Microbes and Environments最新文献

Gene mutations are a fundamental survival strategy in bacteria and often accompany cell growth. We herein demonstrate that Pseudomonas aeruginosa recovered growth under anoxic conditions through a mutation in an essential regulatory gene required for nitrate respiration, overcoming initial growth limitations. Although the open-ocean isolate P. aeruginosa Ocean-1187 possesses denitrification-related genes, it was initially unable to grow under nitrate-respiring conditions due to a defect in the transcriptional regulator NarL, part of the NarX-NarL two-component system that controls the expression of denitrification genes. The growth of this strain was limited by the defective NarL; however, a prolonged incubation under anoxic conditions led to the emergence of spontaneous mutants that regained the ability to grow. Whole-genome sequencing and functional complementation assays revealed that a single amino acid substitution in NarL was sufficient to restore nitrate respiration. Additional variants independently isolated from separate cultures also carried substitutions at the same residue, underscoring its functional importance. The present study shows that a single amino acid substitution in an essential regulatory protein restored metabolic function. These results offer novel insights into the adaptive strategies of bacteria, highlighting the emergence of critical mutations even under restricted growth conditions.

On solid media, Acanthamoeba harboring the endosymbiotic bacterium (Neochlamydia) carries live Escherichia coli on its cell surface without phagocytosing this bacterium, and defends against infection by Legionella pneumophila of a small size in an endosymbiotic bacterium-dependent manner. This implies the presence of an unknown protective mechanism. Therefore, we exami-ned the physical properties of the carried bacteria using transposon insertion mutants that had lost the "backpack" on solid media. A mutant was selected from a library in which the nhaA gene, encoding a Na⁺/H⁺ antiporter, was disrupted. The knockout mutant was longer than the parental strain and was gradually consumed by symbiotic amoebae. Similarly, the NhaA-knockout mutant strain was longer and lacked the backpack. The complementation of nhaA restored bacterial cells to their normal size, and the backpack phenomenon reappeared. Using E. coli elongated by a treatment with mitomycin C, the backpack was not evident, and enlarged bacteria were consumed by symbiotic amoebae. Therefore, symbiotic amoebae protected themselves from intruders by not engulfing small bacteria in an endosymbiosis-dependent manner. The present results propose a novel countermeasure by phagocytic cells against intruders that involves the recognition of bacterial sizes and is dependent on endosymbiosis.

Nitrifying communities in activated sludge play a crucial role in biological nitrogen removal processes in municipal wastewater treatment plants. While extensive research has been conducted in temperate regions, limited information is available on nitrifiers in tropical regions. The present study investigated all currently known nitrifying communities in two full-scale municipal wastewater treatment plants in Malaysia operated under low-dissolved oxygen (DO) (0.2-0.7‍ ‍mg‍ ‍DO‍ ‍L^-1) or high-DO (2.0-5.5‍ ‍mg‍ ‍DO‍ ‍L^-1) conditions at 30°C. The core nitrifiers in the municipal wastewater treatment plants were Nitrosomonas (ammonia-oxidizing bacteria, AOB), Nitrospira (nitrite-oxidizing or complete ammonia-oxidizing, comammox, bacteria), and ammonia-oxidizing archaea (AOA) as identified by a 16S rRNA gene amplicon sequencing ana-lysis and corroborated by 16S rRNA-targeted fluorescence in situ hybridization. A quantitative polymerase chain reaction (qPCR) targeting ammonia monooxygenase subunit A (amoA) genes revealed stable populations of comammox Nitrospira and AOB in both wastewater treatment plants. AOA were detected in only one of the plants and their population sizes fluctuated, with higher temporary abundance under high-DO conditions. These results provide important insights into the composition and dynamics of nitrifying communities in tropical municipal wastewater treatment plants.

The present study exami-ned bacteria that anaerobically degrade the aromatic compound, benzoate, and obtained enrichment cultures from marine sediments under illumination. The enrichment culture contained anoxygenic photosynthetic bacteria and non-photosynthetic bacteria. The photosynthetic strain PS1, a purple sulfur bacterium in the genus Marichromatium, was unable to utilize benzoate; however, when combined with the non-photosynthetic bacterial isolate, Marinobacterium sp. strain BA1, the co-culture grew anaerobically on benzoate in the presence of thiosulfate or tetrathionate. Based on the metabolic profiles of the co-culture and axenic cultures, the following syntrophic interactions were proposed. Strain PS1 oxidizes thiosulfate as the electron source for photosynthesis to produce tetrathionate and relies on carbon dioxide produced through benzoate degradation by strain BA1. Strain BA1 oxidizes benzoate and reduces tetrathionate to provide thiosulfate to strain PS1 for photosynthetic carbon fixation. To the best of our knowledge, this is the first study to report anaerobic benzoate degradation in a photosynthetic co-culture through the syntrophic exchange of sulfur compounds.

