Sorlinia euscelidii gen. nov., sp. nov., a novel acetic acid bacterium isolated from the leafhopper Euscelidius variegatus (Hemiptera: Cicadellidae).

Acetic acid bacteria - belonging to the Acetobacteraceae family - are found in the gut of many sugar-feeding insects. In this study, six strains have been isolated from the hemipteran leafhopper Euscelidius variegatus. While they exhibit high 16S rRNA gene sequence similarities to uncultured members of the Acetobacteraceae family, they could not be unequivocally assigned to any particular type species. Considering the clonality of the six isolates, the EV16PT strain was used as a representative of this group of isolates. The genome sequence of EV16PT is composed of a 2.388 Mbp chromosome, with a DNA G+C content of 57 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis indicate that EV16PT forms a monophyletic clade with the uncultivated endosymbiont of Diaphorina citri, the Candidatus Kirkpatrickella diaphorinae. Such a phylogenetic clade is positioned between those of Asaia-Swaminathania and Kozakia. The genomic distance metrics based on gene and protein sequences support the proposal that EV16PT is a new species belonging to a yet-undescribed genus. It is a rod-shaped Gram-stain-negative bacterium, strictly aerobic, non-motile, non-spore-forming, showing optimal growth without salt (NaCl) at 30 °C and pH of 6-7. The major quinone is Q10, and the dominant cellular fatty acids (>10%) are C18:l ω7c, C19 : 0 cyclo ω6c, C16 : 0 and C19 : 1 2OH. The polar lipid profile comprises diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine, along with unidentified aminophospholipids, glycophospholipids, aminolipids and lipids. Based on a polyphasic approach, including phylogenetic, phylogenomic, genome relatedness, phenotypic and chemotaxonomic characterisations, EV16PT (= KCTC 8296T, = DSM 117028T) is proposed as a representative of a novel species in a novel genus with the proposed name Sorlinia euscelidii gen. nov., sp. nov., in honour of Prof. Claudia Sorlini, an Italian environmental microbiologist at the University of Milan who inspired the research on microbial diversity, including symbiosis in plants and animals.

Mangrovimonas cancribranchiae sp. nov., a novel bacterial species associated with the gills of the fiddler crab Cranuca inversa (Brachyura, Ocypodidae) from Red Sea mangroves.

Two bacteria, UG2_1T and UG2_2, were isolated from the gill tissues of the mangrove fiddler crab Cranuca inversa collected on the east coast of the Red Sea (Thuwal, Saudi Arabia). The cells are Gram-negative, rod-shaped, orange-pigmented, motile by gliding with no flagella, strictly aerobic, and grow at 20-37 °C (optimum, 28-35 °C), at pH 5.0-9.0 (optimum, pH 6.0-7.0), and with 1-11 % (w/v) NaCl (optimum, 2-4 %). They were positive for oxidase and catalase activity. Phylogenetic analysis based on 16S rRNA gene sequences indicated that isolates UG2_1T and UG2_2 belong to the genus Mangrovimonas, showing the highest similarity to Mangrovimonas spongiae HN-E26T (99.4 %). Phylogenomic analysis based on the whole genomes, independently using 49 and 120 concatenated genes, showed that strains UG2_1T and UG2_2 formed a monophyletic lineage in a different cluster from other type strain species within the genus Mangrovimonas. The genome sizes were 3.08 and 3.07 Mbp for UG2_1T and UG2_2, respectively, with a G+C content of 33.8 mol% for both strains. Values of average nucleotide identity and digital DNA-DNA hybridization between the strains and closely related species were 91.0 and 43.5 %, respectively. Chemotaxonomic analysis indicated that both strains had iso-C15 : 0 and iso-C15 : 1 G as dominant fatty acids, and the primary respiratory quinone was identified as MK-6. The major polar lipids comprised phosphatidylethanolamine, one unidentified glycolipid, one unidentified phospholipid, two unidentified aminolipids, and four unidentified lipids. Based on phylogenetic, phylogenomic, genome relatedness, phenotypic, and chemotaxonomical data, the two isolates represent a novel species within the genus Mangrovimonas, with the proposed name Mangrovimonas cancribranchiae sp. nov., and the type strain UG2_1T (=KCTC 102158T=DSM 117025T).

Searching for microbial contribution to micritization of shallow marine sediments.

