Muricauda aurea sp. nov. and Muricauda profundi sp. nov., two marine bacteria isolated from deep sea sediment of Pacific Ocean.

Two novel Gram-stain-negative, aerobic, rod-shaped, carotenoid-pigmented and non-flagellated bacteria, designated BC31-1-A7T and BC31-3-A3T, were isolated from polyethylene-terephthalate-degrading bacterial consortia enriched from deep-sea sediment collected in the Pacific Ocean. Optimal growth of both strains was observed at 28-32 °C, at pH 7.5 and in the presence of 3-4% (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that strains BC31-1-A7T and BC31-3-A3T were closely related to Muricauda aquimarina JCM 11811T, Muricauda lutimaris KCTC 22173T, Muricauda ruestringensis DSM 13258T, Muricauda zhangzhouensis DSM 25030T, Muricauda oceani JCM 33902T and Muricauda oceanensis KCTC 72200T with 96.8-98.9% sequence similarity. The 16S rRNA gene sequence similarity between strains BC31-1-A7T and BC31-3-A3T was 97.5%. The genomic G+C contents of strains BC31-1-A7T and BC31-3-A3T were 42.1 and 41.6 mol%, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain BC31-3-A3T, strain BC31-1-A7T and their six closely related type strains were 77.6-84.3% and 20.5-27.9%, respectively. Menaquinone-6 was detected as the major isoprenoid quinone in all strains. Their major fatty acids were iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH. The major polar lipids of strains BC31-1-A7T and BC31-3-A3T were identified as one phosphatidylethanolamine, some unidentified polar lipids and one aminolipid. Based on their distinct taxonomic characteristics, strains BC31-1-A7T and BC31-3-A3T represent two novel species in the genus Muricauda. The names proposed to accommodate these two strains are Muricauda aurea sp. nov. and Muricauda profundi sp. nov., and the type strains are BC31-1-A7T (=MCCC M23246T=KCTC 82569T) and BC31-3-A3T (=MCCC M23216T=KCTC 82302T), respectively.

Aequorivita iocasae sp. nov., a halophilic bacterium isolated from sediment collected at a cold seep field in the South China Sea.

A moderately halophilic bacterium, designated strain KX20305T, was isolated from sediment collected from a cold seep field in the South China Sea. Cells of strain KX20305T were Gram-stain-negative, rod-shaped, non-motile, facultatively anaerobic, oxidase- and catalase-positive, and grew optimally at 25-30 °C, pH 6.0-8.0 and with 3-6 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain KX20305T grouped with members of the genus Aequorivita, including Aequorivita aquimaris D-24T (98.3 % sequence similarity), Aequorivita vladivostokensis KMM 3516T (98.1 %) and Aequorivita echinoideorum CC-CZW007T (97.5 %). Genome sequencing of strain KX20305T revealed a genome size of 3.35 Mb and a DNA G+C content of 38.71 mol%. Genomic average nucleotide identity (orthoANI) values of strain KX20305T with A. aquimaris D-24T, A. vladivostokensis KMM 3516T and A. echinoideorum JCM 30378T were 83.8, 81.7 and 75.4 %, respectively, while in silico DNA-DNA hybridization (GGDC) values for strain KX20305T with these strains were 27.2, 25.0 and 19.6 %, respectively. The major fatty acids of strain KX20305T were iso-C15 : 0, iso-C17 : 0 3-OH and 10-methyl C16 : 0/iso-C17 : 1 ω9c. The predominant respiratory quinone was menaquinone-6 (MK-6). The polar lipids mainly comprised phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. Based on comparative analysis of phylogenetic, phylogenomic, phenotypic and chemotaxonomic characteristics, strain KX20305T represents a novel species of the genus Aequorivita, for which the name Aequorivita iocasae sp. nov. is proposed. The type strain is KX20305T (=KCTC 82699T=MCCC 1K06238T=JCM 34635T).

