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Background & Aims: University students are prone to changes in their health status and lifestyle due to changes in their living environment and associated stress and anxiety. These changes may affect them in later life. This study utilized a cross-sectional study among Japanese female university students to examine dietary factors affecting their fecal microbiota. Methods: Sixty-eight healthy female university students were evaluated using an eating behavior assessment and diet history questionnaire. The 12-component Japanese diet index (JDI-12) was then calculated. A quantitative real-time PCR method was used to analyze the predominant bacterial species in the gut, and the Firmicutes/Bacteroidetes ratio (F/B ratio) at the phylum level was calculated. The partial correlation between the fecal microbiota and eating behavior abnormality score was assessed, and dietary habits associated with the F/B ratio were analyzed. Results: A significant correlation was identified between F/B ratios and the eating behavior abnormality score (r = 0.26, FDR = 0.064). Additionally, multiple regression analysis identified a negative correlation trend between the F/B ratio and JDI-12 score (β = -0.22; p = 0.091), and exploratory analysis found a negative association between the F/B ratio and consumption of beef and pork, one of the less beneficial JDI-12 components (β = -0.33, FDR = 0.120). Conclusion: In healthy female university students, there was a positive correlation between eating behavior abnormality and the F/B ratio, indicating that adherence to the Japanese diet pattern may be associated with a lower F/B ratio., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The pink-colored and strictly aerobic bacterium strain, designated as TK19036 T , was isolated from mesopelagic layer of the Southwest Indian Ocean. This novel isolate can grow at 10-45 °C (optimum, 30 °C), pH 6.0-8.0 (optimum, pH 7.0), and 2-14% NaCl concentrations (w/v) (optimum, 6%). The predominant respiratory quinone was Menaquinone-7. Major polar lipid profiles contained two aminolipids, aminophospholipid, two glycolipids, phosphatidylethanolamine, and three unknown polar lipids. The preponderant cellular fatty acids were iso-C 15:0 , C 16:1 ω5c and iso-C 17:0 3-OH. Phylogenetic analyses based on 16S rRNA gene sequence uncovered that the strain TK19036 T pertained to the family Catalinimonadaceae under phylum Bacteroidota, and formed a distinct lineage with the closed species Tunicatimonas pelagia NBRC 107804 T . The up-to-bacteria-core gene phylogenetic trees also demonstrated a deep and novel branch formed by the strain TK19036 T within the family Catalinimonadaceae. Based on chemotaxonomic, phylogenetic and genomic features presented above, strain TK19036 T represents a novel species from a novel genus of the family Catalinimonadaceae, for which the name Roseihalotalea indica gen. nov. sp. nov. is proposed. The type strain is TK19036 T (= CGMCC 1.18940 T  = NBRC 116371 T )., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Members of the phylum Bacteroidetes play a key role in the marine carbon cycle through their degradation of polysaccharides via carbohydrate-active enzymes (CAZymes) and polysaccharide utilization loci (PULs). The discovery of novel CAZymes and PULs is important for our understanding of the marine carbon cycle. In this study, we isolated and identified a potential new genus of the family Catalimonadaceae, in the phylum Bacteroidetes, from the southwest Indian Ocean. Strain TK19036, the type strain of the new genus, is predicted to encode CAZymes that are relatively abundant in marine Bacteroidetes genomes. Tunicatimonas pelagia NBRC 107804T, Porifericola rhodea NBRC 107748T and Catalinimonas niigatensis NBRC 109829T, which exhibit 16 S rRNA similarities exceeding 90% with strain TK19036, and belong to the same family, were selected as reference strains. These organisms possess a highly diverse repertoire of CAZymes and PULs, which may enable them to degrade a wide range of polysaccharides, especially pectin and alginate. In addition, some secretory CAZymes in strain TK19036 and its relatives were predicted to be transported by type IX secretion system (T9SS). Further, to the best of our knowledge, we propose the first reported “hybrid” PUL targeting alginates in T. pelagia NBRC 107804T. Our findings provide new insights into the polysaccharide degradation capacity of marine Bacteroidetes, and suggest that T9SS may play a more important role in this process than previously believed.

