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.

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.

Gaoshiqia sediminis gen. nov., sp. nov., isolated from coastal sediment.

A Gram-stain-negative, facultative anaerobic, motile, rod-shaped and orange bacterium, designated A06T, was obtained off the coast of Weihai, PR China. Cells were 0.4-0.5×0.6-1.0 µm in size. Strain A06T grew at 20-40 °C (optimum, 33 °C), at pH 6.0-8.0 (optimum, pH 6.5-7.0) and in the presence of 0-8 % NaCl (w/v) (optimum, 2 %). Cells were oxidase and catalase positive. Menaquinone-7 was detected as the major respiratory quinone. The dominant cellular fatty acids were identified as C15:0 2-OH, iso-C15:0, anteiso-C15:0 and iso-C15:1 ω6c. The DNA G+C content of strain A06T was 46.1 mol%. The polar lipids were phosphatidylethanolamine, one aminolipid, one glycolipid and three unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A06T is a member of the family Prolixibacteraceae and exhibited the highest sequence similarity to Mangrovibacterium diazotrophicum DSM 27148T (94.3 %). Based on its phylogenetic and phenotypic characteristics, strain A06T is considered to represent a novel genus in the family Prolixibacteraceae, for which the name Gaoshiqia gen. nov. is proposed. The type species is Gaoshiqia sediminis sp. nov., with type strain A06T (=KCTC 92029T=MCCC 1H00491T). The identification and acquisition of microbial species and genes in sediments will help broaden the understanding of microbial resources and lay a foundation for its application in biotechnology. Strain A06T uses an enrichment method, so the isolation of strain A06T is of great significance to the enrichment of marine microbial resources.

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.

Sulfuriroseicoccus oceanibius gen. nov., sp. nov., a representative of the phylum Verrucomicrobia with a special cytoplasmic membrane.

Here, we describe a novel bacterial strain, designated T37T, which was isolated from the marine sediment of Xiaoshi Island, PR China. Growth of strain T37T occurs at 15-40 °C (optimum 37 °C), pH 6.0-9.0 (optimum 7.5), and in the presence of 0.5-5.5% (w/v) NaCl (optimum 1.5%). Characteristic biochemical traits of the novel strain include MK-9 as the major menaquinone. The major fatty acids identified were iso-C14:0 and C16:1 ω9c (oleic acid). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphoglycolipids were the major cellular polar lipids. The G + C content of genomic DNA was 58.4 mol%. Unusual outer membrane features deduced from the analysis of cell morphology point towards the formation of an enlarged periplasmic space putatively used for the digestion of macromolecules. Phylogenetic analyses based on 16S rRNA genes and the genome indicated that strain T37T represents a novel species and genus affiliated with a distinct family level lineage of the verrucomicrobial subdivision 1. Our polyphasic taxonomy approach places the novel strain in a new genus within the current family Verrucomicrobiaceae, order Verrucomicrobiales, class Verrucomicrobiae. Strain T37T (= KCTC 72799 T = MCCC 1H00391T) is the type strain of a novel species, for which the name Sulfuriroseicoccus oceanibius gen. nov., sp. nov. is proposed.

Inducible expression of agar-degrading genes in a marine bacterium Catenovulum maritimus Q1T and characterization of a ß-agarase.

Agar-degrading bacteria are crucial drivers for the carbon cycle in the marine environments due to their ability that use algae as a carbon source. Although numerous agar-degrading bacteria and agarases have been reported, little is known about expression levels of agar-degrading genes in wild strains. Here, the genome of an agar-hydrolyzing marine bacterium, Catenovulum maritimus Q1T, was sequenced and annotated with 11 agarase and 2 neoagarooligosaccharide hydrolase genes. Quantitative PCR revealed that all the annotated agar-degrading genes were expressed consistently that initially upregulated and then gradually downregulated under agarose induction. Moreover, the presence of glucose inhibited the agar-degrading ability, in terms of both gene expression and enzymatic activity. These facts indicated the agar-degrading ability of wild bacteria was mainly induced by agarose and repressed by the available carbon source. Additionally, a ß-agarase, AgaQ1, belonging to the GH16 family, with high expression in strain Q1T, was cloned and characterized. Biochemical analysis showed that the recombinant AgaQ1 was substrate-specific, yielding neoagarotetraose and neoagarohexaose as the main products. It exhibited optimal activity at 40 °C, pH 8.0, and an agarose concentration of 1.6% (w/v). Besides, AgaQ1 showed a high-specific activity (757.7 U/mg) and stable enzymatic activity under different ion or agent treatments; thus, AgaQ1 has great potential in industrial applications. KEY POINTS: • The genome of C. maritimus Q1T was sequenced and annotated with 11 agarases and 2 Nabh genes. • The expression of agar-degrading genes in the strain C. maritimus Q1T was induced by agarose. • Glucose was the carbon source utilized prior to agarose for bacterial growth. • A ß-agarase, AgaQ1, with high expression and activity was identified.