Micritization is an early diagenetic process that gradually alters primary carbonate sediment grains through cycles of dissolution and reprecipitation of microcrystalline calcite (micrite). Typically observed in modern shallow marine environments, micritic textures have been recognized as a vital component of storage and flow in hydrocarbon reservoirs, attracting scientific and economic interests. Due to their endolithic activity and the ability to promote nucleation and reprecipitation of carbonate crystals, microorganisms have progressively been shown to be key players in micritization, placing this process at the boundary between the geological and biological realms. However, published research is mainly based on geological and geochemical perspectives, overlooking the biological and ecological complexity of microbial communities of micritized sediments. In this paper, we summarize the state-of-the-art and research gaps in micritization from a microbial ecology perspective. Since a growing body of literature successfully applies in vitro and in situ 'fishing' strategies to unveil elusive microorganisms and expand our knowledge of microbial diversity, we encourage their application to the study of micritization. By employing these strategies in micritization research, we advocate promoting an interdisciplinary approach/perspective to identify and understand the overlooked/neglected microbial players and key pathways governing this phenomenon and their ecology/dynamics, reshaping our comprehension of this process.

Gill-associated bacteria are homogeneously selected in amphibious mangrove crabs to sustain host intertidal adaptation.

BACKGROUND: The transition from water to air is a key event in the evolution of many marine organisms to access new food sources, escape water hypoxia, and exploit the higher and temperature-independent oxygen concentration of air. Despite the importance of microorganisms in host adaptation, their contribution to overcoming the challenges posed by the lifestyle changes from water to land is not well understood. To address this, we examined how microbial association with a key multifunctional organ, the gill, is involved in the intertidal adaptation of fiddler crabs, a dual-breathing organism. RESULTS: Electron microscopy revealed a rod-shaped bacterial layer tightly connected to the gill lamellae of the five crab species sampled across a latitudinal gradient from the central Red Sea to the southern Indian Ocean. The gill bacterial community diversity assessed with 16S rRNA gene amplicon sequencing was consistently low across crab species, and the same actinobacterial group, namely Ilumatobacter, was dominant regardless of the geographic location of the host. Using metagenomics and metatranscriptomics, we detected that these members of actinobacteria are potentially able to convert ammonia to amino acids and may help eliminate toxic sulphur compounds and carbon monoxide to which crabs are constantly exposed. CONCLUSIONS: These results indicate that bacteria selected on gills can play a role in the adaptation of animals in dynamic intertidal ecosystems. Hence, this relationship is likely to be important in the ecological and evolutionary processes of the transition from water to air and deserves further attention, including the ontogenetic onset of this association. Video Abstract.

Bioturbation Intensity Modifies the Sediment Microbiome and Biochemistry and Supports Plant Growth in an Arid Mangrove System.

In intertidal systems, the type and role of interactions among sediment microorganisms, animals, plants and abiotic factors are complex and not well understood. Such interactions are known to promote nutrient provision and cycling, and their dynamics and relationships may be of particular importance in arid microtidal systems characterized by minimal nutrient input. Focusing on an arid mangrove ecosystem on the central Red Sea coast, we investigated the effect of crab bioturbation intensity (comparing natural and manipulated high levels of bioturbation intensity) on biogeochemistry and bacterial communities of mangrove sediments, and on growth performance of Avicennia marina, over a period of 16 months. Along with pronounced seasonal patterns with harsh summer conditions, in which high sediment salinity, sulfate and temperature, and absence of tidal flooding occur, sediment bacterial diversity and composition, sediment physicochemical conditions, and plant performance were significantly affected by crab bioturbation intensity. For instance, bioturbation intensity influenced components of nitrogen, carbon, and phosphate cycling, bacterial relative abundance (i.e., Bacteroidia, Proteobacteria and Rhodothermi) and their predicted functionality (i.e., chemoheterotrophy), likely resulting from enhanced metabolic activity of aerobic bacteria. The complex interactions among bacteria, animals, and sediment chemistry in this arid mangrove positively impact plant growth. We show that a comprehensive approach targeting multiple biological levels provides useful information on the ecological status of mangrove forests. IMPORTANCE Bioturbation is one of the most important processes that governs sediment biocenosis in intertidal systems. By facilitating oxygen penetration into anoxic layers, bioturbation alters the overall sediment biogeochemistry. Here, we investigate how high crab bioturbation intensity modifies the mangrove sediment bacterial community, which is the second largest component of mangrove sediment biomass and plays a significant role in major biogeochemical processes. We show that the increase in crab bioturbation intensity, by ameliorating the anoxic condition of mangrove sediment and promoting sediment bacterial diversity in favor of a beneficial bacterial microbiome, improves mangrove tree growth in arid environments. These findings have significant implications because they show how crabs, by farming the mangrove sediment, can enhance the overall capacity of the system to sustain mangrove growth, fighting climate change.