Efficient recognition and transportation of chitin oligosaccharides are crucial steps for the utilization of chitin by heterotrophic bacteria. In this study, we employed structural biological and biochemical approaches to investigate the substrate recognition and acquisition mechanism of a novel chitin-binding SusD-like protein, AqSusD, which is derived from the chitin utilization gene cluster of a marine Bacteroides strain (Aquimarina sp. SCSIO 21287). We resolved the crystal structures of the AqSusD apo-protein and its complex with chitin oligosaccharides. Our results revealed that some crucial residues (Gln67, Phe87, and Asp276) underwent significant conformational changes to form tighter substrate binding sites for ligand binding. Moreover, we identified the functions of key amino acid residues and discovered that π-π stacking and hydrogen bonding between AqSusD and the ligand played significant roles in recognition of the protein for chitin oligosaccharide binding. Based on our findings and previous investigations, we put forward a model for the mechanism of chitin oligosaccharide recognition, capture, and transport by AqSusD, in collaboration with the membrane protein AqSusC. Our study deepens the understanding of the molecular-level "selfish" use of polysaccharides such as chitin by Bacteroides., (© 2023 Federation of European Biochemical Societies.)

A metagenomic time series from Arctic seawater was obtained from Dease Strait, to analyse the changes in bacterioplankton caused by the summer phytoplankton bloom. Bacterial clades specialized in the metabolism of polysaccharides, such as Bacteroidetes, became dominant along the bloom. These specialized taxa quickly displaced the microbial clades that dominate nutrient-poor waters during early spring, such as Archaea, Alpha-and Gammaproteobacteria. At the functional level, phyla Bacteroidetes, Planctomycetes, and Verrucomicrobia showed higher contents of polysaccharide-degradation functions. The Bacteroidetes community shifted toward species with higher polysaccharide-degrading capabilities, targeting algal polysaccharides in summer. Regarding transporters, Bacteroidetes dominated SusC-TonB transporters and had an exclusive family of glycoside-binding proteins (SusD). These proteins were used to identify polysaccharide-utilization loci that clustered transporters and polysaccharide-active enzymes, showing a higher level of specialization toward polysaccharide use. Altogether, these genomic features point to the genetic adaptations that promote the dominance of Bacteroidetes during phytoplankton blooms.

Gut microbiome dysbiosis has been widely implicated in cognitive impairment, but the identity of the specific bacterial taxa and mechanisms are not fully elucidated. Brain glucose hypometabolism coincides with the cognitive decline. This study explored the link among cognition, gut microbiota and glucose uptake based on the fecal microbiota transplantation from mild cognitive impairment individuals (MCI-FMT) and investigated whether similar mechanisms were involved in 27-hydroxycholesterol (27-OHC)-induced cognitive decline. Our results showed that the MCI-FMT mice exhibited learning and memory decline and morphological lesions in the brain and colon tissues. There were reduced 18 F-fluorodeoxyglucose uptake, downregulated expression of glucose transporters (GLUT1,3,4) and upregulated negative regulator of glucose uptake (TXNIP) in the brain. MCI-FMT altered the bacterial composition and diversity of the recipient mice, and the microbial signatures highlighted by the increased abundance of Bacteroides recapitulated the negative effects of MCI bacterial colonization. However, inhibiting Bacteroidetes or TXNIP increased the expression of GLUT1 and GLUT4, significantly improving brain glucose uptake and cognitive performance in 27-OHC-treated mice. Our study verified that cognitive decline and abnormal cerebral glucose uptake were associated with gut microbiota dysbiosis; we also revealed the involvement of Bacteroidetes and molecular mechanisms of TXNIP-related glucose uptake in cognitive deficits caused by 27-OHC.

Background: Human milk oligosaccharide (HMO) is a unique component of breastmilk. To date, no study has investigated the correlation between HMO and infant nutritional status particularly through the lens of gut microbiota. Therefore, our study aims to investigate the relationships between 2'-Fucosyllactose (2'-FL) in HMO and Firmicutes/Bacteroidetes (F/B) ratio among stunted infants., Methods: A case-control study was conducted among 103 mother-infant pairs in Malang City, Indonesia. The quantification of 2'-FL HMO was assessed using High-Performance Liquid Chromatography (HPLC). The F/B ratio was analyzed with real-time poly-chain reaction (RT-PCR). For bivariate analysis, we employed the Spearman correlation and Mann‒Whitney tests, while for multivariate analysis, we utilized multiple linear regression., Results: The findings showed that the stunted nutritional status was detected in 49 out of 103 infants. In this group, 40.81% of mothers of infants with a stunted nutritional status had a secretor-positive status, while all mothers of infants with appropriate nutritional status tested positive for the secretor status (100%). However, the association between maternal secretor status and infant nutritional status was not statistically significant (p>0.05). The average levels of 2'-FL HMO in breast milk were lower in the group with stunted infants compared to non-stunted infants (1.21 mg/L vs 1.40 mg/L). The regression analysis revealed a significant association of 2'-FL HMO levels with the presence of Bacteroidetes and value of the F/B ratio (p>0.05)., Conclusions: The breast milk component 2'-FL HMO significantly influences the gut microbiota of stunted infants. Future research aimed at elucidating the mechanisms by which 2'-FL HMO modulates infant gut microbiota should consider not only concentration and specific bacterial taxa but also intake levels (Tab. 2, Fig. 1, Ref. 37). Text in PDF www.elis.sk Keywords: 2'-fucosyllactose, human milk, oligosaccharide, firmicutes, bacteroidetes, stunting, infant.

