Cultivating the uncultured: Harnessing the "sandwich agar plate" approach to isolate heme-dependent bacteria from marine sediment.

In the classical microbial isolation technique, the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival. In this study, we designed a simple coculture technique named the "sandwich agar plate method," which maintains microbial interactions throughout the isolation and pure culture processes. The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group. Many uncultured species displayed commensal lifestyles. Further study found that heme was the growth-promoting factor of some marine commensal bacteria. Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa. Moreover, our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably. These findings highlight that cocultivation based on the "sandwich agar plate method" could be developed and used to isolate more uncultured bacteria.

Direct Degradation of Fresh and Dried Macroalgae by Agarivorans albus B2Z047.

Marine macroalgae are increasingly recognized for their significant biological and economic potential. The key to unlocking this potential lies in the efficient degradation of all carbohydrates from the macroalgae biomass. However, a variety of polysaccharides (alginate, cellulose, fucoidan, and laminarin), are difficult to degrade simultaneously in a short time. In this study, the brown alga Saccharina japonica was found to be rapidly and thoroughly degraded by the marine bacterium Agarivorans albus B2Z047. This strain harbors a broad spectrum of carbohydrate-active enzymes capable of degrading various polysaccharides, making it uniquely equipped to efficiently break down both fresh and dried kelp, achieving a hydrolysis rate of up to 52%. A transcriptomic analysis elucidated the presence of pivotal enzyme genes implicated in the degradation pathways of alginate, cellulose, fucoidan, and laminarin. This discovery highlights the bacterium's capability for the efficient and comprehensive conversion of kelp biomass, indicating its significant potential in biotechnological applications for macroalgae resource utilization.

Crohn's disease treatment and memory T-cell subset changes: insights from a case series.

Background: Crohn's disease (CD) is a chronic inflammatory bowel disease with significant morbidity, affecting millions worldwide. The intricacies of immune responses in CD, especially post-treatment, remain a vital area of exploration. While memory T (Tm)-cell subsets play a pivotal role in adaptive immunity, their specific function in patients with CD after treatment is not well-understood. This study aims to investigate the effect and function of Tm-cell subsets in these patients, addressing a crucial knowledge gap in the context of CD therapeutics. Methods: A total of eight patients diagnosed with CD were selected based on predefined inclusion criteria. All patients were treated with either anti-inflammatory agents, immunosuppressive drugs, or a combination of both. For comparison, healthy donors were enrolled based on exclusion of autoimmune or inflammatory diseases. Peripheral blood mononuclear cells (PBMCs) and lymphocytes were isolated from blood and lymph node tissue respectively. The phenotype and cytokine production of T lymphocytes from both CD patients and healthy donors were analyzed using flow cytometry. Statistical comparisons of the outcomes between CD patients and healthy donors were made using Mann-Whitney test (two-tailed) and Student t-test. Results: Post-treatment CD patients exhibited an altered T cell distribution with a notable increase in CD8+ T cells in PBMCs (P=0.0005), and altered frequencies of CD4+ and CD8+ T cells in mesenteric lymph nodes (MLNs). Tm cells showed decreased interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production, with significant alterations in the frequency of IFN-γ-producing CD8+ stem cell-like Tm (Tscm) cells in lesions of the MLNs from patients with CD (CD-M-Lys) compared to healthy MLNs from patients with CD (N-M-Lys) (P=0.0152). Differences in tissue-resident Tm (Trm)-cell subset frequencies were observed between the MLNs and small intestinal mucosa in CD patients. Conclusions: The treatments with anti-inflammatory agents and/or immunosuppressive drugs have a significant effect on the frequency and function of Tm-cell subsets. Clinically, these findings suggest a potential therapeutic avenue in modulating Tm-cell responses, which might be particularly beneficial for conditions where immune response modulation is crucial. Further clinical studies are warranted to explore the full therapeutic implications of these findings.

Short-chain fatty acids (SCFAs) as potential resuscitation factors that promote the isolation and culture of uncultured bacteria in marine sediments.