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.

Thauera lacus sp. nov., isolated from a saline lake in Inner Mongolia.

A Gram-stain-negative, facultatively anaerobic, motile and rod-shaped bacterium, designated D20T, was isolated from the saline Lake Dai in Inner Mongolia, PR China. Growth of strain D20T occurred at 25-45 °C (optimum, 40 °C), pH 4.0-12.0 (optimum, 8.0) and with 0-3 % NaCl (w/v); (optimum, 0-1 %). The results of 16S rRNA gene sequence analysis revealed that strain D20T was most closely related to three Thauera species, Thaueraselenatis AXT, Thaueraaminoaromatica S2T and Thaueraaromatica K172T, with a similarity value of 96.2 %. The major respiratory quinone of strain D20T was ubiquinone-8 (Q-8), and the dominant fatty acids (>10 %) were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 39.8 %), C16 : 0 (30.9 %) and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c; 13.5 %). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one aminophospholipid and five unidentified lipids. The DNA G+C content was 67.2 mol% (data from the genome sequence). The estimated genome size was 3.7 Mb. The phenotypic, genotypic and chemotaxonomic differences between strain D20T and its phylogenetic relatives indicated that strain D20T should be regarded as a novel species in the genus Thauera, for which the name Thaueralacus sp. nov. is proposed. The type strain is D20T (=MCCC 1H00305T=KCTC 62586T).

Metatranscriptomic and comparative genomic insights into resuscitation mechanisms during enrichment culturing.

BACKGROUND: The pure culture of prokaryotes remains essential to elucidating the role of these organisms. Scientists have reasoned that hard to cultivate microorganisms might grow in pure culture if provided with the chemical components of their natural environment. However, most microbial species in the biosphere that would otherwise be "culturable" may fail to grow because of their growth state in nature, such as dormancy. That means even if scientist would provide microorganisms with the natural environment, such dormant microorganisms probably still remain in a dormant state. RESULTS: We constructed an enrichment culture system for high-efficiency isolation of uncultured strains from marine sediment. Degree of enrichment analysis, dormant and active taxa calculation, viable but non-culturable bacteria resuscitation analysis, combined with metatranscriptomic and comparative genomic analyses of the interactions between microbial communications during enrichment culture showed that the so-called enrichment method could culture the "uncultured" not only through enriching the abundance of "uncultured," but also through the resuscitation mechanism. In addition, the enrichment culture was a complicated mixed culture system, which contains the competition, cooperation, or coordination among bacterial communities, compared with pure cultures. CONCLUSIONS: Considering that cultivation techniques must evolve further-from axenic to mixed cultures-for us to fully understand the microbial world, we should redevelop an understanding of the classic enrichment culture method. Enrichment culture methods can be developed and used to construct a model for analyzing mixed cultures and exploring microbial dark matter. This study provides a new train of thought to mining marine microbial dark matter based on mixed cultures.

Hanstruepera crassostreae sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from an oyster.

A Gram-stain-negative, rod-shaped, non-motile, aerobic and orange-pigmented marine bacterium, designated strain L53T, was isolated from an oyster sample collected from the coast of Weihai, China (122.0° E 37.5° N). Growth of strain L53T occurred at 4-40 °C (optimum, 33 °C), at pH 6.5-8.0 (optimum, pH 7.5) and with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain L53T was closely related to Bizionia echini KCTC 22015T (96.9 %) and Hanstruepera neustonica JCM 19743T (96.1 %). Strain L53T was located in a distinct phyletic lineage in a discrete clade associated with H. neustonica JCM 19743T. The DNA G+C content of strain L53T was 33.5 mol%. The sole menaquinone was MK-6. The polar lipids comprised one phosphatidylethanolamine, four unidentified aminolipids and four unidentified lipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 0 G. Based on morphological, physiological and molecular properties as well as on phylogenetic distinctiveness, strain L53T should be placed in the genus Hanstruepera as representing a novel species, for which the name Hanstruepera crassostreae sp. nov. is proposed. The type strain is L53T (=KCTC 62247T=MCCC 1H00246T).