Salegentibacter lacus sp. nov. and Salegentibacter tibetensis sp. nov., isolated from hypersaline lakes on the Tibetan Plateau.

Two Gram-stain-negative, catalase- and oxidase-positive, rod-shaped and non-motile strains (LM13ST and JZCK2T) were isolated from hypersaline lakes in China. The colonies of both strains were yellow-pigmented and convex. Both strains could grow at 4-34 °C, pH 6.5-9.0 and with 1.0-13.0 % (w/v) NaCl. Comparisons based on 16S rRNA gene sequences showed that strains LM13ST and JZCK2T share less than 98.3 % similarity with species of the genus Salegentibacter. The phylogenetic tree reconstructed based on 16S rRNA gene sequences also showed that Salegentibacter species are the most closely related neighbours of strains LM13ST and JZCK2T. The sequenced draft genome sizes of strains LM13ST and JZCK2T are 4.06 and 4.22 Mbp with G+C contents of 37.0 and 37.8 mol%, respectively. The phylogenomic tree reconstructed using the Up-to-date Bacterial Core Gene set pipeline also demonstrated that both strains belong to the genus Salegentibacter. The calculated pairwise average nucleotide identity values and digital DNA-DNA hybridization values between strains LM13ST and JZCK2T and Salegentibacter species were less than 86.4 and 32.0 %, respectively. The respiratory quinone in both strains was MK-6. Their major fatty acids were iso-C12 : 0, iso-C14 : 0, C15 : 1 ω10c, iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C17 : 1 ω10c. Their major polar lipids included phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid, but strain LM13ST also contained one more unidentified aminolipid, one more unidentified lipid and one unidentified phospholipid. Combining the above descriptions, strains LM13ST and JZCK2T should represent two independent novel species of the genus Salegentibacter, for which the names Salegentibacter lacus sp. nov. (type strain LM13ST=GDMCC 1.2643T=KCTC 82861T) and Salegentibacter tibetensis sp. nov. (type strain JZCK2T=GDMCC 1.2621T=KCTC 82862T) are proposed.

Constantimarinum furrinae gen. nov., sp. nov., a marine bacterium isolated from saline volcanic rock aquifer (lava seawater) at Jeju Island, Republic of Korea.

A Gram-stain-negative, aerobic, rod-shaped (0.3-0.5 × 1.0-1.9 µm), non-motile marine bacterium designated as ALE3EIT was isolated from a saline volcanic rock aquifer (lava sea-water) on Jeju Island, Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain ALE3EIT showed high similarity to 'Altibacter lentus' JLT2010T (97.2%), followed by Marixanthomonas ophiurae KMM 3046T (94.5%). Growth was observed at 10-41°C (optimum, 30°C), at pH 6.0-8.5 (optimum, pH 7.5) and at 0.5-8% (optimum, 4.0%) NaCl. The predominant cellular fatty acids were iso-C15:0 (23.5%), iso-C16:0 (10.2%), iso-C16:0 3OH (10.5%), and iso-C17:0 3OH (16.8%). The DNA G + C contents was 40.4 mol%. The major respiratory quinone was MK-6. The major polar lipids were determined to be phosphatidylethanolamine, two unidentified glycolipids, and two unidentified aminolipids. Several phenotypic characteristics such as production of acetoin, activities of arginine dihydrolase and acid phosphatase, and utilization pattern of carbon sources differentiate strain ALE3EIT from 'A. lentus' JLT2010T. Activities of the lipase, trypsin, α-chymotrypsin and gelatinase and utilization pattern of carbon sources differentiate strain ALE3EIT from M. ophiurae KMM 3046T. The genome of strain ALE3EIT is 3.0 Mbp long and its ANI and AAI values against 'A. lentus' JLT2010T were 76.58 and 72.76, respectively, however, AAI values against members in other genera were lower than 72%. The phylogenomic tree inferred by PhyloPhlAn clearly differentiated the strain ALE3EIT together with strain JLT2010T from other genera in the Falvobacteriaceae. This polyphasic taxonomic data indicates that strain ALE3EIT should be identified as a novel species in the genus 'Altibacter', however, the name has not been validated. Therefore, the strain is classified as a novel genus and is proposed as Constantimarinum furrinae gen. nov., sp. nov. The type strain is ALE3EIT (= KCCM 43303T = JCM 33022T).

