Discovery of Terminal Oxazole-Bearing Natural Products by a Targeted Metabologenomic Approach.

A targeted metabologenomic method was developed to selectively discover terminal oxazole-bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole-bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene-targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H-13C coupled-HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl-oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl-oxazolomycins A and B (4 and 5) selectively showed anti-proliferative activity against estrogen receptor-positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

Freshwater Viral Metagenome Analyses Targeting dsDNA Viruses.

Viral metagenomics is one of the most widely used approaches to study viral population genomics. With the recent development of bioinformatic tools, the number of molecular biological methods, programs, and software to analyze viral metagenome data have greatly increased. Here, we describe the basic analysis workflow along with bioinformatic tools that can be used to analyze viral metagenome data. Although this chapter assumes that the viral metagenome data are prepared from the freshwater samples and are subjected to dsDNA sequencing, the protocol can be applied and modified for other types of metagenome data collected from a variety of sources.

Metagenome sequencing and recovery of 444 metagenome-assembled genomes from the biofloc aquaculture system.

Biofloc technology is increasingly recognised as a sustainable aquaculture method. In this technique, bioflocs are generated as microbial aggregates that play pivotal roles in assimilating toxic nitrogenous substances, thereby ensuring high water quality. Despite the crucial roles of the floc-associated bacterial (FAB) community in pathogen control and animal health, earlier microbiota studies have primarily relied on the metataxonomic approaches. Here, we employed shotgun sequencing on eight biofloc metagenomes from a commercial aquaculture system. This resulted in the generation of 106.6 Gbp, and the reconstruction of 444 metagenome-assembled genomes (MAGs). Among the recovered MAGs, 230 were high-quality (≥90% completeness, ≤5% contamination), and 214 were medium-quality (≥50% completeness, ≤10% contamination). Phylogenetic analysis unveiled Rhodobacteraceae as dominant members of the FAB community. The reported metagenomes and MAGs are crucial for elucidating the roles of diverse microorganisms and their functional genes in key processes such as nitrification, denitrification, and remineralization. This study will contribute to scientific understanding of phylogenetic diversity and metabolic capabilities of microbial taxa in aquaculture environments.

Metagenomic data from surface seawater of the east coast of South Korea.

The East Sea, also known as the Sea of Japan, is a marginal sea located in the western Pacific Ocean, displaying comparable characteristics to Earth's oceans, thereby meriting its recognition as a "miniature ocean". The East Sea exhibits a range of annually-recurring biogeochemical features in accordance with seasonal fluctuations, such as phytoplankton blooms during the spring and autumn seasons. Despite ongoing monitoring efforts focused on water quality and physicochemical parameters, the investigation of prokaryotic assemblages in the East Sea, encompassing seasonal variations, has been infrequently pursued. Here, we present a monthly time-series metagenomic dataset spanning a one-year period in 2009, obtained from surface (10 m) seawater samples collected off the coast of the East Sea. The dataset encompasses 12 metagenomes, amounting 195 Gbp, with 14.73-22.52 Gbp per sample. This dataset is accompanied by concurrently measured physicochemical parameters. Our anticipation is that these metagenomes will facilitate extensive investigations aimed at elucidating various aspects of the marine microbial ecosystems in the East Sea.

Zwartia vadi sp. nov., a Novel Species of the GKS98 Cluster Isolated from a Stream, and the Reclassification of 'Achromobacter Panacis' as Zwartia panacis comb. nov.

A Gram-stain-negative, aerobic, motile by gliding, and rod-shaped bacterium, designated IMCC34845T, was isolated from a freshwater stream in the Republic of Korea. The results of 16S rRNA gene-based phylogenetic analyses showed that strain IMCC34845T was affiliated with the genus Zwartia and was most closely related to 'Achromobacter panacis' DCY105T (100%) and Zwartia hollandica LF4-65T (98.9%). The whole-genome sequence of strain IMCC34845T was 3.2 Mbp in size with a 51.5% DNA G+C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain IMCC34845T and 'Achromobacter panacis' KCTC 42751T were 77.7% and 20.7%, respectively, revealing that they are independent species. Moreover, the strains IMCC34845T and KCTC 42751T exhibited ≤ 72.5% ANI and ≤18.5% dDDH values with closely related species Zwartia hollandica LF4-65T, further supporting that the two strains represent each novel species of the genus. The major respiratory quinone of strain IMCC34845T was ubiquinone-8 (Q-8), and the predominant cellular fatty acids were C16:0 (41.3%) and C17:0 cyclo (34.5%). The major polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipids, and unidentified aminolipids. Based on the phylogenetic, genomic, physiological, and chemotaxonomic characteristics, strain IMCC34845T was considered to represent a novel species within the genus Zwartia, for which the name Zwartia vadi sp. nov. is proposed. The type of strain is IMCC34845T (=KCTC 92920T = NBRC 114902T). Furthermore, based on the taxonomic data, 'Achromobacter panacis' is proposed to be reclassified as Zwartia panacis comb. nov.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Cultivation of marine bacteria of the SAR202 clade.

