Wiley

Serpentinization is a geologic process that produces highly reduced, hydrogen‐rich fluids that support microbial communities under high pH conditions. We investigated the activity of microbes capable of extracellular electron transfer in a terrestrial serpentinizing system known as ‘The Cedars’. Measuring current generation with an on‐site two‐electrode system, we observed daily oscillations in current with the current maxima and minima occurring during daylight hours. Distinct members of the microbial community were enriched. Current generation in lab‐scale electrochemical reactors did not oscillate, but was correlated with carbohydrate amendment in Cedars‐specific minimal media. Gammaproteobacteria and Firmicutes were consistently enriched from lab electrochemical systems on δ‐MnO₂ and amorphous Fe(OH)₃ at pH 11. However, isolation of an electrogenic strain proved difficult as transfer cultures failed to grow after multiple rounds of media transfer. Lowering the bulk pH in the media allowed us to isolate a Firmicutes strain (Paenibacillus sp.). This strain was capable of electrode and mineral reduction (including magnetite) at pH 9. This report provides evidence of the in situ activity of microbes using extracellular substrates as sinks for electrons at The Cedars, but also highlights the potential importance of community dynamics for supporting microbial life through either carbon fixation, and/or moderating pH stress.

We constructed and characterized a plasmid‐based genetic system that reports the expression of a toluene‐responsive promoter (P_tbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the P_tbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site‐specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80–100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 µm toluene for one to three hours. Compared to the response of A506 containing a plasmid‐borne P_tbuA1‐gfp fusion, the recombination‐based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the P_tbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high P_tbuA1‐promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant‐ or rhizosphere microbial community‐derived inducers of the P_tbuA1 promoter. This recombination‐based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment.

Các cộng đồng vi khuẩn methanotrophic đã được nghiên cứu trong một số loại đất gleyic bão hòa nước theo chu kỳ. Khi thu mẫu, mỗi loại đất đều có một lớp bề mặt ôxy và tiêu thụ metan từ khí quyển (tại 1.75 ppmv). Trong hầu hết các loại đất gleyic, giá trị K_m(app) cho metan nằm trong khoảng từ 70 đến 800 ppmv. Những giá trị này cao hơn hầu hết các giá trị quan sát được trong đất đồi khô, nhưng thấp hơn so với các giá trị đo được trong đất ngập nước. Dựa trên việc thu nhận độc lập và định lượng gen pmoA mà không cần nuôi cấy, các vi khuẩn methanotroph loại II (Alphaproteobacteria) thuộc chi Methylocystis spp. đã được phát hiện phong phú (> 10⁷ phân tử mục tiêu pmoA trên mỗi gram đất khô). Các vi khuẩn methanotroph loại I (Gammaproteobacteria) liên quan đến các chi Methylobacter và Methylocaldum/Methylococcus đã được phát hiện trong một số loại đất. Sáu kiểu chuỗi pmoA không liên quan chặt chẽ đến các chuỗi từ vi khuẩn methanotroph nuôi cấy cũng đã được phát hiện, cho thấy rằng có nhiều vi khuẩn methanotroph không thể nuôi cấy hiện diện. Ba loại Gleysols đã được ủ với các tỷ lệ phối trộn khác nhau của metan đánh dấu ¹³C để kiểm tra sự tích hợp ¹³C vào axit béo phospholipid (PLFAs). Các axit béo phospholipid đặc trưng cho vi khuẩn methanotroph loại II, 16:0 và 18:1ω7c, đã được gán nhãn với ¹³C trong tất cả các loại đất sau khi ủ trong bầu không khí chứa 30 ppmv metan. Việc ủ dưới 500 ppmv metan dẫn đến việc gán nhãn thêm các PLFA ngoài 16:0 và 18:1ω7c, gợi ý rằng thành phần của cộng đồng vi khuẩn methanotroph hoạt động đã thay đổi để phản ứng với việc cung cấp metan tăng lên. Trong hai loại đất, các PLFA 16:1 đặc trưng cho vi khuẩn methanotroph loại I đã được gán nhãn mạnh sau khi ủ chỉ dưới tỷ lệ phối trộn metan cao. Các vi khuẩn methanotroph loại II có khả năng chịu trách nhiệm cho việc hấp thụ metan trong khí quyển ở những loại đất này, trong khi các vi khuẩn methanotroph loại I sẽ hoạt động khi metan được sản xuất trong đất.

Antagonistic interactions are frequently observed among bacteria in the environment and result in complex networks, which could promote co‐existence, and therefore promote biodiversity. We analysed interactions of aquatic bacteria isolated by their ability to grow in Pseudomonas isolation agar from Churince, Cuatro Ciénegas, Mexico. In the resulting network, highly antagonistic and highly sensitive strains could be distinguished, forming a largely hierarchical structure. Most of the highly antagonistic strains belonged to the genus Pseudomonas. The network was sender‐determined, which means that the antagonist strains had a larger influence on its structure than the sensitive ones. Very few interactions were necessary to connect all strains, implying that the network was ‘small world’. The network was highly nested, having a core of highly interacting strains, with which the less antagonistic or highly sensitive interact. A probabilistic model was built, which captured most features of the network. Biological interpretation of the model implied a state in which many different antagonistic mechanisms were present, and most strains were resistant to them. Our work shows that strains of Pseudomonas from the water column at Cuatro Ciénegas have the potential to interact antagonistically with many closely related strains and that these interactions are usually not reciprocal.