The Integrative and Conjugative Element ICECspPOL2 Contributes to the Outbreak of Multi-Antibiotic-Resistant Bacteria for Chryseobacterium Spp. and Elizabethkingia Spp.

Antibiotic resistance genes (ARGs) and horizontal transfer of ARGs among bacterial species in the environment can have serious clinical implications as such transfers can lead to disease outbreaks from multidrug-resistant (MDR) bacteria. Infections due to antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 was isolated from the wastewater of a livestock farm. Whole-genome sequencing and annotation revealed that the POL2 genome encodes dozens of ARGs. The integrative and conjugative element (ICE) ICECspPOL2, which encodes ARGs associated with four types of antibiotics, including carbapenem, was identified in the POL2 genome, and phylogenetic affiliation analysis suggested that ICECspPOL2 evolved from related ICEEas of Elizabethkingia spp. Conjugation assays verified that ICECspPOL2 can horizontally transfer to Elizabethkingia species, suggesting that ICECspPOL2 contributes to the dissemination of multiple ARGs among Chryseobacterium spp. and Elizabethkingia spp. Because Elizabethkingia spp. is associated with clinically significant infections and high mortality, there would be challenges to clinical treatment if these bacteria acquire ICECspPOL2 with its multiple ARGs, especially the carbapenem resistance gene. Therefore, the results of this study support the need for monitoring the dissemination of this type of ICE in Chryseobacterium and Elizabethkingia strains to prevent further outbreaks of MDR bacteria. IMPORTANCE Infections with multiple antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the mobile integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and ICECspPOL2 can horizontally transfer to Elizabethkingia species with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern.

SNPPar: identifying convergent evolution and other homoplasies from microbial whole-genome alignments.

Homoplasic SNPs are considered important signatures of strong (positive) selective pressure, and hence of adaptive evolution for clinically relevant traits such as antibiotic resistance and virulence. Here we present a new tool, SNPPar, for efficient detection and analysis of homoplasic SNPs from large whole genome sequencing datasets (>1000 isolates and/or >100 000 SNPs). SNPPar takes as input an SNP alignment, tree and annotated reference genome, and uses a combination of simple monophyly tests and ancestral state reconstruction (ASR, via TreeTime) to assign mutation events to branches and identify homoplasies. Mutations are annotated at the level of codon and gene, to facilitate analysis of convergent evolution. Testing on simulated data (120 Mycobacterium tuberculosis alignments representing local and global samples) showed SNPPar can detect homoplasic SNPs with very high specificity (zero false-positives in all tests) and high sensitivity (zero false-negatives in 89 % of tests). SNPPar analysis of three empirically sampled datasets (Elizabethkingia anophelis, Burkholderia dolosa and M. tuberculosis) produced results that were in concordance with previous studies, in terms of both individual homoplasies and evidence of convergence at the codon and gene levels. SNPPar analysis of a simulated alignment of ~64 000 genome-wide SNPs from 2000 M. tuberculosis genomes took ~23 min and ~2.6 GB of RAM to generate complete annotated results on a laptop. This analysis required ASR be conducted for only 1.25 % of SNPs, and the ASR step took ~23 s and 0.4 GB of RAM. SNPPar automates the detection and annotation of homoplasic SNPs efficiently and accurately from large SNP alignments. As demonstrated by the examples included here, this information can be readily used to explore the role of homoplasy in parallel and/or convergent evolution at the level of nucleotide, codon and/or gene.

Algibacter onchidii sp. nov., a symbiotic bacterium isolated from a marine invertebrate.