Bacteria of the SAR202 clade, within the phylum Chloroflexota, are ubiquitously distributed in the ocean but have not yet been cultivated in the lab. It has been proposed that ancient expansions of catabolic enzyme paralogs broadened the spectrum of organic compounds that SAR202 bacteria could oxidize, leading to transformations of the Earth's carbon cycle. Here, we report the successful cultivation of SAR202 bacteria from surface seawater using dilution-to-extinction culturing. The growth of these strains is very slow (0.18-0.24 day-1) and is inhibited by exposure to light. The genomes, of ca. 3.08 Mbp, encode archaella (archaeal motility structures) and multiple sets of enzyme paralogs, including 80 genes coding for enolase superfamily enzymes and 44 genes encoding NAD(P)-dependent dehydrogenases. We propose that these enzyme paralogs participate in multiple parallel pathways for non-phosphorylative catabolism of sugars and sugar acids. Indeed, we demonstrate that SAR202 strains can utilize several substrates that are metabolized through the predicted pathways, such as sugars ʟ-fucose and ʟ-rhamnose, as well as their lactone and acid forms.

Proposal of Flavihumibacter fluvii sp. nov. as a replacement name for the effectively published but invalidated epithet Flavihumibacter fluminis Park et al. 2022.

The name Flavihumibacter fluminis Park et al. 2022, which was effectively published but invalidated, is an illegitimate homonymic epithet of Flavihumibacter fluminis Guo et al. 2023. The low 16S rRNA gene sequence similarity and genomic relatedness between the type strains IMCC34837T and RY-1T of the two homonymic species indicated that they are different species. To avoid further confusion, we propose a new name Flavihumibacter fluvii sp. nov. to replace the effectively published but invalidated homonymic epithet Flavihumibacter fluminis Park et al. 2022.

Ten Novel Species Belonging to the Genus Flavobacterium, Isolated from Freshwater Environments: F. praedii sp. nov., F. marginilacus sp. nov., F. aestivum sp. nov., F. flavigenum sp. nov., F. luteolum sp. nov., F. gelatinilyticum sp. nov., F. aquiphilum sp. nov., F. limnophilum sp. nov., F. lacustre sp. nov., and F. eburneipallidum sp. nov.

Eleven bacterial strains were isolated from freshwater environments and identified as Flavobacterium based on 16S rRNA gene sequence analyses. Complete genome sequences of the 11 strains ranged from 3.45 to 5.83 Mb with G + C contents of 33.41-37.31%. The average nucleotide identity (ANI) values showed that strains IMCC34515T and IMCC34518 belonged to the same species, while the other nine strains represented each separate species. The ANI values between the strains and their closest Flavobacterium species exhibited ≤ 91.76%, indicating they represent each novel species. All strains had similar characteristics such as being Gram-stain-negative, rod-shaped, and contained iso-C15:0 as the predominant fatty acid, menaquinone-6 as the respiratory quinone, and phosphatidylethanolamine and aminolipids as major polar lipids. Genomic, phylogenetic, and phenotypic characterization confirmed that the 11 strains were distinct from previously recognized Flavobacterium species. Therefore, Flavobacterium praedii sp. nov. (IMCC34515T = KACC 22282 T = NBRC 114937 T), Flavobacterium marginilacus sp. nov. (IMCC34673T = KACC 22284 T = NBRC 114940 T), Flavobacterium aestivum sp. nov. (IMCC34774T = KACC 22285 T = NBRC 114941 T), Flavobacterium flavigenum sp. nov. (IMCC34775T = KACC 22286 T = NBRC 114942 T), Flavobacterium luteolum sp. nov. (IMCC34776T = KACC 22287 T = NBRC 114943 T), Flavobacterium gelatinilyticum sp. nov. (IMCC34777T = KACC 22288 T = NBRC 114944 T), Flavobacterium aquiphilum sp. nov. (IMCC34779T = KACC 22289 T = NBRC 114945 T), Flavobacterium limnophilum sp. nov. (IMCC36791T = KACC 22290 T = NBRC 114947 T), Flavobacterium lacustre sp. nov. (IMCC36792T = KACC 22291 T = NBRC 114948 T), and Flavobacterium eburneipallidum sp. nov. (IMCC36793T = KACC 22292 T = NBRC 114949 T) are proposed as novel species.