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe‐defined filamentous bacteria directly in a complex system. The technique, QMAR‐FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria ‘Candidatus Meganema perideroedes’ and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium‐labelled substrates revealed an average cell‐specific uptake rate of 4.1 ¥ 10⁻¹⁵ mol of acetate cell−1 h⁻¹ and 3.1 ¥ 10⁻¹⁵ mol of acetate cell−1 h⁻¹ for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe‐defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (K_s) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The K_s values of the ‘Candidatus Meganema perideroedes’ and the Thiothrix filamentous bacteria were determined to be 1.8 µM and 2.4 µM acetate respectively.

Trong kỷ nguyên hậu di truyền, trọng tâm của nhiều nhà nghiên cứu đã chuyển sang việc nghiên cứu các sản phẩm chức năng của quá trình biểu hiện gene. Trong vi sinh vật học, những phương pháp ‘omics’ này chủ yếu được giới hạn ở các nuôi cấy đơn thuần của vi sinh vật. Do đó, chúng không cung cấp thông tin về biểu hiện gene trong hỗn hợp phức tạp của vi sinh vật tồn tại trong môi trường. Phương pháp của chúng tôi đã có thể tách và tinh lọc thành công toàn bộ proteome từ một hệ thống bùn hoạt tính quy mô phòng thí nghiệm tối ưu hoá cho việc loại bỏ photpho sinh học nâng cao, thực hiện phân tách bằng điện di gel polyacrylamide hai chiều và lập bản đồ của metaproteome này. Các đốm protein biểu hiện cao đã được tách ra và xác định bằng phương pháp khối phổ bốn cực thời gian bay với chuỗi peptide de novo. Các protein được phân lập đã được nhận diện sơ bộ là protein màng ngoài (porin), acetyl coenzyme A acetyltransferase và một thành phần protein của hệ thống vận chuyển amino acid dạng nhánh ABC. Các protein này có thể có nguồn gốc từ các tổ chức tích lũy polyphosphate kiểu Rhodocyclus thống trị và chưa được nuôi cấy trong bùn hoạt tính. Chúng tôi đề xuất thuật ngữ ‘metaproteomics’ cho việc đặc trưng trên diện rộng toàn bộ hệ protein của vi sinh vật môi trường tại một thời điểm nhất định.

Domestication of dogs from wolves is the oldest known example of ongoing animal selection, responsible for generating more than 300 dog breeds worldwide. In order to investigate the taxonomic and functional evolution of the canine gut microbiota, a multi‐omics approach was applied to six wild wolves and 169 dog faecal samples, the latter encompassing 51 breeds, which fully covers currently known canine genetic biodiversity. Specifically, 16S rRNA gene and bifidobacterial Internally Transcribed Spacer (ITS) profiling were employed to reconstruct and then compare the canine core gut microbiota to those of wolves and humans, revealing that artificial selection and subsequent cohabitation of dogs with their owners influenced the microbial population of canine gut through loss and acquisition of specific bacterial taxa. Moreover, comparative analysis of the intestinal bacterial population of dogs fed on Bones and Raw Food (BARF) or commercial food (CF) diet, coupled with shotgun metagenomics, highlighted that both bacterial composition and metabolic repertoire of the canine gut microbiota have evolved to adapt to high‐protein or high‐carbohydrates intake. Altogether, these data indicate that artificial selection and domestication not only affected the canine genome, but also shaped extensively the bacterial population harboured by the canine gut.

Fungal sexual reproduction requires complex cellular differentiation processes of hyphal cells. The plant pathogenic fungus Fusarium graminearum produces fruiting bodies called perithecia via sexual reproduction, and perithecia forcibly discharge ascospores into the air for disease initiation and propagation. Lipid metabolism and accumulation are closely related to perithecium formation, yet the molecular mechanisms that regulate these processes are largely unknown. Here, we report that a novel fungal specific bZIP transcription factor, F. graminearum perithecium overproducing 1 (Fpo1), plays a role as a global transcriptional repressor during perithecium production and maturation in F. graminearum. Deletion of FPO1 resulted in reduced vegetative growth, asexual sporulation and virulence and overproduced perithecium, which reached maturity earlier, compared with the wild type. Intriguingly, the hyphae of the fpo1 mutant accumulated excess lipids during perithecium production. Using a combination of molecular biological, transcriptomic and biochemical approaches, we demonstrate that repression of FPO1 after sexual induction leads to reprogramming of carbon metabolism, particularly fatty acid production, which affects sexual reproduction of this fungus. This is the first report of a perithecium‐overproducing F. graminearum mutant, and the findings provide comprehensive insight into the role of modulation of carbon metabolism in the sexual reproduction of fungi.

Fusarium graminearum is a prominent plant pathogenic fungus causing Fusarium head blight in major cereal crops worldwide. To understand the molecular mechanisms underlying fungal development and virulence, large collections of F. graminearum mutants have been constructed. Cytochrome P450 monooxygenases (P450s) are widely distributed in organisms and are involved in a diverse array of molecular/metabolic processes; however, no systematic functional analysis of P450s has been attempted in filamentous fungi. In this study, we constructed a genome‐wide deletion mutant set covering 102 P450s and analyzed these mutants for changes in 38 phenotypic categories, including fungal development, stress responses and responses to several xenobiotics, to build a comprehensive phenotypic dataset. Most P450 mutants showing defective phenotypes were impaired in a single phenotypic trait, demonstrating that our mutant library is a good genetic resource for further fungal genetic studies. In particular, we identified novel P450s specifically involved in virulence (5) and both asexual (1) and sexual development (2). Most P450s seem to play redundant roles in the degradation of xenobiotics in F. graminearum. This study is the first phenome‐based functional analysis of P450s, and it provides a valuable genetic resource for further basic and applied biological research in filamentous fungi and other plant pathogens.