consortia regulate Symbiodiniaceae productivity and are in turn central to the health of corals. Indeed, we propose that this bacteria-Symbiodiniaceae-coral relationship underpins the coral holobiont's nutrition, stress tolerance and potentially influences the future survival of coral reef ecosystems under changing environmental conditions. Resolving Symbiodiniaceae-bacteria associations is therefore a logical next step towards understanding the complex multi-partner interactions occurring in the coral holobiont.

Nitratireductor alexandrii sp. nov., from phycosphere microbiota of toxic marine dinoflagellate Alexandrium tamarense.

A taxonomic study was carried out on a novel algae-associated bacterial strain Z3-1T, which was isolated from phycosphere microbiota of toxic marine dinoflagellate Alexandrium tamarense 880. Cells of strain Z3-1T were Gram-stain-negative, rod-shaped and strictly aerobic and were motile by means of flagella. Strain Z3-1T grew at 25-42 °C, pH 5.0-10.0 and 1.0-5.0 % (w/v) NaCl. Strain Z3-1T reduced nitrate to nitrite, but did not reduce nitrite to nitrogen gas. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Z3-1T belongs to the genus Nitratireductor showing the highest sequence similarity (97.0 %) to Nitratireductor basaltis JCM 14935T. The average nucleotide identity and digital DNA-DNA hybridization relatedness between strain Z3-1T and type strains of genus Nitratireductor with available genome sequences were in the ranges of 72.4-74.4 % and 22.7-23.3 %, respectively. The major fatty acids were summed in feature 8 (C18:1 ω7c and/or C18:1 ω6c), C19:0 ω8c cyclo, C18:1 ω7c 11-metyl and iso-C17:0. The major polar lipids were determined as diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids and four unidentified polar lipids. The genomic DNA G+C content calculated from genome sequence was 65.6 mol%. Based on genotypic, chemotaxonomic and phenotypic data obtained, strain Z3-1T represents a novel species of the genus Nitratireductor, for which the name Nitratireductor alexandrii sp. nov. is proposed with the type strain Z3-1T (=KCTC 62458T=CCTCC AB 2017227T).

Saccharospirillum alexandrii sp. nov., isolated from the toxigenic marine dinoflagellate Alexandrium catenella LZT09.

A Gram-stain-negative, strictly aerobic, non-motile and non-pigmented spirillum, designated strain LZ-5T, was isolated from cultures of the paralytic shellfish poisoning (PSP) toxin-producing marine dinoflagellate Alexandrium catenella LZT09 collected from the Zhoushan sea area in the East China Sea during an algal bloom. The isolate grew at 4-40 °C (optimum, 25-33 °C) and pH 5.0-9.0 (optimum, 7.5) in the presence of 0.5-10 % (w/v) NaCl (optimum, 4.0 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LZ-5T clearly belonged to the genus Saccharospirillum of the family Saccharospirillaceae. Strain LZ-5T shared highest 16S rRNA gene sequence similarity with Saccharospirillum impatiens EL-105T (98.9 %), Saccharospirillum mangrovi HK-33T (97.2 %), Saccharospirillum correiae CPA1T (96.8 %), Saccharospirillum salsuginis YIM-Y25T (96.8 %) and Saccharospirillum aestuarii IMCC 4453T (95.1 %). The average nucleotide identity and in silico DNA-DNA hybridization between strain LZ-5T and the two most closely related Saccharospirillum strains, S. impatiens EL-105T and S. mangrovi HK-33T, were 82.2 and 19.3 %, and 72.2 and 13.2 %, respectively. The predominant respiratory quinone of strain LZ-5T was Q-8, and the major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipids of strain LZ-5T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), glycolipid (GL), two unidentified glycophospholipids (GPLs), three unidentified aminophospholipids (APLs) and two unidentified lipids. The genomic DNA G+C content was 57.2 mol%. On the basis of this polyphasic characterization, strain LZ-5T represents a novel species of the genus Saccharospirillum, for which the name Saccharospirillum alexandrii sp. nov. is proposed. The type strain is LZ-5T (=KCTC 62460T=CCTCC AB2017232T).