Metabolic Versatility of the Family Halieaceae Revealed by the Genomics of Novel Cultured Isolates.

The family Halieaceae (OM60/NOR5 clade) is a gammaproteobacterial group abundant and cosmopolitan in coastal seawaters and plays an important role in response to phytoplankton blooms. However, the ecophysiology of this family remains understudied because of the vast gap between phylogenetic diversity and cultured representatives. Here, using six pure cultured strains isolated from coastal seawaters, we performed in-depth genomic analyses to provide an overview of the phylogeny and metabolic capabilities of this family. The combined analyses of 16S rRNA genes, genome sequences, and functional genes relevant to taxonomy demonstrated that each strain represents a novel species. Notably, two strains belonged to the hitherto-uncultured NOR5-4 and NOR5-12 subclades. Metabolic reconstructions revealed that the six strains likely have aerobic chemo- or photoheterotrophic lifestyles; five of them possess genes for proteorhodopsin or aerobic anoxygenic phototrophy. The presence of blue- or green-tuned proteorhodopsin in Halieaceae suggested their ability to adapt to light conditions varying with depth or coastal-to-open ocean transition. In addition to the genes of anaplerotic CO2 fixation, genes encoding a complete reductive glycine pathway for CO2 fixation were found in three strains. Putative polysaccharide utilization loci were detected in three strains, suggesting the association with phytoplankton blooms. Read mapping of various metagenomes and metatranscriptomes showed that the six strains are widely distributed and transcriptionally active in marine environments. Overall, the six strains genomically characterized in this study expand the phylogenetic and metabolic diversity of Halieaceae and likely serve as a culture resource for investigating the ecophysiological features of this environmentally relevant bacterial group. IMPORTANCE Although the family Halieaceae (OM60/NOR5 clade) is an abundant and cosmopolitan clade widely found in coastal seas and involved in interactions with phytoplankton, a limited number of cultured isolates are available. In this study, we isolated six pure cultured Halieaceae strains from coastal seawaters and performed a comparative physiological and genomic analysis to give insights into the phylogeny and metabolic potential of this family. The cultured strains exhibited diverse metabolic potential by harboring genes for anaplerotic CO2 fixation, proteorhodopsin, and aerobic anoxygenic phototrophy. Polysaccharide utilization loci detected in some of these strains also indicated an association with phytoplankton blooms. The cultivation of novel strains of Halieaceae and their genomic characteristics largely expanded the phylogenetic and metabolic diversity, which is important for future ecophysiological studies.

Thermomonas paludicola sp. nov., isolated from a lotus wetland.

A Gram-stain-negative, aerobic, rod-shaped and motile bacterium, designated IMCC34681T, was isolated from a lotus wetland in the Republic of Korea. Cellular growth occurred at 10-37 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7) and with 0-2 % (w/v) NaCl (optimum, 0.5 % NaCl). The results of 16S rRNA gene sequence analysis indicated that IMCC34681T represented a member of the genus Thermomonas, sharing 95.3-96.9 % similarities with type strains of species of the genus. The whole-genome sequence of IMCC34681T was 2.72 Mbp in size with 66.2 % DNA G+C content. The IMCC34681T genome shared the highest average nucleotide identity (ANI) value, 82.8 %, with that of Thermomonas brevis KACC 16975T among species of the genus Thermomonas, indicating that the strain represents a novel genomic species. The major respiratory quinone of the strain was ubiquinone-8 (Q-8) and the predominant cellular fatty acids were iso-C15 : 0 (25.7 %) and iso-C14 : 0 (20.8 %). The strain harboured diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid as major fatty polar lipids. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characteristics, IMCC34681T was assigned to the genus Thermomonas as the type strain of a novel species, for which the name Thermomonas paludicola sp. nov. is proposed. The type strain is IMCC34681T (=KACC 21793T=NBRC 114635T).

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Exploring bacterioplankton communities and their temporal dynamics in the rearing water of a biofloc-based shrimp (Litopenaeus vannamei) aquaculture system.