Many marine bacteria are difficult to culture because they are dormant, rare or found in low-abundances. Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes. However, many more mechanisms remain uncertain. Here, based on 16S rRNA gene high-throughput sequencing and metabolomics technology, it was found that the short-chain fatty acids (SCFAs) in metabolites were significantly correlated with uncultured bacterial groups during enrichment cultures. A pure culture analysis showed that the addition of SCFAs to media also resulted in high efficiency for the isolation of uncultured strains from marine sediments. As a result, 238 strains belonging to 10 phyla, 26 families and 82 species were successfully isolated. Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured. Amongst the newly isolated uncultured microbes, most genomes, e.g. bacteria, possess SCFA oxidative degradation genes, and these features might aid these microbes in better adapting to the culture media. A further resuscitation analysis of a viable but non-culturable (VBNC) Marinilabiliales strain verified that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days, and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate. Overall, this study provides new insights into SCFAs, which were first studied as resuscitation factors in uncultured marine bacteria. Thus, this study can help improve the utilisation and excavation of marine microbial resources, especially for the most-wanted or key players. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00187-w.

Association of Long Noncoding RNA Expression Signatures with Stress-Induced Myocardial Perfusion Defects.

Stress-induced myocardial perfusion defects found in dipyridamole-thallium-201 single-photon emission computed tomography imaging may indicate vascular perfusion abnormalities and risk of obstructive or nonobstructive coronary heart disease. Besides nuclear imaging and subsequent coronary angiography (CAG), no blood test can indicate whether dysregulated homeostasis is associated with stress-induced myocardial perfusion defects. This study investigated the expression signature of long noncoding RNAs (lncRNAs) and genes involved in vascular inflammation and stress response in the blood of patients with stress-induced myocardial perfusion abnormalities (n = 27). The results revealed an expression signature consisting of the upregulation of RMRP (p < 0.01) and downregulations of THRIL (p < 0.01) and HIF1A (p < 0.01) among patients with a positive thallium stress test and no significant coronary artery stenosis within 6 months after baseline treatment. We developed a scoring system based on the expression signatures of RMRP, MIAT, NTT, MALAT1, HSPA1A, and NLRP3 to predict the need for further CAG among patients with moderate-to-significant stress-induced myocardial perfusion defects (area under the receiver operating characteristic curve = 0.963). Therefore, we identified a dysregulated expression profile of lncRNA-based genes in the blood that could be valuable for the early detection of vascular homeostasis imbalance and personalized therapy.

Niche Modification by Sulfate-Reducing Bacteria Drives Microbial Community Assembly in Anoxic Marine Sediments.

Sulfate-reducing bacteria (SRB) are essential functional microbial taxa for degrading organic matter (OM) in anoxic marine environments. However, there are little experimental data regarding how SRB regulates microbial communities. Here, we applied a top-down microbial community management approach by inhibiting SRB to elucidate their contributions to the microbial community during OM degradation. Based on the highly replicated microcosms (n = 20) of five different incubation stages, we found that many microbial community properties were influenced after inhibiting SRB, including the composition, structure, network, and community assembly processes. We also found a strong coexistence pattern between SRB and other abundant phylogenetic lineages via positive frequency-dependent selection. The relative abundances of the families Synergistaceae, Peptostreptococcaceae, Dethiosulfatibacteraceae, Prolixibacteraceae, Marinilabiliaceae, and Marinifilaceae were simultaneously suppressed after inhibiting SRB during OM degradation. A close association between SRB and the order Marinilabiliales among coexisting taxa was most prominent. They contributed to preserved modules during network successions, were keystone nodes mediating the networked community, and contributed to homogeneous ecological selection. The molybdate tolerance test of the isolated strains of Marinilabiliales showed that inhibited SRB (not the inhibitor of SRB itself) triggered a decrease in the relative abundance of Marinilabiliales. We also found that inhibiting SRB resulted in reduced pH, which is unsuitable for the growth of most Marinilabiliales strains, while the addition of pH buffer (HEPES) in SRB-inhibited treatment microcosms restored the pH and the relative abundances of these bacteria. These data supported that SRB could modify niches to affect species coexistence. IMPORTANCE Our model offers insight into the ecological properties of SRB and identifies a previously undocumented dimension of OM degradation. This targeted inhibition approach could provide a novel framework for illustrating how functional microbial taxa associate the composition and structure of the microbial community, molecular ecological network, and community assembly processes. These findings emphasize the importance of SRB during OM degradation. Our results proved the feasibility of the proposed study framework, inhibiting functional taxa at the community level, for illustrating when and to what extent functional taxa can contribute to ecosystem services.