A novel symbiotic bacterium, designated strain XY-114T, was isolated from the cerata of an Onchidium marine invertebrate species collected in the South China Sea. Strain XY-114T was an aerobic, Gram-stain-negative, non-motile and short rod-shaped bacterium (0.5-0.8 µm wide and 1.0-1.5 µm long) without flagellum. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XY-114T belonged to the genus Algibacter with the highest similarity of 97.2 % to the closest phylogenetic relative Algibacter aestuarii KYW371T. Cells grew at 15-37 °C (optimum, 30 °C), at pH 5.5-9.0 (optimum 7.0-8.0) and at NaCl concentrations of 0.5-5.0 % (w/v; optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant respiratory quinone was MK-6. Flexirubin-type pigments were absent. The genome size of strain XY-114T was 3.4 Mbp, with 34.9 mol% of DNA G+C content. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain XY-114T and A. aestuarii KYW371T were 74.5 %, 17.0±1.8 % and 73.9 %. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain XY-114T represents a novel species of the genus Algibacter, for which the name Algibacter onchidii sp. nov. is proposed. The type strain is XY-114T (=KCTC 72217T=MCCC 1K03606T).

Hanstruepera crassostreae He et al. 2018 is a later heterotypic synonym of Pseudobizionia ponticola Park et al. 2018.

Hanstruepera crassostreae L53T was compared with Pseudobizionia ponticola MM-14T to examine the taxonomic relationship between the two type strains. The 16S rRNA gene sequence of H. crassostreae L53T had complete similarity (100.0%) to that of P. ponticola MM-14T. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the two strains formed a tight cluster within the genus Pseudobizionia. Draft genomic comparison between the two strains revealed an average nucleotide identity of 96.9 % and a digital DNA-DNA hybridization estimate of 75.3±2.8 %, strongly indicating that the two strains represented a single species. In addition, neither strain displayed any striking difference in metabolic, physiological or chemotaxonomic features. Therefore, we propose that Hanstruepera crassostreae is a later heterotypic synonym of Pseudobizionia ponticola.

Muricauda onchidii sp. nov., isolated from a marine invertebrate from South China Sea, and transfers of Flagellimonas algicola, Flagellimonas pacifica and Flagellimonas maritima to Muricauda algicola comb. nov., Muricauda parva nom. nov. and Muricauda aurantiaca nom. nov., respectively, and emended description of the genus Muricauda.

An aerobic, Gram-stain-negative, rod-shaped and non-motile strain (XY-359T) was isolated from the mouth of a marine invertebrate Onchidium species from the South China Sea. It grew at pH 6.0-8.5 (optimum, pH 7.5), at 15-37 °C (optimum, 30 °C) and in the presence of 0.5-4.5 % (w/v) NaCl (optimum, 2.5 %). It could not hydrolyse Tweens 20, 40, 60 or 80 and no flexirubin-type pigments were produced. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, six unidentified phospholipids and two unidentified polar lipids. The major fatty acids were iso-C17:0 3-OH, iso-C15:1 G and iso-C15:0 3-OH. The respiratory quinone was MK-6. Strain XY-359T showed the greatest degree of 16S rRNA sequence similarity to Flagellimonas algicola AsT0115T (96.54 %), followed by Muricauda flava DSM 22638T (96.27 %). Phylogenetic analysis based on 16S rRNA gene sequences and 31 core genes indicated that strain XY-359T belongs to the genus Muricauda. The genome size of strain XY-359T was 4 207 872 bp, with 39.1 mol% of DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between strain XY-359T and F. algicola AsT0115T were 74.58 % and 18.5 %, respectively, and those between strain XY-359T and M. flava DSM 22638T were 74.2 % and 18.3 %. The combined phenotypic, chemotaxonomic and phylogenetic data suggest that strain XY-359T represents a novel species of the genus Muricauda, for which the name Muricauda onchidii sp. nov. is proposed. The type strain is XY-359T (=MCCC 1K03658T =KCTC 72218T). Moreover, based on the proposal of nesting Spongiibacterium and Flagellimonas within Muricauda by García (Validation List No. 193) and the analyses of phylogenetic trees and average amino acid identities in this study, the transfers of F. algicola, F. pacifica and F. maritima to the genus Muricauda as Muricauda algicola comb. nov., Muricauda parva nom. nov. and M. aurantiaca nom. nov., respectively, are proposed, with an emended description of the genus Muricauda.