Nioella ostreopsis sp. nov., isolated from toxic dinoflagellate, Ostreopsis lenticularis.

A novel short-rod-shaped bacterial strain with poly-ß-hydroxybutyric acid granules inside, designated as Z7-4T, was isolated from a culture of a marine dinoflagellate with palytoxin-producing capacity, Ostreopsis lenticularis OS06, collected from the East China Sea. Cells of Z7-4T were Gram-stain-negative, non-motile, strictly aerobic, 0.9-1.2 µm wide and 2.0-3.9 µm long. Growth occurred in 1-4 % (w/v) NaCl, at 15-37 °C and at pH 5.0-10.0, with optimum growth in 3.5 % (w/v) NaCl, at 30 °C and at pH 7.0. Analysis of 16S rRNA gene sequence revealed that Z7-4T shared the highest 16S rRNA gene sequence similarities with Nioella aestuarii JCM 30752T (98.8 %), followed by Nioella sediminis KCTC 42144T (98.6 %) and Nioella nitratireducens KCTC 32417T (96.9 %). Phylogenetic analysis based on nearly complete 16S rRNA gene sequences revealed that Z7-4T clearly represented a member of the genus Nioella within the family Rhodobacteraceae. The respiratory quinone of Z7-4T was identified as Q-10. Polar lipids of Z7-4T were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, three unidentified aminophospholipids and one unidentified phospholipid. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The DNA G+C content of Z7-4T was 63.0 mol%. DNA-DNA hybridization values of the isolate against the closely related type strains were far below the 70 % limit for species delineation. The average nucleotide identity and in silico DNA-DNA genome hybridization relatedness between Z7-4T and the closely related members of the genus Nioella, N. sediminis KCTC 42144T and N. nitratireducens KCTC 32417T, were 91.1 and 46.3 %, and 79.3 and 19.4 %, respectively. On the basis of the results of polyphasic analysis, Z7-4T is proposed to represent a novel species of the genus Nioella, for which the name Nioella ostreopsis sp. nov. is proposed. The type strain of Nioella ostreopsis is Z7-4T (=KCTC 62459T=CCTCC AB 2017231T).

Haliea alexandrii sp. nov., isolated from phycosphere microbiota of the toxin-producing dinoflagellate Alexandrium catenella.

A Gram-negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterium, named strain LZ-16-2T, was isolated from the phycosphere microbiota of the paralytic shellfish poisoning toxin-producing marine dinoflagellate Alexandrium catenella LZT09. Strain LZ-16-2T grew optimally at 28 °C at pH 6.5 and with 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain LZ-16-2T fell within the genus Haliea and was most closely related to Haliea salexigens DSM 19537T, with which the new isolate exhibited 98.5 % 16S rRNA gene sequence similarity. The major respiratory quinone was Q-8. The predominant cellular fatty acids were C17 : 1 ω8c, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C17 : 1 ω6c, C11 : 0 3-OH and C17 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The average nucleotide identity and in silico DNA-DNA genome hybridization relatedness values between strain LZ-16-2T and its closest relative, H. salexigens DSM 19537T, were 92.8 and 55.1 %, respectively. The DNA G+C content was 61.3 mol%. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LZ-16-2T from all other members of the genus Haliea. On the basis of the polyphasic characterization, strain LZ-16-2T represents a novel species of the genus Haliea, for which the name Haliea alexandrii sp. nov. is proposed. The type strain is LZ-16-2T (=KCTC 62344T=CCTCC AB2017229T).

Mesorhizobium alexandrii sp. nov., isolated from phycosphere microbiota of PSTs-producing marine dinoflagellate Alexandrium minutum amtk4.