Simultaneous determination of serum tryptophan metabolites in an older Chinese population.

Tryptophan (TRP) and its metabolites exhibit significant biological effects and are strongly associated with age-related disease and mortality. However, reports on quantitatively analyzing these metabolites in older individuals are not available. We used ultra-high-performance liquid chromatography-tandem mass spectrometry to optimize and validate a method for isotope dilution analysis of TRP metabolites in older individuals. The targeted analytes are TRP, serotonin or 5-hydroxytryptamine, kynurenine, kynurenic acid, xanthurenic acid, indole-3-acetic acid, indole-3-propionic acid, and tryptamine. The serum sample was purified using solid-phase extraction and was separated on a Waters HSS T3 column (100 mm × 2.1 mm, 1.8 µm). The analytes were detected in the multiple reaction monitoring mode under positive ionization. TRP was confirmed and measured after being diluted 100 times. This method exhibited satisfactory linearity (r > 0.99). The intrabatch and interbatch accuracies (85.7-114%) and precisions (<15%) were acceptable. The standard-normalized matrix effects ranged from 51.6 to 145%. This method was successfully applied to a cohort of 1021 older Chinese individuals, and this study may enable further understanding of the metabolic phenotypes associated with TRP in other populations.

Microvenator marinus gen. nov., sp. nov., isolated from marine sediment, and description of Microvenatoraceae fam. nov. and Lujinxingiaceae fam. nov.

A Gram-stain-negative, facultatively anaerobic, oxidase-negative and catalase-positive predatory bacillus, designated strain V1718T, was isolated from Xiaoshi Island, PR China. Strain V1718T was found to be closely related to Lujinxingia sediminis SEH01T, with 89.8 % similarity in the 16S rRNA gene sequence, followed by Bradymonas sediminis FA350T with a similarity of 88.4 %. Strain V1718T had the ability to prey on other bacteria, and selective predation on members of Algoriphagus, Nocardioides and Bacillus occurred with the strain. Growth was observed within the range of 20-45 °C (optimal at 37 °C), pH 6.5-9.0 (optimal at pH 8.0) and 1-10 % NaCl (optimal at 3-4 %, w/v). The predominant cellular fatty acids in strain V1718T were iso-C15 : 0 (53.0 %) and C16 : 0 (19.1 %). The major polar lipids present in the strain were phosphatidylglycerol and phosphatidylethanolamine, and the respiratory quinone was menaquinone MK-7. The complete genome sequence of strain V1718T was 5 847 748 bp with a G+C content of 55.2 mol%. The topology of the phylogenomic tree indicated that strain V1718T forms a separate branch in the same clade with the genus Lujinxingia and the family Bradymonadaceae. The average nucleotide identity and average amino acid identity values were 66.4 and 48.6 %, respectively, with Bradymonas sediminis FA350T (type species of Bradymonas) and 66.8 % and 48.9 % with Lujinxingia litoralis B210T (type species of Lujinxingia). The genes related to biosynthesis pathways of several important chemical compounds could not be found in the genome of strain V1718T, which was predicted to be the intrinsic reason for predation in this group. The physiological, biochemical and phylogenetic properties of strain V1718T suggest that it belongs to a novel family distinct from other culturable bradymonabacteria. The name Microvenator marinus gen. nov., sp. nov. is proposed, with strain V1718T (=KCTC 72082T=MCCC 1H00380T) as type strain; the name Microvenatoraceae fam. nov. is also proposed. Meanwhile, the genus Lujinxingia can also be taxonomic classified as Lujinxingiaceae fam. nov. Thus, two novel families and a novel genus of the order Bradymonadales are proposed in this paper.

