Microbial associations of shallow-water Mediterranean marine cave Solenogastres (Mollusca)

The first cave-dwelling Solenogastres—marine shell-less worm-like mollusks—were sampled from Mediterranean marine caves floor silt in the Marseille area. The mollusks were 1.5 mm in length, had a transparent body with shiny spicules and appear to represent a new Tegulaherpia species. Electron microscopy revealed a high number of microbial cells, located on the surface of the spicules as well as in the cuticle of Tegulaherpia sp. The observed microbial cells varied in morphology and were unequally distributed through the cuticle, reaching a highest density on the dorsal and lateral sides and being practically absent on the ventral side. Next Generation Sequencing (NGS) of V4 region of 16S rRNA gene amplicons, obtained from the DNA samples of whole bodies of Tegulaherpia sp. revealed three dominating microorganisms, two of which were bacteria of Bacteroidetes and Nitrospirae phyla, while the third one represented archaea of Thaumarchaeota phylum. The Operational Taxonomic Unit (OTU), affiliated with Bacteroidetes was an uncultured bacteria of the family Saprospiraceae (93–95% of Bacteroidetes and 25–44% of the total community, depending on sample), OTU, affiliated with Nitrospirae belonged to the genus Nitrospira (8–30% of the community), while the thaumarchaeal OTU was classified as Candidatus Nitrosopumilus (11–15% of the community). Members of these three microbial taxa are known to form associations with various marine animals such as sponges or snails where they contribute to nitrogen metabolism or the decomposition of biopolymers. A similar role is assumed to be played by the microorganisms associated with Tegulaherpia sp.

MINTIA: a metagenomic INserT integrated assembly and annotation tool

The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes. This approach is based on the insertion of large metagenomic DNA fragments into a vector and transformation of a host to express heterologous genes. Metagenomic libraries are then screened for activities of interest, and the metagenomic DNA inserts of active clones are extracted to be sequenced and analysed to identify genes that are responsible for the detected activity. Hundreds of metagenomics sequences found using this strategy have already been published in public databases. Here we present the MINTIA software package enabling biologists to easily generate and analyze large metagenomic sequence sets, retrieved after activity-based screening. It filters reads, performs assembly, removes cloning vector, annotates open reading frames and generates user friendly reports as well as files ready for submission to international sequence repositories. The software package can be downloaded from https://github.com/Bios4Biol/MINTIA.

Biodiversity and Phylogenetic Relationship of Total Hydrocarbon Degrading Genes in Selected Bacteria Species

Hydrocarbons which forms the bulk of soil and water pollutants in the Niger Delta region of Nigeria differs in their susceptibility to microbial attack and degradation. Considering the importance of hydrocarbons in the economy of any nation, a complete knowledge of the hydrocarbons degrading capabilities of associated microbial species becomes increasingly important and indispensable. The research was aimed at assessing the biodiversity and phylogenetic relationship of hydrocarbon degrading genes in selected bacteria species. Sequences of nucleotides and amino acids of hydrocarbons degrading genes in 12 species of bacteria such as Pseudomonas aeruginosa I & II, P. stutgeni, Thalassosqir spp. I & II, Alcavorox spp., Arthrobacter spp., Martellela spp., P. taenensis, Aneuribacillus species, Rhodococcus spp. and Uncultured bacteria, were retrieved from the National Center for Biotechnology Information (NCBI) and analysed for their variability in physicochemical properties, percentage identity and similarity, G-C content, secondary and tertiary structures, their biodiversity and their phylogenetic relationship using MEGA 6 soft-wares, GOR IV, Phyre, Genscan and SIB Protparam. The analysis showed great genetic diversity and polymorphism in the hydrocarbon degrading genes present in the bacteria species. The phylogenetic tree showed that the genes in most species had undergone mutation. Martella spp had the highest probability or chance of mutating with E-value of 7e-129. Only two of the bacteria species had low instability index indicating that majority of the bacteria species contains or possessed the unstable nature of the hydrocarbon degrading genes. The biodiversity of bacterial species and their genetic distance was ascertained.