Morphological characteristics and abundance of prokaryotes associated with gills in mangrove brachyuran crabs living along a tidal gradient.

Due to the chemico-physical differences between air and water, the transition from aquatic life to the land poses several challenges for animal evolution, necessitating morphological, physiological and behavioural adaptations. Microbial symbiosis is known to have played an important role in eukaryote evolution, favouring host adaptation under changing environmental conditions. We selected mangrove brachyuran crabs as a model group to investigate the prokaryotes associated with the gill of crabs dwelling at different tidal levels (subtidal, intertidal and supratidal). In these animals, the gill undergoes a high selective pressure, finely regulating multiple physiological functions during both animal submersion under and emersion from the periodical tidal events. We hypothesize that similarly to other marine animals, the gills of tidal crabs are consistently colonized by prokaryotes that may quantitatively change along the environmental gradient driven by the tides. Using electron microscopy techniques, we found a thick layer of prokaryotes over the gill surfaces of all of 12 crab species from the mangrove forests of Saudi Arabia, Kenya and South Africa. We consistently observed two distinct morphotypes (rod- and spherical-shaped), positioned horizontally and/or perpendicularly to the gill surface. The presence of replicating cells indicated that the prokaryote layer is actively growing on the gill surface. Quantitative analysis of scanning electron microscopy images and the quantification of the bacterial 16S rRNA gene by qPCR revealed a higher specific abundance of prokaryote cells per gill surface area in the subtidal species than those living in the supratidal zone. Our results revealed a correlation between prokaryote colonization of the gill surfaces and the host lifestyle. This finding indicates a possible role of prokaryote partnership within the crab gills, with potential effects on animal adaptation to different levels of the intertidal gradient present in the mangrove ecosystem.

Label-Free Proteomic Approach to Study the Non-lethal Effects of Silver Nanoparticles on a Gut Bacterium.

Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs) have become one of the most commonly utilized because of their antimicrobial properties. Despite their common use, the effects of sublethal concentrations of AgNPs, especially on gut biofilms, have been poorly investigated. To address this issue, we investigated in vitro the proteomic response of a monospecies Escherichia coli gut biofilm to chronic and acute exposures in sublethal concentrations of AgNPs. We used a new gel- and label-free proteomic approach based on shotgun nanoflow liquid chromatography-tandem mass spectrometry. This approach allows a quantification of the whole proteome at a dynamic range that is higher than the traditional proteomic investigation. To assess all different possible exposure scenarios, we compared the biofilm proteome of four treatments: (i) untreated cells for the control treatment, (ii) cells treated with 1 µg/ml AgNPs for 24 h for the acute treatment, (iii) cells grown with 1 µg/ml AgNPs for 96 h for the chronic treatment, and (iv) cells grown in the presence of 1 µg/ml AgNPs for 72 h and then further treated for 24 h with 10 µg/ml AgNPs for the chronic + acute treatment. Among the 1,917 proteins identified, 212 were significantly differentially expressed proteins. Several pathways were altered including biofilm formation, bacterial adhesion, stress response to reactive oxygen species, and glucose utilization.

Impacts of dietary silver nanoparticles and probiotic administration on the microbiota of an in-vitro gut model.