Degradation of Alginate by a Newly Isolated Marine Bacterium Agarivorans sp. B2Z047.

Alginate is the main component of brown algae, which is an important primary production in marine ecosystems and represents a huge marine biomass. The efficient utilization of alginate depends on alginate lyases to catalyze the degradation, and remains to be further explored. In this study, 354 strains were isolated from the gut of adult abalones, which mainly feed on brown algae. Among them, 100 alginate-degrading strains were gained and the majority belonged to the Gammaproteobacteria, followed by the Bacteroidetes and Alphaproteobacteria. A marine bacterium, Agarivorans sp. B2Z047, had the strongest degradation ability of alginate with the largest degradation circle and the highest enzyme activity. The optimal alginate lyase production medium of strain B2Z047 was determined as 1.1% sodium alginate, 0.3% yeast extract, 1% NaCl, and 0.1% MgSO4 in artificial seawater (pH 7.0). Cells of strain B2Z047 were Gram-stain-negative, aerobic, motile by flagella, short rod-shaped, and approximately 0.7-0.9 µm width and 1.2-1.9 µm length. The optimal growth conditions were determined to be at 30 °C, pH 7.0-8.0, and in 3% (w/v) NaCl. A total of 12 potential alginate lyase genes were identified through whole genome sequencing and prediction, which belonged to polysaccharide lyase family 6, 7, 17, and 38 (PL6, PL7, PL17, and PL38, respectively). Furthermore, the degradation products of nine alginate lyases were detected, among which Aly38A was the first alginate lyase belonging to the PL38 family that has been found to degrade alginate. The combination of alginate lyases functioning in the alginate-degrading process was further demonstrated by the growth curve and alginate lyase production of strain B2Z047 cultivated with or without sodium alginate, as well as the content changes of total sugar and reducing sugar and the transcript levels of alginate lyase genes. A simplified model was proposed to explain the alginate utilization process of Agarivorans sp. B2Z047.

Psychrobacter halodurans sp. nov. and Psychrobacter coccoides sp. nov., two new slightly halophilic bacteria isolated from marine sediment.

In this study, two bacterial strains designated F2608T and F1192T, isolated from marine sediment sampled in Weihai, PR China, were characterized using a polyphasic approach. Strains were aerobic, Gram-stain-negative and motile. According to the results of phylogenetic analyses based on their 16S rRNA genes, these two strains should be classified under the genus Psychrobacter and they both show <98.5% sequence similarity to their closest relative, Psychrobacter celer JCM 12601T. Moreover, strain F2608T showed 97.5% sequence similarity to strain F1192T. Strain F2608T grew at 4-37 °C (optimum, 30-33 °C) and at pH 6.0-9.0 (optimum, pH 6.5-7.0) in the presence of 0-12% (w/v) NaCl (optimum, 4.0-5.0%). Strain F1192T grew at 4-37 °C (optimum, 30 °C) and at pH 5.5-9.0 (optimum, pH 7.0-7.5) in the presence of 0.5-12% (w/v) NaCl (optimum, 3.0-4.0%). The genomic DNA G+C contents of strain F2608T and strain F1192T were 47.4 and 44.9 %, respectively. Genomic characteristics including average nucleotide identity and digital DNA-DNA hybridization values clearly separated strain F2608T from strain F1192T. The sole isoprenoid quinone in these two strains was ubiquinone 8 and the major cellular fatty acids (>10.0%) were C18:1 ω9c and C17:1 ω8c. The major polar lipids of these two strains were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Based on the results of polyphasic analysis, the two strains represent two novel species of the genus Psychrobacter, for which the names Psychrobacter halodurans sp. nov. and Psychrobacter coccoides sp. nov. are proposed. The type strains are F2608T (=MCCC 1K05774T=KCTC 82766T) and F1192T (=MCCC 1K05775T=KCTC 82765T), respectively.

Effect of Restricted Feeding on Metabolic Health and Sleep-Wake Rhythms in Aging Mice.