An aerobic, Gram-stain-negative, motile and rod-shaped bacterial strain, designated as Z1-4T, was isolated from the phycosphere microbiota of marine dinoflagellate Alexandrium minutum that produces paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolate belongs to the genus Mesorhizobium, and it was closely related to Mesorhizobium waimense LMG 28228T and Mesorhizobium amorphae LMG 18977T with both 16S rRNA gene sequence similarities of 97.3%. The values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness between strain Z1-4T and its relatives are both well below the thresholds used for the delineation of a new species. A genome-based phylogenetic tree constructed by up-to-date bacterial core gene set (UBCG) indicates that strain Z1-4T forms an independent branch within the genus Mesorhizobium. The respiratory quinone of strain Z1-4T was Q-10. The major fatty acids were similar to other members of the genus Mesorhizobium containing the summed feature 8, C16:0, C19:0cycloω8c, C17:0 and summed feature 3. The polar lipids are phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, five glycolipids and seven unknown polar lipids. The DNA G + C content was determined to be 62.1 mol % based on its genomic sequence. Combined evidences based on the genotypic, chemotaxonomic and phenotypic characteristics clearly indicates that strain Z1-4T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium alexandrii sp. nov. is proposed. The type strain is Z1-4T (= KCTC 72512T = CCTCC AB 2019101T).

Tropicibacter alexandrii sp. nov., a novel marine bacterium isolated from the phycosphere of a dinoflagellate, Alexandrium minutum.

An aerobic, Gram-negative, rod-shaped, non-motile bacterium was isolated from a liquid culture of the dinoflagellate Alexandrium minutum and designated as strain LMIT003T. Analyses of 16S rRNA gene sequences revealed that this strain is affiliated with the genus Tropicibacter in the family Rhodobacteraceae of the class Alphaproteobacteria and shows high similarity (97.3%) with the type species Tropicibacter naphthalenivorans C02T. Phylogenetic analysis based on core genes showed that the isolate groups with members of the genus Tropicibacter. The G + C content of strain LMIT003T was determined to be 61.9 mol%. The major fatty acids identified included summed feature 8 (C18:1 ω7c/C18:1 ω6c), C18:0, C12:1 3-OH and C16:0. The sole respiratory lipoquinone was found to be ubiquinone-10. The major polar lipids were found to be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and four unidentified phospholipids. The draft genome size of strain LMIT003T was determined to be 4.8 Mbp. The average nucleotide identity values between strain LMIT003T and reference Tropicibacter species were determined to be 78.7% (T. naphthalenivorans) and 74.2% (Tropicibacter phthalicicus). Based on physiological, chemotaxonomic and phylogenetic analysis, strain LMIT003T is concluded to represent a novel species in the genus Tropicibacter, for which the name Tropicibacter alexandrii sp. nov., is proposed. The type strain is LMIT003T (= CICC 24660T = KCTC 62895T).

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

The response of Prorocentrum sigmoides and its associated culturable bacteria to metals and organic pollutants.

This study investigates the effect of metals (cadmium, lead, mercury, and tellurium) and organic pollutants (benzene, diesel, lindane, and xylene) on a dinoflagellate-Prorocentrum sigmoides Böhm-and its associated culturable bacteria. Two bacterial cultures (Bacillus subtilis strain PD005 and B. xiamensis strain PD006) were isolated from P. sigmoides and characterized by scanning electron microscopy, 16S ribosomal RNA sequencing, biochemical analyses, and growth curve studies. This study points to a mutualistic relationship between P. sigmoides and its associated Bacillus isolates. P. sigmoides enhanced the growth of its associated Bacillus spp., through the secretion of extracellular exudates. In return, both Bacillus isolates contributed to the resistance of P. sigmoides to metals and organic pollutants. P. sigmoides and both Bacillus isolates exhibited concentration-dependent responses to metals and organic pollutants. An intriguing feature was the similar response of P. sigmoides and its associated Bacillus isolates to mercury and cadmium, indicating a co-selection of mercury and cadmium resistance. This provides support to the "dinoflagellate host-phycosphere bacteria" behaving as a single functional unit. However, the sensitivity profiles of P. sigmoides and its associated Bacillus isolates are different with respect to metals versus organic pollutants. These aspects need to be addressed in future studies to unravel the effect of metal and organic pollutants on dinoflagellates, an important component of the phytoplankton community, and to discern the influence of associated "phycosphere" bacteria on the response of dinoflagellates to pollutants.