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

Palleronia sediminis sp. nov. and Flavivirga algicola sp. nov., two marine bacteria isolated from offshore areas near Weihai.

Two bacterial strains, designated SS33T and Y03T, were isolated from marine sediment and marine red alga collected on the coast of Weihai, PR China. Based on the results of 16S rRNA gene sequence analysis, strain SS33T was found to be closely related to Primorskyibacter marinus PX7T, Pelagivirga dicentrarchi YLY04T, Palleronia marisminoris DSM 26347T and Maribius pontilimi GH1-23T with 94.8, 94.6, 94.5 and 94.5 % sequence similarity; strain Y03T was found to be closest to Flavivirga aquimarina EC2D5T, Flavivirga eckloniae ECD14T and Flavivirga amylovorans JC2681T with 96.4, 96.1 and 96.0 % sequence similarity. Strain SS33T grew at 4-37 °C (optimum, 33 °C), at pH 6.0-9.5 (optimum, pH 7.5-8.0) and in the presence of 0-10 % (w/v) NaCl (optimum, 3.0 %). Chemotaxonomic analysis of strain SS33T showed that the predominant respiratory quinone was ubiquinone-10. The major fatty acids (>10.0 %) included C18 : 1 ω7c and C16 : 0. The major polar lipids included phosphatidylglycerol, phosphatidylcholine, one unidentified phospholipid, one unidentified glycolipid, one unidentified polar lipid and two unidentified aminolipids. Strain Y03T grew at 15-40 °C (optimum, 28 °C), at pH 6.5-8.0 (optimum, pH 7.0) and in the presence of 0.5-9.0 % (w/v) NaCl (optimum, 2.0%). Chemotaxonomic analysis showed that the predominant respiratory quinone was menaquinone-6. The major fatty acids (>10.0 %) included iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids included phosphatidylethanolamine, one unidentified phospholipid, one unidentified aminolipid and four unidentified polar lipids. Based on the polyphasic data, strain SS33T is considered to represent a novel species of the genus Palleronia, for which the name Palleronia sediminis sp. nov. is proposed, with the type strain SS33T (=KCTC 62986T=MCCC 1H00387T). Strain Y03T is considered to represent a novel species of the genus Flavivirga, for which the name Flavivirga algicola sp. nov. is proposed, with the type strain Y03T (=KCTC 72001T=MCCC 1H00386T).

Marixanthomonas spongiae sp. nov., isolated from marine sponge.

A novel bacterial strain, designated as HN-E44T, was isolated from marine sponge collected from Yangpu Bay, Hainan, PR China. Strain HN-E44T was Gram-stain-negative, non-motile, catalase-positive, oxidase-negative, rod-shaped and yellow-pigmented. Growth occurred at 4-37 °C (optimum, 28 °C), at pH 6-8 (pH 7) and in 0.5-14 % (w/v) NaCl (3-5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HN-E44T formed an independent cluster with Marixanthomonas ophiurae JCM 14121T within the family Flavobacteriaceae and had the highest sequence similarity of 93.6 % to the closest type strain M. ophiurae JCM 14121T. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C15 : 1 G. The polar lipids comprised phosphatidylethanolamine, sphingolipid, four unidentified phospholipids, an unidentified aminophospholipid and an unidentified lipid. The respiratory quinone was identified as MK-6. The genomic DNA G+C content was determined to be 40.6 mol%. The average nucleotide identity (ANI) and average amino acid identity (AAI) values between strain HN-E44T and closest type strain M. ophiurae JCM 14121T were, respectively, 79.6 and 85.2 %, both of which were below thresholds for species delineation (95-96 % ANI and 95-96 % AAI), but were over thresholds for genus delineation (73.98 % ANI and 70-76 % AAI). The combined genotypic and phenotypic distinctiveness demonstrated that strain HN-E44T could be differentiated from closely related genera. Therefore, it is proposed that strain HN-E44T represents a novel species of the genus Marixanthomonas, for which the name Marixanthomonas spongiae sp. nov. is proposed, with the type strain HN-E44T (=MCCC 1K03332T=LMG 30459T).

Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov., two flavobacteria from the epiphytic microbiota of the brown alga Ascophyllum nodosum, and emended description of the genus Zobellia.

Four marine bacterial strains were isolated from a thallus of the brown alga Ascophyllum nodosum collected in Roscoff, France. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, gliding, rod-shaped and grew optimally at 25-30 °C, at pH 7-8 and with 2-4 % NaCl. Phylogenetic analyses of their 16S rRNA gene sequences showed that the bacteria were affiliated to the genus Zobellia (family Flavobacteriaceae, phylum Bacteroidetes). The four strains exhibited 97.8-100 % 16S rRNA gene sequence similarity values among themselves, 97.9-99.1 % to the type strains of Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T, and less than 99 % to other species of the genus Zobellia. The DNA G+C content of the four strains ranged from 36.7 to 37.7 mol%. Average nucleotide identity and digital DNA-DNA hybridization calculations between the new strains and other members of the genus Zobellia resulted in values of 76.4-88.9 % and below 38.5 %, respectively. Phenotypic, phylogenetic and genomic analyses showed that the four strains are distinct from species of the genus Zobellia with validly published names. They represent two novel species of the genus Zobellia, for which the names Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov. are proposed with Asnod1-F08T (RCC6906T=KMM 6823T=CIP 111902T) and Asnod2-B07-BT (RCC6908T=KMM 6825T=CIP 111904T), respectively, as the type strains.

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea.

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.

Mesonia hitae sp. nov., isolated from the seawater of the South Atlantic Ocean.

A Gram-negative, non-motile, non-spore-forming, aerobic and short rod-shaped bacterial strain R32T, was isolated from seawater of the South Atlantic Ocean. Strain R32T grew at 10-40 °C (optimum 28 °C), at pH 6.0-8.0 (optimum 7.0), and in the presence of 3-8 % NaCl (w/v) (optimum 5 %). Cells were oxidase- and catalase-positive. The 16S rRNA gene sequence of strain R32T shared the highest similarities with Mesonia oceanica (98.3 %), followed by Salegentibacter salarius (93.0 %), Salegentibacter mishustinae (92.8 %), Salegentibacter salegens (92.5 %) and Mesonia maritima (92.4 %). The dominant fatty acids were iso-C15 : 0 (32.7 %) and iso-C17 : 0 3-OH (21.1 %). Menaquinone-6 (MK-6) was detected as the sole respiratory quinone. The polar lipids found were phosphatidylethanolamine, three aminolipids and three unidentified lipids. The DNA G+C content was 35.0 mol%. The ANI value and dDDH value between strain R32T and the Salegentibacter and Mesonia species were 70.5-85.8 % and 18.7-30.5 %, respectively. Based on the results of the polyphasic characterization, strain R32T is considered to represent a novel species of the genus Mesonia, for which the name Mesonia hitae sp. nov. is proposed. The type strain is R32T (=MCCC 1A09780T=KCTC 72004T).

Proteorhodopsin Phototrophy in Antarctic Coastal Waters.