Aging, an irreversible and unavoidable physiological process in all organisms, is often accompanied by obesity, diabetes, cardiovascular diseases, sleep disorders, and fatigue. Thus, older adults are more likely to experience metabolic symptoms and sleep disturbances than are younger adults. Restricted feeding (RF) is a dietary regimen aimed at improving metabolic health and extending longevity, as well as reorganizing sleep-wake cycles. However, the potential of RF to improve metabolic health and sleep quality in older adults who are known to show a tendency toward increased weight gain and decreased sleep is unknown. To elucidate this issue, aged mice were assigned to an RF protocol during the active phase for 2 h per day for 2 weeks. Sleep-wake cycles were recorded during the RF regime in RF group and control mice. At the end of this period, body weight and blood biochemistry profiles, including blood glucose, cholesterol, and enzyme activity, in addition to dopamine concentrations in the brain, were measured in the RF group and age-matched controls. RF for 2 weeks improved the metabolic health of aged mice by reducing their body weights and blood glucose and cholesterol levels. At the beginning of the RF regime, sleep decreased in the dark period but not in the light period. After stable food entrainment was achieved (7 days post-RF commencement), the amount of time spent in wakefulness during the light period dramatically increased for 2 h before food availability, thereby increasing the mean duration of awake episodes and decreasing the number of wakefulness episodes. There was no significant difference in the sleep-wake time during the dark period in the RF group, with similar total amounts of wakefulness and sleep in a 24-h period to those of the controls. During the RF regime, dopamine levels in the midbrain increased in the RF group, pointing to its potential as the mechanism mediating metabolic symptoms and sleep-wake regulation during RF. In conclusion, our study suggested that RF during aging might prohibit or delay the onset of age-related diseases by improving metabolic health, without having a severe deleterious effect on sleep.

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).

Aquimarina algicola sp. nov., isolated from the surface of a marine red alga.

A bacterial strain, designated M625T, was isolated from the surface of a marine red alga. Phylogenetic trees were reconstructed based on the 16S rRNA gene and RpoB protein sequences, which indicated that the strain belongs to the genus Aquimarina within the family Flavobacteriaceae. Strain M625T showed high sequence similarities to A. aggregata RZW4-3-2 T (95.7%), A. seongsanensis CBA3208T (95.3%) and A. versatilis CBA3207T (95.0%). The AAI and POCP values between strain M625T and A. muelleri DSM 19832 T were 71.8% and 57.9% respectively. The dDDH and ANI values between strain M625T and A. aggregata were 19.5% and 74.6% respectively. The strain was Gram-stain negative, strictly aerobic, non-motile and long rod-shaped, and positive for hydrolysis of starch, cellulose, alginate, DNA and Tween 20. The dominant respiratory quinone was MK-6. The major fatty acids were iso-C15:0, iso-C17:0 3-OH, and iso-C15:1 G, and the polar lipids consisted of phosphatidylethanolamine, one unidentified phospholipid, two unidentified aminolipids, and seven unidentified lipids. Based on the polyphasic comparisons, strain M625T is proposed to represent a novel species within the genus Aquimarina, for which the name Aquimarina algicola sp. nov. (type strain M625T = MCCC 1H00399T = KCTC 72685 T) was proposed.

Planococcus maritimus ML1206 Isolated from Wild Oysters Enhances the Survival of Caenorhabditis elegans against Vibrio anguillarum.