Ingestion of silver nanoparticles (AgNPs) is inevitable linked to their widespread use in food, medicines and other consumer products. However, their effects on human microbiota at non-lethal concentrations remain poorly understood. In this study, the interactions among 1 µg mL-1 AgNPs, the intestinal microbiota, and the probiotic Bacillus subtilis (BS) were tested using in-vitro batch fermentation models inoculated with human fecal matter. Results from metagenomic investigations revealed that the core bacterial community was not affected by the exposure of AgNPs and BS at the later stage of fermentation, while the proportions of rare species changed drastically with the treatments. Furthermore, shifts in the Firmicutes/Bacteriodetes (F/B) ratios were observed after 24 h with an increase in the relative abundance of Firmicutes species and a decrease in Bacteroidetes in all fermentation cultures. The co-exposure to AgNPs and BS led to the lowest F/B ratio. Fluorescent in-situ hybridization analyses indicated that non-lethal concentration of AgNPs negatively affected the relative percentage of Faecalibacterium prausnitzii and Clostridium coccoides/Eubacterium rectales taxa in the fermentation cultures after 24 h. However, exposure to single and combined treatments of AgNPs and BS did not change the overall diversity of the fecal microflora. Functional differences in cell motility, translation, transport, and xenobiotics degradation occurred in AgNPs-treated fermentation cultures but not in AgNPs+BS-treated samples. Compared to the control samples, treated fecal cultures showed no significant statistical differences in terms of short-chain fatty acids profiles, cytotoxic and genotoxic effects on Caco-2 cell monolayers. Overall, AgNPs did not affect the composition and diversity of the core fecal microflora and its metabolic and toxic profiles. This work indicated a chemopreventive role of probiotic on fecal microflora against AgNPs, which were shown by the decrease of F/B ratio and the unaltered state of some key metabolic pathways.

Effects of Sub-lethal Concentrations of Silver Nanoparticles on a Simulated Intestinal Prokaryotic-Eukaryotic Interface.

Nanotechnology applications are expected to bring a range of benefits to the food sector, aiming to provide better quality and conservation. In this research, the physiological response of both an Escherichia coli mono-species biofilm and Caco-2 intestinal cells to sub-lethal concentrations of silver nanoparticles (AgNPs) has been investigated. In order to simulate the anaerobic and aerobic compartments required for bacteria and intestinal cells growth, a simplified semi-batch model based on a transwell permeable support was developed. Interaction between the two compartments was obtained by exposing Caco-2 intestinal cells to the metabolites secreted by E. coli biofilm after its exposure to AgNPs. To the best of the authors' knowledge, this study is the first to investigate the effect of AgNPs on Caco-2 cells that takes into consideration previous AgNP-intestinal biofilm interactions, and at concentrations mimicking real human exposure. Our data show that 1 µg/mL AgNPs in anaerobic conditions (i) promote biofilm formation up to 2.3 ± 0.3 fold in the first 72 h of treatment; (ii) increase reactive oxygen species (ROS) production to 84 ± 21% and change the physiological status of microbial cells after 96 h of treatment; (iii) seriously affect a 72-h old established biofilm, increasing the level of oxidative stress to 86 ± 21%. Moreover, the results indicate that oxygen renders the biofilm more adequate to counteract AgNP effects. Comet assays on Caco-2 cells demonstrated a protective role of biofilm against the genotoxic effect of 1 µg/mL AgNPs on intestinal epithelial cells.

Effects of sublethal concentrations of silver nanoparticles on Escherichia coli and Bacillus subtilis under aerobic and anaerobic conditions.

The present work is aimed at comparing the effects of sublethal concentrations of silver nanoparticles (AgNPs) on the growth kinetic, adhesion ability, oxidative stress, and phenotypic changes of model bacteria (Escherichia coli and Bacillus subtilis) under both aerobic and anaerobic conditions. Growth kinetic tests conducted in 96-well microtiter plates revealed that sublethal concentrations of AgNPs do not affect E. coli growth, whereas 1 µg/ml AgNPs increased B. subtilis growth rate under aerobic conditions. At the same concentration, AgNPs promoted B. subtilis adhesion, while it discouraged E. coli attachment to the surface in the presence of oxygen. As determined by 2,7-dichlorofluorescein-diacetate assays, AgNPs increased the formation of intracellular reactive oxygen species, but not at the highest concentrations, suggesting the activation of scavenging systems. Finally, motility assays revealed that 0.01 and 1 µg/ml AgNPs, respectively, promoted surface movement in E. coli and B. subtilis under aerobic and anaerobic conditions. The results demonstrate that E. coli and B. subtilis react differently from AgNPs over a wide range of sublethal concentrations examined under both aerobic and anaerobic conditions. These findings will help elucidate the behavior and impact of engineered nanoparticles on microbial ecosystems.