Large-scale phage cultivation for commensal human gut bacteria.

Phages are highly abundant in the human gut, yet most of them remain uncultured. Here, we present a gut phage isolate collection (GPIC) containing 209 phages for 42 commensal human gut bacterial species. Genome analysis of the phages identified 34 undescribed genera. We discovered 22 phages from the Salasmaviridae family that have small genomes (∼10-20 kbp) and infect Gram-positive bacteria. Two phages from a candidate family, Paboviridae, with high prevalence in the human gut were also identified. Infection assays showed that Bacteroides and Parabacteroides phages are specific to a bacterial species, and strains of the same species also exhibit substantial variations in phage susceptibility. A cocktail of 8 phages with a broad host range for Bacteroides fragilis strains effectively reduced their abundance in complex host-derived communities in vitro. Our study expands the diversity of cultured human gut bacterial phages and provides a valuable resource for human microbiome engineering.

Effects of microbial community structure and its co-occurrence on the dynamic changes of physicochemical properties and free amino acids in the Cantonese soy sauce fermentation process.

The soy sauce produced by Cantonese fermentation has a unique flavor, among which brine fermentation plays an important role. In this fermentation process, 61 volatile compounds, including 19 esters, 10 aldehydes, 9 alcohols, 5 phenols, and 18 others, were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry. Seventeen kinds of free amino acids were detected by high-performance liquid chromatography. Results showed that Touyou, which comprised 1.5 g/100 g total nitrogen, 1.0 g/100 mL amino acid nitrogen, 3.66 g/100 g reducing sugar, 1.44 g/100 mL total acid, 17.04 g/100 mL salt content, and 27.3% umami free amino acids, had excellent quality. High-throughput sequencing was used to identify microorganisms. The top 3 of bacteria were Weissella, Staphylococcus, and Lactobacillus, and the top 3 fungi were Aspergillus, Zygosaccharomyces, and Candida. The co-occurrence network analysis of microorganisms showed that the top-ranked microorganisms were Plectosphaerella, Aureobasidium, unidentified_Mortierellales_sp, Glutinomyces, Faecalibacterium, and Cladophialophora. Then, eight microorganisms (VIP[pred] > 1) were obtained by two-way orthogonal partial least squares model, namely, Staphylococcus, Candida, Weissella, Aspergillus, Zygosaccharomyces, Lactobacillus, Monilinia, and Clavispora. Correlation analysis showed that these microorganisms were strongly related to flavor metabolites. This study explored the dynamics of traditional Cantonese fermentation, which has positive implications for optimizing this traditional fermentation process.

Quantitative proteomic analysis reveals the metabolic characteristics and adaptive mechanism of Cupriavidus oxalaticus T2 in the process of simultaneous nitrogen and phenol removal.

Phenol and ammonia in wastewater pose a serious threat to ecosystems and human health. However, the currently limited studies on single bacterium simultaneously removing phenol and nitrogen pollution have not fully elucidated the relevant metabolic mechanisms. The differences in proteomic profile after supplementing with phenol and ammonia for 6 and 24 h, respectively, were evaluated to explore the metabolic characteristics and adaptive mechanism of Cupriavidus oxalaticus T2 during the simultaneous removal process of phenol and nitrogen. Results revealed that a new potential phenol para-degradation pathway appeared in T2. Phenol induced changes in nitrogen metabolism, resulting in increased denitrification and decreased synthesis of glutamate from ammonia at 6 h. In addition, phenol exposure enhanced the expression of cytochrome oxidases with high oxygen affinity and increased ATP synthesis. The increase in chemotaxis and flagellar assembly was conducive to the uptake and utilization of phenol. The synthesis of lipoic acid and biotin was also promoted to resist phenol toxicity. Moreover, phenol triggered cellular stress response, thereby leading to the upregulation of anti-stress proteins, such as universal stress protein, iron­sulfur cluster protein, and chaperones. This study contributes to revealing the metabolic characteristics and adaptive mechanism of T2 during simultaneous nitrogen and phenol removal. SIGNIFICANCE: Phenol and ammonia often co-exist in wastewater, causing serious environmental problems. The information on the metabolic mechanism of simultaneously removing these two pollutants by bacteria is insufficient at present. Moreover, phenol is toxic to microbial and causes cells damage. Therefore, exploring the response mechanism of bacteria to phenol stress is conducive to understand their tolerance mechanism to aromatic compounds. In this study, the metabolic characteristics and adaptive mechanism of C. oxalaticus T2 during the simultaneous removal of phenol and nitrogen process were evaluated by comparing the proteome profiles at different stages. The results revealed the degradation pathways of phenol and nitrogen by strain T2. A variety of phenol response mechanisms were determined, including enhanced energy production, improved cell motility, increased the synthesis of lipoic acid and biotin, and combined action of multiple anti-stress proteins. This study is potentially useful to future phenol and nitrogen co-pollution bioremediation strategies and provides insight into the phenolic compound resistance mechanism in bacteria.

Characterization of DinJ-YafQ toxin-antitoxin module in Tetragenococcus halophilus: activity, interplay, and evolution.

Tetragenococcus halophilus is a moderately halophilic lactic acid bacterium widely used in high-salt food fermentation because of its coping ability under various stress conditions. Bacterial toxin-antitoxin (TA) modules are widely distributed and play important roles in stress response, but those specific for genus Tetragenococcus have never been explored. Here, a bona fide TA module named DinJ1-YafQ1tha was characterized in T. halophilus. The toxin protein YafQ1tha acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ1tha, indicating that YafQ1tha activity is blocked by the formed DinJ1-YafQ1tha complex. In vivo and in vitro assays showed that DinJ1tha alone or DinJ1-YafQ1tha complex can repress the transcription of dinJ1-yafQ1tha operon by binding directly to the promoter sequence. In addition, dinJ1-yafQ1tha is involved in plasmid maintenance and stress response, and its transcriptional level is regulated by various stresses. These findings reveal the possible roles of DinJ1-YafQ1tha system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ2tha without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ1tha, indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ2tha can cross-interact with noncognate toxin YafQ1tha and cross-regulate with dinJ1-yafQ1tha operon. In summary, DinJ-YafQtha characterization may be helpful in investigating the key roles of TA systems in T. halophilus and serves as a foundation for further research. KEY POINTS: • dinJ1-yafQ1tha is the first functional TA module characterized in T. halophilus and upregulated significantly upon osmotic and acidic stress. • DinJ2tha can exhibit physical and transcriptional interplay with DinJ1-YafQ1tha. • dinJ2tha may be acquired from bacteria in distant affiliation and inserted into the T. halophilus genome through horizontal gene transfer.