Biofloc technology (BFT) has recently gained considerable attention as a sustainable method in shrimp aquaculture. In a successful BFT system, microbial communities are considered a crucial component in their ability to both improve water quality and control microbial pathogens. Yet, bacterioplankton diversity in rearing water and how bacterioplankton community composition changes with shrimp growth are rarely documented. In this study, the Pacific white shrimp, Litopenaeus vannamei was cultivated in a greenhouse-enclosed BFT system. Rearing water samples were collected on a weekly basis for 5 months (152 days) and water quality variables such as physicochemical parameters and inorganic nutrients were monitored. In parallel, 16S rRNA gene pyrosequencing was employed to investigate the temporal patterns of rearing-water microbiota. The productivity, survival rate, and feed conversion ratio were 3.2-4.4 kg/m3, 74%-89%, and 1.2-1.3, respectively, representing successful super-intensive cultures. The metataxonomic results indicated a highly dynamic bacterioplankton community, with two major shifts over the culture. Members of the phylum Planctomycetes dominated in rearing water during the early stages, while Actinobacteria dominated during the middle stages, and Chloroflexi and TM7 dominated during the late stages of culture. The bacterioplankton community fluctuated more in the beginning but stabilized as the culture progressed. Intriguingly, we observed that certain bacterioplankton groups dominated in a culture-stage-specific manner; these groups include Rhodobacteraceae, Flavobacteriaceae, Actinobacteria, and Chloroflexi, which either contribute to water quality regulation or possess probiotic potential. Altogether, our results indicate that an operationally successful BFT-based aquaculture system favors the growth and dynamics of specific microbial communities in rearing water. Our study expands the scientific understanding of the practical utilization of microbes in sustainable aquaculture. A thorough understanding of rearing-water microbiota and factors influencing their dynamics will help to establish effective management strategies.

Epoxinnamide: An Epoxy Cinnamoyl-Containing Nonribosomal Peptide from an Intertidal Mudflat-Derived Streptomyces sp.

Cinnamoyl-containing nonribosomal peptides (CCNPs) form a unique family of actinobacterial secondary metabolites and display various biological activities. A new CCNP named epoxinnamide (1) was discovered from intertidal mudflat-derived Streptomyces sp. OID44. The structure of 1 was determined by the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data along with a mass spectrum. The absolute configuration of 1 was assigned by the combination of advanced Marfey's method, 3JHH and rotating-frame overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis. The putative biosynthetic pathway of epoxinnamide (1) was identified through the whole-genome sequencing of Streptomyces sp. OID44. In particular, the thioesterase domain in the nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster was proposed as a bifunctional enzyme, which catalyzes both epimerization and macrocyclization. Epoxinnamide (1) induced quinone reductase (QR) activity in murine Hepa-1c1c7 cells by 1.6-fold at 5 µM. It also exhibited effective antiangiogenesis activity in human umbilical vein endothelial cells (IC50 = 13.4 µM).

Flavihumibacter fluminis sp. nov. and Flavihumibacter rivuli sp. nov., isolated from a freshwater stream.

Two Gram-stain-positive, aerobic, chemoheterotrophic, non-motile, rod-shaped, and yellow-pigmented bacterial strains, designated IMCC34837T and IMCC34838T, were isolated from a freshwater stream. Results of 16S rRNA gene-based phylogenetic analyses showed that strains IMCC34837T and IMCC34838T shared 96.3% sequence similarity and were most closely related to Flavihumibacter profundi Chu64-6-1T (99.6%) and Flavihumibacter cheonanensis WS16T (96.4%), respectively. Complete whole-genome sequences of strains IMCC34837T and IMCC34838T were 5.0 Mbp and 4.3 Mbp of genome size with 44.5% and 47.9% of DNA G + C contents, respectively. Average nucleotide identity (ANI) and digital DNA- DNA hybridization (dDDH) values between the two strains were 70.0% and 17.9%, repectively, revealing that they are independent species. The two strains showed ≤ 75.2% ANI and ≤ 19.3% dDDH values to each closely related species of the genus Flavihumibacter, indicating that the two strains represent each novel species. Major fatty acid constituents of strain IMCC34837T were iso-C15:0, iso-C15:1 G and anteiso-C15:0 and those of strain IMCC34838T were iso-C15:0 and iso-C15:1 G. The predominant isoprenoid quinone detected in both strains was menaquinone-7 (MK-7). Major polar lipids of both strains were phosphatidylethanolamine, aminolipids, and glycolipids. Based on the phylogenetic and phenotypic characterization, strains IMCC34837T and IMCC34838T were considered to represent two novel species within the genus Flavihumibacter, for which the names Flavihumibacter fluminis sp. nov. and Flavihumibacter rivuli sp. nov. are proposed with IMCC34837T (= KACC 21752T = NBRC 115292T) and IMCC34838T (= KACC 21753T = NBRC 115293T) as the type strains, respectively.

Taeanamides A and B, Nonribosomal Lipo-Decapeptides Isolated from an Intertidal-Mudflat-Derived Streptomyces sp.