Frankia spp. are multicellular actinobacteria with the ability to fix atmospheric dinitrogen (N₂). Frankia fixes N₂ not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species called actinorhizal plants. In the present study, we isolated mutants of the pyrE (orotate phosphoribosyltransferase) and pyrF (orotidine-5'-phosphate decarboxylase) genes in Frankia casuarinae strain CcI3 using gamma rays as a mutagen and systematically identified the types of mutations that occurred in these genes. pyrEF mutants were isolated as uracil auxotrophs using the antimetabolite 5-fluoroorotic acid. We elucidated the nucleotide sequences of the pyrEF genes in 32 uracil auxotrophs, and detected eight substitutions, 17 single-nucleotide deletions, and seven large insertions. Large insertions were insertion sequences (IS elements); four belonged to the IS4 family, two to the IS66 family, and one to the IS110 family. This is the first study to demonstrate the mobilization of IS elements in the Frankia genome.

The present study proposes a categorization of bacteria that leak from activated sludge processes to secondary treated water (STW). Bacterial populations in primary treated water (PTW), activated sludge (AS), STW, and the 0.2‍ ‍μm-filtrate of STW (FSTW) in a full-scale wastewater treatment plant with two treatment trains were observed for a period of one year using a 16S rRNA ana-lysis approach. The taxonomic groups detected were categorized as different "leak types" based on the read occupancies in PTW, AS, STW, and FSTW, where a leak type indicates the likelihood of a taxonomic group to leak to STW. Five leak types were introduced: "LTE", "LTE-I", "LTEF", "LTF", and "NLT", with "LT" for leak type, "E" for high read occupancy in STW or the effluent of secondary settling tanks, "I" for high read occupancy in PTW or influent to the AS process, "F" for high read occupancy in FSTW, and "NLT" for a smaller likelihood to leak. Representative taxonomic groups for each leak type were Neisseria and ABY1 for "LTE" Parcubacteria for "LTEF", Campylobacterota for "LTE-I", and Saccharimonadia, Bdellovibrionota, and some lineages in Comamonadaceae for "LTF". Although some taxonomic groups, such as Comamonadaceae, included different leak types, the categorization assigned to each taxonomic group was mostly consistent between the two treatment trains. The categorization scheme proposed herein may become a useful key for understanding the characteristics of bacteria that appear in AS and STW.

Naturally occurring caves are sites of significant cultural value, while also displaying the unique biodiversity of associated microbiomes that may provide an untapped source of potentially beneficial organisms. However, the touristic exploitation of show caves may ultimately result in the biodeterioration of speleothems, primarily through the introduction and establishment of alien microbiota or the uncontrolled growth of indigenous species, exacerbated by the use of artificial lighting. These habitat characteristics are present in the Dictean cave, also known as "Diktaion Andron", a highly visited cave in eastern Crete, Greece, which was regarded in ancient Greek mythology as one of the putative sites of the birth of Zeus. Therefore, an efficient approach to controlling these ecological niches without irreversibly disturbing microbial diversity is needed, and essential oils are currently being investigated as a mild cleaning method. The present study exami-ned the microbial diversity of the Dictean cave using 16S and 18S rRNA gene amplicon sequencing and methods for quantitative metabolic activity estimations and also investigated the application of a formulation containing specific essential oils as a mild cleaning method. Amplicon sequencing ana-lyses revealed distinct profiles among the different sample sites, with species of the genera Pseudomonas, Sporosarcina, Butiauxella, Glutamicibacter, Paenibacillus, Mortierella, and Jenufa being the most abundant, while uncharacterized microorganisms were also detected. The single simultaneous application of a formulation of 0.2% (v/v) oregano and 0.4% (v/v) cinnamon essential oils was effective at significantly reducing microbial metabolic activity by up to 89.2% within 24 h, without adversely affecting the coloration of speleothems.