Comparison of culture-based strategies for isolating bacteria from the porcine intestine

The isolation of bacteria that represent the diversity of autochthonous taxa in the gastrointestinal tract is necessary to fully ascertain function, but the majority of bacterial species inhabiting the intestines of mammals are fastidious, and thus challenging to isolate. The goal of the current study was to isolate a diverse assemblage of bacteria from the intestine of pigs as a model animal, and to comparatively examine various novel and traditional isolation strategies. Methods used included long-term enrichments, direct plating, a modified Ichip, as well as ethanol and Tyndallization treatment of samples to select for endospore-forming taxa. A total of 234 taxa (91 previously uncultured) comprising 80 genera and seven phyla were isolated from mucosal and luminal samples from the ileum, cecum, ascending colon, and spiral colon removed from animals under anesthesia. The diversity of bacteria isolated from the large intestine was less than detected by next-generation sequence analysis. Long-term enrichments yielded the greatest diversity of recovered bacteria (Shannon’s index [SI] = 4.7). Methods designed to isolate endospore-forming bacteria produced the lowest diversity (SI ≤ 2.7), with Tyndallization yielding a lower diversity than the ethanol method. However, the isolation frequency of previously uncultured bacteria was greatest for ethanol-treated samples (41.9%) and the Ichip method (32.5%). The goal of recovering a diverse collection of enteric bacteria was achieved. Importantly, study findings demonstrate that it is necessary to use a combination of methods in concert to isolate bacteria that are representative of the diversity within the intestines of mammals. IMPORTANCE This work determined that using a combination of isolation methods is necessary to increase the diversity of bacteria recovered from the intestines of monogastric mammals. Direct plating methods have traditionally been used to isolate enteric bacteria, and recent methods (e.g. diffusion methods [i.e. Ichip] or differential isolation of endospore-forming bacteria) have been suggested to be superior at increasing diversity, including the recovery of previously uncultured taxa. We showed that long-term enrichment of samples using a variety of media isolated the most diverse and novel bacteria. Application of the Ichip method delivered similar diversity of bacteria to enrichment and direct plating methods. Methods that selected for endospore-forming bacteria generated collections that differed in composition to other methods with reduced diversity. However, the ethanol treatment frequently isolated novel bacteria. By using a combination of methods in concert, a diverse collection of enteric bacteria was generated for ancillary experimentation.

Revealing of Non-Cultivable Bacteria Associated with the Mycelium of Fungi in the Kerosene-Degrading Community Isolated from the Contaminated Jet Fuel

Fuel (especially kerosene) biodamage is a challenge for global industry. In aviation, where kerosene is a widely used type of fuel, its biodeterioration leads to significant damage. Six isolates of micromycetes from the TS-1 aviation kerosene samples were obtained. Their ability to grow on the fuel was studied, and the difference between biodegradation ability was shown. Micromycetes belonged to the Talaromyces, Penicillium, and Aspergillus genera. It was impossible to obtain bacterial isolates associated with their mycelium. However, 16S rRNA metabarcoding and microscopic observations revealed the presence of bacteria in the micromycete isolates. It seems to be that kerosene-degrading fungi were associated with uncultured bacteria. Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were abundant in the fungal cultures isolated from the TS-1 jet fuel samples. Most genera among these phyla are known as hydrocarbon degraders. Only bacteria-containing micromycete isolates were able to grow on the kerosene. Most likely, kerosene degradation mechanisms are based on synergism of bacteria and fungi.

New Microbial Biodiversity in Marine Sediments

Microbes in marine sediments represent a large portion of the biosphere, and resolving their ecology is crucial for understanding global ocean processes. Single-gene diversity surveys have revealed several uncultured lineages that are widespread in ocean sediments and whose ecological roles are unknown, and advancements in the computational analysis of increasingly large genomic data sets have made it possible to reconstruct individual genomes from complex microbial communities. Using these metagenomic approaches to characterize sediments is transforming our view of microbial communities on the ocean floor and the biodiversity of the planet. In recent years, marine sediments have been a prominent source of new lineages in the tree of life. The incorporation of these lineages into existing phylogenies has revealed that many belong to distinct phyla, including archaeal phyla that are advancing our understanding of the origins of cellular complexity and eukaryotes. Detailed comparisons of the metabolic potentials of these new lineages have made it clear that uncultured bacteria and archaea are capable of mediating key previously undescribed steps in carbon and nutrient cycling.