Microbial proton-pumping rhodopsins are considered the simplest strategy among phototrophs to conserve energy from light. Proteorhodopsins are the most studied rhodopsins thus far because of their ubiquitous presence in the ocean, except in Antarctica, where they remain understudied. We analyzed proteorhodopsin abundance and transcriptional activity in the Western Antarctic coastal seawaters. Combining quantitative PCR (qPCR) and metagenomics, the relative abundance of proteorhodopsin-bearing bacteria accounted on average for 17, 3.5, and 29.7% of the bacterial community in Chile Bay (South Shetland Islands) during 2014, 2016, and 2017 summer-autumn, respectively. The abundance of proteorhodopsin-bearing bacteria changed in relation to environmental conditions such as chlorophyll a and temperature. Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were the main bacteria that transcribed the proteorhodopsin gene during day and night. Although green light-absorbing proteorhodopsin genes were more abundant than blue-absorbing ones, the latter were transcribed more intensely, resulting in >50% of the proteorhodopsin transcripts during the day and night. Flavobacteriia were the most abundant proteorhodopsin-bearing bacteria in the metagenomes; however, Alphaproteobacteria and Gammaproteobacteria were more represented in the metatranscriptomes, with qPCR quantification suggesting the dominance of the active SAR11 clade. Our results show that proteorhodopsin-bearing bacteria are prevalent in Antarctic coastal waters in late austral summer and early autumn, and their ecological relevance needs to be elucidated to better understand how sunlight energy is used in this marine ecosystem. IMPORTANCE Proteorhodopsin-bearing microorganisms in the Southern Ocean have been overlooked since their discovery in 2000. The present study identify taxonomy and quantify the relative abundance of proteorhodopsin-bearing bacteria and proteorhodopsin gene transcription in the West Antarctic Peninsula's coastal waters. This information is crucial to understand better how sunlight enters this marine environment through alternative ways unrelated to chlorophyll-based strategies. The relative abundance of proteorhodopsin-bearing bacteria seems to be related to environmental parameters (e.g., chlorophyll a, temperature) that change yearly at the coastal water of the West Antarctic Peninsula during the austral late summers and early autumns. Proteorhodopsin-bearing bacteria from Antarctic coastal waters are potentially able to exploit both the green and blue spectrum of sunlight and are a prevalent group during the summer in this polar environment.

Muriicola soli sp. nov., isolated from soil.

A Gram-stain-negative, oxidase-positive, catalase-positive, aerobic, orange-pigmented, rod-shaped and non-motile bacterium designated strain MMS17-SY002T was isolated from island soil. The isolate grew at 20-37 °C (optimum, 30 °C), at pH 6.0-9.5 (optimum, pH 7) and in the presence of 0.5-4.0 % (w/v) NaCl (optimum, 2.0 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MMS17-SY002T was mostly related to the genus Muriicola of the family Flavobacteriaceae and had highest sequence similarity of 96.82 % to Muriicola marianensis A6B8T and Muriicola jejuensis EM44T, but formed a distinct phylogenetic line within the genus. Chemotaxonomic analyses showed that menaquinone 6 was the predominant isoprenoid quinone, the major fatty acids were iso-C15 : 1 G and iso-C15 : 0, and the diagnostic polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 42.4 mol%. Strain MMS17-SY002T could be distinguished from related species by the combination of trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, ß-galactosidase and ß-glucosidase activities. The orthologous average nucleotide identity between the genomes of strain MMS17-SY002T and M. jejuensis and that between the strain and M. marianensis A6B8T were 73.26 and 73.33%, respectively, thus confirming the separation of the strain from related species at species level. Based on the phenotypic, phylogenetic, chemotaxonomic and genomic characterization, MMS17-SY002T should be recognized as a novel species of the genus Muriicola, for which the name Muriicola soli sp. nov. is proposed. The type strain is MMS17-SY002T (=KCTC 62790T=JCM 32370T).

Kordia aestuariivivens sp. nov. and Olleya sediminilitoris sp. nov., isolated from a tidal flat.