Two new lipo-decapeptides, namely taeanamides A and B (1 and 2), were discovered from the Gram-positive bacterium Streptomyces sp. AMD43, which was isolated from a mudflat sample from Anmyeondo, Korea. The exact molecular masses of 1 and 2 were revealed by high-resolution mass spectrometry, and the planar structures of 1 and 2 were elucidated using NMR spectroscopy. The absolute configurations of 1 and 2 were determined using a combined analysis of 1H-1H coupling constants and ROESY correlations, the advanced Marfey's method, and bioinformatics. The putative nonribosomal peptide synthetase pathway for the taeanamides was identified by analyzing the full genome sequence data of Streptomyces sp. AMD43. We also found that taeanamide A exhibited mild anti-tuberculosis bioactivity, whereas taeanamide B showed significant bioactivity against several cancer cell lines.

Heme auxotrophy in abundant aquatic microbial lineages.

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms. Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment. These heme auxotroph groups are largely limited to pathogens, symbionts, or microorganisms living in nutrient-replete conditions, whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms. Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme, based on the experimental or genomic evidence. We found that two recently cultivated acI isolates require exogenous heme for their growth. One of the cultured acI isolates also exhibited auxotrophy for riboflavin. According to whole-genome analyses, all (n = 20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and Marine groups IIb and III of Euryarchaeota Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity.

A sulfate-reducing bacterial genus, Desulfosediminicola gen. nov., comprising two novel species cultivated from tidal-flat sediments.

Tidal-flat sediments harbor a diverse array of sulfate-reducing bacteria. To isolate novel sulfate-reducing bacteria and determine their abundance, a tidal-flat sediment sample collected off Ganghwa Island (Korea) was investigated using cultivation-based and culture-independent approaches. Two Gram-stain-negative, strictly anaerobic, rod-shaped, sulfate-reducing bacteria, designated IMCC35004T and IMCC35005T, were isolated from the sample. The two strains reduced sulfate, sulfite, elemental sulfur, thiosulfate, Fe(III) citrate, and Mn(IV) oxide by utilizing several carbon sources, including acetate. The 16S rRNA gene amplicon sequencing revealed that the tidal-flat sediment contained diverse members of the phylum Desulfobacterota, and the phylotypes related to IMCC35004T and IMCC35005T were < 1%. The two strains shared 97.6% similarity in 16S rRNA gene sequence and were closely related to Desulfopila aestuarii DSM 18488T (96.1-96.5%). The average nucleotide identity, level of digital DNA-DNA hybridization, average amino acid identity, and percentages of conserved proteins determined analyzing the whole-genome sequences, as well as the chemotaxonomic data showed that the two strains belong to two novel species of a novel genus. Additionally, genes related to dissimilatory sulfate reduction were detected in the genomes of the two strains. Unlike the genera Desulfopila and Desulfotalea, IMCC35004T and IMCC35005T contained menaquinone-5 as the major respiratory quinone. Collectively, IMCC35004T and IMCC35005T were concluded to represent two novel species of a novel genus within the family Desulfocapsaceae, for which the names Desulfosediminicola ganghwensis gen. nov., sp. nov. (IMCC35004T = KCTC 15826T = NBRC 114003T) and Desulfosediminicola flagellatus sp. nov. (IMCC35005T = KCTC 15827T = NBRC 114004T) are proposed.

Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3-20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

Uliginosibacterium aquaticum sp. nov., Isolated from a Freshwater Lake.

A Gram-stain-negative, aerobic, chemoheterotrophic, rod-shaped bacterium motile by a polar flagellum, designated IMCC34675T, was isolated from Chungju Lake, an artificial freshwater reservoir in Republic of Korea. The 16S rRNA gene sequence analysis indicated that strain IMCC34675T belongs to the genus Uliginosibacterium, sharing ≤ 97.1% sequence similarities with the type strains of the genus. Whole genome sequencing of strain IMCC34675T revealed a 4.1 Mbp of genome size with 62.4% of the DNA G + C content. The IMCC34675T genome shared 73.3% of average nucleotide identity and 19.9% of digital DNA-DNA hybridization values to the genome of Uliginosibacterium gangwonense DSM 18521T, the type species of the genus. The major fatty acids of strain IMCC34675T were summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c) and C16:0. The respiratory quinone detected in the strains was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one aminolipid, and five unidentified lipids. Based on the phylogenetic and phenotypic characterization, strain IMCC34675T was considered to represent a novel species within the genus Uliginosibacterium, for which the name Uliginosibacterium aquaticum sp. nov. is proposed with IMCC34675T (= KACC 21758T = NBRC 114418T) as the type strain.