Despite its ecological importance, the gut microbiota of whale sharks (Rhincodon typus) remains poorly understood. Therefore, the present study exami-ned how environmental differences affect the fecal microbiota by comparing long-term captive and newly captured individuals. Fecal samples were collected over time from four long-term captive and two newly captured whale sharks, with seawater also being sampled from their respective tanks. Using 16S rRNA sequencing, 12,497 amplicon sequence variants (ASVs) were identified, including 6,976 classified as major ASVs. There were no significant differences in alpha diversity indexes between long-term captive and newly captured sharks; however, the latter showed slightly larger variance in four indexes. The ASV count per individual was slightly lower in long-term captive sharks than in their newly captured counterparts. In long-term captive individuals, Photobacterium was highly abundant. Conversely, Ureaplasma was dominant in newly captured individuals, but was barely detected in long-term captive sharks. Although alpha diversity did not differ significantly between the groups, a beta diversity ana-lysis showed clear distinctions. The high abundance of Ureaplasma in newly captured sharks suggests its involvement in nitrogen metabolism, possibly through urea recycling. Although further research is needed to clarify the taxonomic position and ecological functions of these Ureaplasma populations, the present study provides key insights for the conservation of wild whale sharks and improving health management for captive individuals.

Nitrous oxide (N₂O) is a key atmospheric greenhouse gas that contributes to global warming, with anthropogenic N₂O emissions from agriculture being a particular concern. Among agricultural sources, unknown soil organisms in the legume rhizosphere emit N₂O from degraded root nodules. To discriminate between fungal and bacterial N₂O emissions, we adopted an isotopomer ana-lysis, which provides site preference values (the difference in ¹⁵N abundance of the central and terminal N atoms in the N₂O molecule). The addition of nitrite instead of nitrate to soybean nodulated roots significantly increased SP_N2O from -3.5‰ to 4.2‰ in a pot system. Moreover, a mutation of the nirK gene (encoding dissimilatory nitrite reductase) in symbiotic bradyrhizobia significantly increased SP_N2O from 4.2‰ to 13.9‰ with nitrite. These results suggest that nitrite-utilizing N₂O emissions via fungal denitrification occurred in the model pot system of the soybean rhizosphere. Microscopic observations showed fungal hyphae and crescent spores around N₂O-emitting nodules. Therefore, we isolated single spores from soybean nodules under a microscope. A phylogenetic ana-lysis revealed that all 12 fungal isolates were Fusarium species, which exist in soybean field soil. When these isolates were cultivated in glycerol-peptone medium supplemented with nitrate or nitrite (1‍ ‍mM), 11 of the 12 isolates strongly converted nitrite to N₂O; however, no N₂O emissions were noted in the presence of nitrate. A ¹⁵N-nitrite tracer experiment revealed that one N₂O molecule was derived exclusively from two molecules of nitrite (NO₂^-) in the fungal culture. These results suggest that nitrite-utilizing Fusarium fungi mediate N₂O emissions in the soybean rhizosphere.

The anammox process using marine anammox bacteria is a promising nitrogen removal process for recirculating aquaculture system wastewater. Marine anammox bacteria are typically found in oxygen-deficient zones and coastal areas under low phosphate concentrations. The optimal phosphate concentration for marine anammox bacteria remains unknown because most laboratory studies on these bacteria have been conducted under high phosphate concentrations. Therefore, the present study investigated the long-term effects of varying phosphate concentrations on the marine anammox bacteria, Candidatus Scalindua sp., to identify the optimal range of phosphate. Anammox activity and average growth rates were evaluated under seven phosphate concentrations (0, 0.23, 0.46, 0.68, 1.14, 6.15 [control], and 15.48‍ ‍mg P L^-1) over a period of 70 days. After 50 days of reactor operation, reactor performance under phosphate concentrations ranging from 0.23 to 6.15‍ ‍mg P L^-1 stabilized at 70% of total nitrogen removal efficiency, indicating the successful establishment of the anammox process. Conversely, anammox reactor performance under conditions without phosphate addition (0‍ ‍mg P L^-1) and the highest phosphate concentration (15.48‍ ‍mg P L^-1) did not reach 70% of total nitrogen removal efficiency, indicating a suboptimal phosphate concentration for normal anammox activity. Average growth rates calculated from total biomass samples varied from 0.0006 to 0.0012 h^-1. These results indicate that Ca. Scalindua need to be kept at phosphate concentrations between 0.23 and 6.15‍ ‍mg P L^-1 for optimal functioning in wastewater treatment ecosystems.