Two Gram-stain-negative and non-flagellated bacteria, YSTF-M3T and YSTF-M6T, were isolated from a tidal flat from Yellow Sea, Republic of Korea, and subjected to a polyphasic taxonomic study. Neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strains YSTF-M3T and YSTF-M6T belong to the genera Kordia and Olleya of the family Flavobacteriaceae, respectively. The 16S rRNA gene sequence similarities between strain YSTF-M3T and the type strains of Kordia species and between strain YSTF-M6T and the type strains of Olleya species were 94.1-98.4 and 97.3-98.3 %, respectively. The ANI and dDDH values between genomic sequences of strain YSTF-M3T and the type strains of five Kordia species and between those of strain YSTF-M6T and the type strains of three Olleya species were in ranges of 77.0-83.2 and 20.7-27.1 % and 79.4-81.5 and 22.3-23.9 %, respectively. The DNA G+C contents of strain YSTF-M3T and YSTF-M6T from genomic sequences were 34.1 and 31.1 %, respectively. Both strains contained MK-6 as predominant menaquinone and phosphatidylethanolamine as only major phospholipid identified. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strains YSTF-M3T and YSTF-M6T are separated from recognized species of the genera Kordia and Olleya, respectively. On the basis of the data presented, strains YSTF-M3T (=KACC 21639T=NBRC 114499T) and YSTF-M6T (=KACC 21640T=NBRC 114500T) are considered to represent novel species of the genera Kordia and Olleya, respectively, for which the names Kordia aestuariivivens sp. nov. and Olleya sediminilitoris sp. nov. are proposed.

Salegentibacter maritimus sp. nov., isolated from marine coastal sediment.

Two bacterial strains were isolated from a marine sediment sample taken from Jingzi Wharf, Weihai, China. These two strains were characterized at the phenotypic, chemotaxonomic, and genomic level. The two strains possessed almost identical 16S rRNA gene sequences (99.9 %). However, RAPD-PCR fingerprint patterns discriminated that they were not from one clonal origin. The average nucleotide identity (ANI) value and the digital DNA-DNA hybridization (dDDH) value between the two strains were 98.3 % and 85.4 %, respectively, suggestingthat they belonged to the same species. On the basis of the result of phylogenetic analysis of the 16S rRNA gene sequences, the two strains belonged to the genus Salegentibacter and were closely related to S. holothuriorum KCTC 12371T (98.6 %) and S. salegens DSM 5424T (98.2-98.3 %). The ANI and dDDH clearly separated strains F63223T and F60176 from the the most related type strains with values below the thresholds for species. The genome sizes of strains F63223T and F60176 were approximate 3.89 and 3.59 Mbp, respectively. The strain F63223T had 3,335 predicted genes with DNA G + C content of 35.6 %. The major respiratory quinone was MK-6 and the major polar lipids were phosphatidylethanolamine and one unidentified lipid. According to the results of the phenotypic, chemotaxonomic characterization, phylogenetic classification and genome analysis, the two isolates could be considered to represent a novel species of the genus Salegentibacter, for which the name Salegentibacter maritimus sp. nov., is proposed, with F63223T (=MCCC 1H00433T = KCTC 82417T) as the type strain.

Genome-Wide Analysis of PL7 Alginate Lyases in the Genus Zobellia.

We carried out a detailed investigation of PL7 alginate lyases across the Zobellia genus. The main findings were obtained using the methods of comparative genomics and spatial structure modeling, as well as a phylogenomic approach. Initially, in order to elucidate the alginolytic potential of Zobellia, we calculated the content of polysaccharide lyase (PL) genes in each genome. The genus-specific PLs were PL1, PL6, PL7 (the most abundant), PL14, PL17, and PL40. We revealed that PL7 belongs to subfamilies 3, 5, and 6. They may be involved in local and horizontal gene transfer and gene duplication processes. Most likely, an individual evolution of PL7 genes promotes the genetic variability of the Alginate Utilization System across Zobellia. Apparently, the PL7 alginate lyases may acquire a sub-functionalization due to diversification between in-paralogs.