Metabolic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes. However, few complete BGCs from uncultivated bacteria have been recovered, making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly complete BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. The phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota, but also the actinobacterial classes Acidimicrobiia, Thermoleophilia, and the gammaproteobacterial order UBA7966, were found to encode a large number of highly divergent BGCs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped reservoir of specialised metabolites of the uncultured majority of microbes.

Metagenomic Analysis Reveals the Mechanism for the Observed Increase in Antibacterial Activity of Penicillin against Uncultured Bacteria Candidatus Liberibacter asiaticus Relative to Oxytetracycline in Planta

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.

A Novel Culturing Chip (cChip) Can Facilitate Culturing of Unculturable Bacteria From Aquatic Environment

Background: Culturing the unculturable microorganisms is an important aspect of microbiology. Once cultured the unculturable microorganisms can be a source of useful antibiotics, enzymes etc. Several studies have been conducted on culturing the unculturable microorganisms from soil. But little knowledge exists about culturing such bacteria from aquatic environment. Therefore, in this study we designed a novel culturing chip (cChip) to facilitate the growth of unculturable aquatic bacterial community. cChip was optimized for microbial growth using known bacteria in the lab, later, microbes from a freshwater lake were concentrated (instead of using the traditional dilution method) and inoculated in cChip before incubating the chip in simulated lake environment. Then further sub-culturing was done on laboratory media. The field samples were also analyzed using traditional culturing and metagenomics for a comparison. Results: Metagenomics analysis showed that 832 microbial species were present in the samples belonging to five different phyla, that is, Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Verrucomicrobia. However, traditional culturing yielded only 36 isolates that belonged to four different phyla, that is, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Culturing through cChip yielded 154 isolates belonging to five different phyla, that is, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia and Firmicutes. Out of these 154 cChip isolates, 45 were previously uncultured bacteria having a 16S rRNA gene similarity from 91.35 % to 98.7 % to their closest relatives according to NCBI GenBank. Conclusion: This study shows that culturing microorganisms in the cChip from aquatic environment using concentration process before performing the traditional petri plate culturing can result in the successful growth of unculturable bacteria. To the best of our knowledge this is the first study conducted for successful exploration of unculturable aquatic microbial community. This study can have a significant impact on our understanding of the techniques that can be applied for exploring the unexplored microbiome from diverse environments. We also hypothesize that certain modifications in the manufacturing material, keeping the design and techniques of this study intact can result in the application of this technique from gut microbiome to extremophiles in the environment.

Hot in Cold: Microbial Life in the Hottest Springs in Permafrost

Chukotka is an arctic region located in the continuous permafrost zone, but thermal springs are abundant there. In this study, for the first time, the microbial communities of the Chukotka hot springs (CHS) biofilms and sediments with temperatures 54–94 °C were investigated and analyzed by NGS sequencing of 16S rRNA gene amplicons. In microbial mats (54–75 °C), phototrophic bacteria of genus Chloroflexus dominated (up to 89% of all prokaryotes), while Aquificae were the most numerous at higher temperatures in Fe-rich sediments and filamentous “streamers” (up to 92%). The electron donors typical for Aquificae, such as H2S and H2, are absent or present only in trace amounts, and the prevalence of Aquificae might be connected with their ability to oxidize the ferrous iron present in CHS sediments. Armatimonadetes, Proteobacteria, Deinococcus-Thermus, Dictyoglomi, and Thermotogae, as well as uncultured bacteria (candidate divisions Oct-Spa1-106, GAL15, and OPB56), were numerous, and Cyanobacteria were present in low numbers. Archaea (less than 8% of the total community of each tested spring) belonged to Bathyarchaeota, Aigarchaeota, and Thaumarchaeota. The geographical location and the predominantly autotrophic microbial community, built on mechanisms other than the sulfur cycle-based ones, make CHS a special and unique terrestrial geothermal ecosystem.