Salegentibacter sediminis sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from coastal sediment.

A Gram-stain-negative, aerobic, non-motile, rod-shaped (0.2-0.3×0.8-1.4 µm) and yellow-pigmented bacterium, designated K5023T, was isolated from marine sediment obtained off the coast of Weihai, China. Strain K5023T was found to grow at 16-37 °C (optimum, 33 °C), at pH 6.5-8.0 (optimum, 7.0-7.5) and in the presence of 1-10 % (w/v) NaCl (optimum, 5 %). Cells were oxidase-positive and catalase-negative. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain K5023T is a member of the genus Salegentibacter and exhibited the hightest sequence similarity to Salegentibacter flavus DSM 22702T (97.0 %). Menaquinone-6 (MK-6) was detected as the major respiratory quinone. The dominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The DNA G+C content of strain K5023T was 40.1 mol%. The polar lipids included one phosphatidylethanolamine, three phospholipids and four unidentified lipids. Based on the phylogenetic and phenotypic characteristics, strain K5023T is considered to represent a novel species of the genus Salegentibacter, for which the name Salegentibacter sediminis sp. nov. is proposed. The type strain is K5023T (=KCTC 52477T=MCCC 1H00173T).

Pistricoccus aurantiacus gen. nov., sp. nov., a moderately halophilic bacterium isolated from a shark.

A novel Gram-stain negative, non-motile, moderately halophilic, facultatively anaerobic and spherical bacterium designated strain SS9T was isolated from the gill homogenate of a shark. Cells of SS9T were observed to be 0.8-1.2 µm in diameter. The strain was found to grow optimally at 33 °C, pH 7.0-8.0 and in the presence of 6.0 % (w/v) NaCl. On the basis of 16S rRNA gene phylogeny, strain SS9T can be affiliated with the family Halomonadaceae and is closely related to Chromohalobacter marismortui NBRC 103155T (95.6 % sequence similarity), Halomonas ilicicola SP8T (95.6 %) and Chromohalobacter salexigens DSM 3043T (95.5 %). Multilocus sequence analysis of strain SS9T using the housekeeping genes 16S rRNA, 23S rRNA, gyrB, rpoD and secA revealed the strain's distinct phylogenetic position, separate from other known genera of the family Halomonadaceae. Strain SS9T was found to contain ubiquinone-9 (Q-9) as the predominant ubiquinone and C18:1 ω7c, C16:0 and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) as the major fatty acids. The major polar lipids of strain SS9T were identified as phosphatidylglycerol and phosphatidylethanolamine. The DNA G + C content of strain SS9T was determined to be 60.4 mol%. It is evident from phylogenetic, genotypic, phenotypic and chemotaxonomic results that strain SS9T represents a novel species in a new genus, for which the name Pistricoccus aurantiacus gen. nov., sp. nov. is proposed. The type strain is SS9T (=KCTC 42586T = MCCC 1H00111T).

Thalassotalea sediminis sp. nov., isolated from coastal sediment.

A novel Gram-stain negative, facultatively anaerobic, rod-shaped and yellowish-white-pigmented marine bacterium, designated strain N211(T), was isolated from marine sediment obtained off the coastal area of Weihai, China. The cells are approximately 0.4-0.9 × 1.8-3.5 µm in size and motile by means of a polar flagellum. The strain grows optimally at 33 °C, pH 7.5-8.5 and in the presence of 3.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences indicated that strain N211(T) fell within the clade comprising the type strains of species of the genus Thalassotalea. Strain N211(T) was most closely related to Thalassotalea ganghwensis DSM 15355(T) (96.4 %) based on the 16S rRNA gene sequence, and shared 94.4-96.4 % similarity with type strains of all members of the genus Thalassotalea. The predominant isoprenoid quinone of strain N211(T) was identified as ubiquinone-8 (Q-8). The major polar lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and an unidentified lipid. C17:1 w8c, summed feature 3 (C16:1 w7c and/or iso-C 15:0 2-OH) and C16:0 were found to be main cellular fatty acids. The G+C content of the genomic DNA is 39.1 mol%. It is evident from phenotypic data and phylogenetic inference that strain N211(T) represents a novel species of the genus Thalassotalea, for which the name T. sediminis sp. nov. is proposed. The type strain is N211(T) (=KCTC 42588(T) = MCCC 1H00116(T)).