Algicidal Activity of Novel Marine Bacterium Paracoccus sp. Strain Y42 against a Harmful Algal-Bloom-Causing Dinoflagellate, Prorocentrum donghaiense.

Prorocentrum donghaiense blooms occur frequently in the Yangtze River estuary and the adjacent East China Sea. These blooms have damaged marine ecosystems and caused enormous economic losses over the past 2 decades. Thus, highly efficient, low-cost, ecofriendly approaches must be developed to control P. donghaiense blooms. In this study, a bacterial strain (strain Y42) was identified as Paracoccus sp. and was used to lyse P. donghaiense The supernatant of the strain Y42 culture was able to lyse P. donghaiense, and the algicidal activity of this Y42 supernatant was stable with different temperatures and durations of light exposure and over a wide pH range. In addition to P. donghaiense, Y42 showed high algicidal activity against Alexandrium minutum, Scrippsiella trochoidea, and Skeletonema costatum, suggesting that it targets primarily Pyrrophyta. To clarify the algicidal effects of Y42, we assessed algal lysis and determined the chlorophyll a contents, photosynthetic activity, and malondialdehyde contents of P. donghaiense after exposure to the Y42 supernatant. Scanning electron microscopy and transmission electron microscopy analyses showed that the Y42 supernatant disrupted membrane integrity and caused algal cell breakage at the megacytic zone. Photosynthetic pigment loss and significant declines in both photosynthetic efficiency and the electron transport rate indicated that the Y42 supernatant damaged the photosynthetic system of P. donghaiense Malondialdehyde overproduction indicated that the Y42 supernatant caused lipid peroxidation and oxidative damage to membrane systems in the algal cell, ultimately leading to death. The findings of this study reveal the potential of Y42 to remove algal cells from P. donghaiense blooms.IMPORTANCEP. donghaiense is one of the most common dinoflagellate species that form harmful algal blooms, which frequently cause serious ecological pollution and pose health hazards to humans and other animals. Screening for bacteria with high algicidal activity against P. donghaiense and studying their algicidal processes and characteristics will contribute to an understanding of their algicidal effects and provide a theoretical basis for preventing algal blooms and reducing their harm to the environment. This study reports the algicidal activity and characteristics of Paracoccus against P. donghaiense The stability of the algicidal activity of Paracoccus in different environments (including different temperature, pH, and sunlight conditions) indicates its potential for use in the control of P. donghaiense blooms.

Ponticoccus alexandrii sp. nov., a novel bacterium isolated from the marine toxigenic dinoflagellate Alexandrium minutum.

During an investigation of the biodiversity of the cultivable bacterial community associated with paralytic shellfish poisoning toxin-producing marine dinoflagellate, Alexandrium minutum a novel algal-associated bacterium, designated strain AT2-AT was isolated. 16S rRNA gene sequence similarity analysis showed that the strain is a member of the genus Ponticoccus, with high sequence similarity to Ponticoccus litoralis DSM 18986T (97.9%) and Ponticoccus lacteus JCM 30379T (96.0%). However, based on the data obtained for the physiological and biochemical characteristics, and low level of DNA-DNA relatedness analysis, the strain could be genotypically and phenotypically differentiated from two type strains of the genus Ponticoccus. Therefore, this algal-associated bacterial strain is concluded to represent a novel species of the genus Ponticoccus, for which the name Ponticoccus alexandrii sp. nov. is proposed. The type strain is AT2-AT (CCTCC AB 2016296T = KCTC 62340T) [corrected].