With the widespread occurrence of aquaculture diseases and the broad application of antibiotics, drug-resistant pathogens have increasingly affected aquatic animals' health. Marine probiotics, which live under high pressure in a saltwater environment, show high potential as a substitute for antibiotics in the field of aquatic disease control. In this study, twenty strains of non-hemolytic bacteria were isolated from the intestine of wild oysters and perch, and a model of Caenorhabditis elegans infected by Vibrio anguillarum was established. Based on the model, ML1206, which showed a 99% similarity of 16S rRNA sequence to Planococcus maritimus, was selected as a potential marine probiotic, with strong antibacterial capabilities and great acid and bile salt tolerance, to protect Caenorhabditis elegans from being damaged by Vibrio anguillarum. Combined with plate counting and transmission electron microscopy, it was found that strain ML1206 could significantly inhibit Vibrio anguillarum colonization in the intestinal tract of Caenorhabditis elegans. Acute oral toxicity tests in mice showed that ML1206 was safe and non-toxic. The real-time qPCR results showed a higher expression level of genes related to the antibacterial peptide (ilys-3) and detoxification (ugt-22, cyp-35A3, and cyp-14A3) in the group of Caenorhabditis elegans protected by ML1206 compared to the control group. It is speculated that ML1206, as a potential probiotic, may inhibit the infection caused by Vibrio anguillarum through stimulating Caenorhabditis elegans to secrete antibacterial effectors and detoxification proteins. This paper provides a new direction for screening marine probiotics and an experimental basis to support the potential application of ML1206 as a marine probiotic in aquaculture.

Tenacibaculum pelagium sp. nov., isolated from marine sediment.

A novel bright yellow pigmented, Gram-stain-negative, gliding, aerobic and rod-shaped marine bacterium, designated strain S7007T, was isolated from a marine sediment sample taken from Jingzi Wharf, Weihai, China. The bacterium was able to grow at 4-33 °C (optimum 28 °C), at pH 6.5-9.0 (optimum 7.0) and with 2.0-4.0% (w/v) NaCl (optimum 3.0%). According to the phylogenetic analysis based on the 16S rRNA gene sequences, strain S7007T was associated with the genus Tenacibaculum and showed highest similarity to Tenacibaculum adriaticum JCM 14633T (98.0%). The average nucleotide identity (ANI) scores of strain S7007T with T. adriaticum JCM 14633T and T. maritimum NBRC 110778T were 78.3% and 77.1%, respectively and the Genome-to-Genome Distance Calculator (dDDH) scores were 20.5% and 19.9%, respectively. The sole isoprenoid quinone was MK-6 and the major cellular fatty acids (> 10.0%) were iso-C15:0, iso-C15:0 3-OH, iso-C15: 1 G and summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c). The major polar lipids of strain S7007T were phosphatidylethanolamine, phosphatidyldimethylethanolamine, one unidentified lipid and two unidentified aminolipids. The genomic DNA G + C content was 30.9 mol %. The combined phenotypic data and phylogenetic inference that strain S7007T should be classified as a novel species in the genus Tenacibaculum, for which the name Tenacibaculum pelagium sp. nov. is proposed. The type strain is S7007T (= MCCC 1H00428T = KCTC 72941T).

Strategies for culturing active/dormant marine microbes.

Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service. However, many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture. Although many novel techniques have brought previously uncultured microbes into laboratory culture, there are still many most-wanted or key players that need to be cultured from marine environments. This review discusses possible reasons for 'unculturable microbes' and categorizes uncultured bacteria into three groups: dominant active bacteria, rare active bacteria, and dormant bacteria. This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria. Simulating the natural environment is an effective strategy for isolating dominant active bacteria, whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria. For dormant bacteria, resuscitation culture is an appropriate strategy. Furthermore, the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments. The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.

Bradymonabacteria, a novel bacterial predator group with versatile survival strategies in saline environments.

BACKGROUND: Bacterial predation is an important selective force in microbial community structure and dynamics. However, only a limited number of predatory bacteria have been reported, and their predatory strategies and evolutionary adaptations remain elusive. We recently isolated a novel group of bacterial predators, Bradymonabacteria, representative of the novel order Bradymonadales in δ-Proteobacteria. Compared with those of other bacterial predators (e.g., Myxococcales and Bdellovibrionales), the predatory and living strategies of Bradymonadales are still largely unknown. RESULTS: Based on individual coculture of Bradymonabacteria with 281 prey bacteria, Bradymonabacteria preyed on diverse bacteria but had a high preference for Bacteroidetes. Genomic analysis of 13 recently sequenced Bradymonabacteria indicated that these bacteria had conspicuous metabolic deficiencies, but they could synthesize many polymers, such as polyphosphate and polyhydroxyalkanoates. Dual transcriptome analysis of cocultures of Bradymonabacteria and prey suggested a potential contact-dependent predation mechanism. Comparative genomic analysis with 24 other bacterial predators indicated that Bradymonabacteria had different predatory and living strategies. Furthermore, we identified Bradymonadales from 1552 publicly available 16S rRNA amplicon sequencing samples, indicating that Bradymonadales was widely distributed and highly abundant in saline environments. Phylogenetic analysis showed that there may be six subgroups in this order; each subgroup occupied a different habitat. CONCLUSIONS: Bradymonabacteria have unique living strategies that are transitional between the "obligate" and the so-called facultative predators. Thus, we propose a framework to categorize the current bacterial predators into 3 groups: (i) obligate predators (completely prey-dependent), (ii) facultative predators (facultatively prey-dependent), and (iii) opportunistic predators (prey-independent). Our findings provide an ecological and evolutionary framework for Bradymonadales and highlight their potential ecological roles in saline environments. Video abstract.

Tichowtungia aerotolerans gen. nov., sp. nov., a novel representative of the phylum Kiritimatiellaeota and proposal of Tichowtungiaceae fam. nov., Tichowtungiales ord. nov. and Tichowtungiia class. nov.

Kiritimatiellaeota is widespread and ecologically important in various anoxic environments. However, the portion of culturable bacteria within this phylum is quite low and, in fact, there is only one currently described species. In this study, a novel anaerobic, non-motile, coccoid, Gram-stain-negative bacterial strain, designated S-5007T, was isolated from surface marine sediment. The 16S rRNA gene sequence was found to have very low 16S rRNA gene sequence similarity to the nearest known type strain, Kiritimatiella glycovorans L21-Fru-ABT (84.9 %). The taxonomic position of the novel isolate was investigated using a polyphasic approach and comparative genomic analysis. Phylogenetic analyses based on 16S rRNA genes and genomes indicated that strain S-5007T branched within the radiation of the phylum Kiritimatiellaeota. Different from the type strain, strain S-5007T can grow under microaerobic conditions, and the genomes of strain S-5007T and the other strains in its branch have many more antioxidant-related genes. Meanwhile, other different metabolic features deduced from genome analysis supported the separate evolution of the proposed class (strain S-5007T branch) and K. glycovorans L21-Fru-ABT. Based on phylogenetic and phenotypic characterization studies, Tichowtungia aerotolerans gen. nov., sp. nov. is proposed with S-5007T (=MCCC 1H00402T=KCTC 15876T) as the type strain, as the first representative of novel taxa, Tichowtungiales ord. nov., Tichowtungiaceae fam. nov. in Tichowtungiia class. nov.

Pelagivirga dicentrarchi sp. nov., a member of the family Rhodobacteraceae isolated from the gut microflora of sea bass (Dicentrarchus labrax L.).

A Gram-stain negative, aerobic, non-flagellated, non-gliding, rod-shaped bacterium, designated strain YLY04T, was isolated from the gut microflora of a sea bass (Dicentrarchus labrax L.) collected from the coast of Yuanyao Wharf, Weihai, China. Growth was found to occur at pH 6.0-9.0 (optimum, 7.0-8.0), 4-37 °C (optimum, 28-30 °C) in the presence of 0-11.0% (w/v) NaCl (optimum, 3.0-4.0%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YLY04T is closely related to Pelagivirga sediminicola BH-SD19T and Roseovarius antarcticus M-S13-148T. Strain YLY04T contains ubiquinone-10 as the sole respiratory quinone and summed feature 8 (C18:1ω7c and/or C18:1ω6c), cyclo-C19:0ω8c, C16:0 and 11-methyl-C18:1ω7c as the major fatty acids. The polar lipids of strain YLY04T were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and three unidentified lipids. The DNA G+C content was determined to be 62.7 mol%. The phenotypic, chemotaxonomic and phylogenetic properties, and genome analysis, indicated that strain YLY04T represents a novel species within the genus Pelagivirga, for which the name Pelagivirga dicentrarchi sp. nov. is proposed. The type strain is YLY04T (= MCCC 1H00334T = KCTC 62452T).