The aim of the study was to examine serum levels of liver and intestinal fatty acid-binding proteins (L-FABP and I-FABP), fecal numbers of Bacteroidetes (B) and Firmicutes (F) in patients with metabolic-associated fatty liver disease (MAFLD) in combination with type 2 diabetes mellitus (Т2DM) and small intestinal bacterial overgrowth (SIBO) syndrome. Materials and methods. The prospective, interventional, randomized study included 51 patients with MAFLD in combination with Т2DM, who were examined and divided into 2 groups. Group 1 consisted of 24 patients with MAFLD and Т2DM without SIBO. Group 2 was comprised of 27 patients with MAFLD in combination with Т2DM and SIBO. The control group included 20 apparently healthy individuals. Serum levels of L-FABP and I-FABP were measured by ELISA method using the Human L-FABP and I-FABP ELISA Kit test systems, respectively (Elabscience, USA). Fecal numbers of B and F were determined by real-time PCR. Bacterial DNA was detected in a thermal cycler Rotor-Gene 6000 (QIAGEN, Germany) using DNA 16S rRNA primers and NanoDrop ND-8000 reagents (Thermo Scientific, USA). Results. To assess the state of intestinal permeability, serum levels of L-FABP and I-FABP were examined and numbers of phylum F and В as well as their ratio were calculated. Patients of both groups have been found to have increased serum levels of L-FABP, I-FABP, numbers of B in fecal samples and decreased numbers of F and F/B ratio. Conclusions. The study results obtained have revealed increased intestinal permeability and demonstrated an important diagnostic value of serum L-FABP and I-FABP as a biomarker of intestinal permeability in diabetic MAFLD patients with or without SIBO. Increased fecal numbers of Bacteroidetes, decreased numbers of Firmicutes and F/B ratio have been detected in diabetic MAFLD patients with or without SIBO.

Gut microbiome dysbiosis has been widely implicated in cognitive impairment, but the identity of the specific bacterial taxa and mechanisms are not fully elucidated. Brain glucose hypometabolism coincides with the cognitive decline. This study explored the link among cognition, gut microbiota and glucose uptake based on the fecal microbiota transplantation from mild cognitive impairment individuals (MCI-FMT) and investigated whether similar mechanisms were involved in 27-hydroxycholesterol (27-OHC)-induced cognitive decline. Our results showed that the MCI-FMT mice exhibited learning and memory decline and morphological lesions in the brain and colon tissues. There were reduced 18F-fluorodeoxyglucose uptake, downregulated expression of glucose transporters (GLUT1,3,4) and upregulated negative regulator of glucose uptake (TXNIP) in the brain. MCI-FMT altered the bacterial composition and diversity of the recipient mice, and the microbial signatures highlighted by the increased abundance of Bacteroides recapitulated the negative effects of MCI bacterial colonization. However, inhibiting Bacteroidetes or TXNIP increased the expression of GLUT1 and GLUT4, significantly improving brain glucose uptake and cognitive performance in 27-OHC-treated mice. Our study verified that cognitive decline and abnormal cerebral glucose uptake were associated with gut microbiota dysbiosis; we also revealed the involvement of Bacteroidetes and molecular mechanisms of TXNIP-related glucose uptake in cognitive deficits caused by 27-OHC.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 β-sandwich (β1-β7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands β3 and β4 ('motif Lβ3β4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS , which shares the same general topology as in Porphyromonas CTDs. However, motif Lβ3β4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Gut microbiota plays a crucial role in gastrointestinal tumors. Additionally, gut microbes influence the progression of esophageal cancer. However, the major bacterial genera that affect the invasion and metastasis of esophageal cancer remain unknown, and the underlying mechanisms remain unclear. Here, we investigated the gut flora and metabolites of patients with esophageal squamous cell carcinoma and found abundant Bacteroides and increased secretion and entry of the surface antigen lipopolysaccharide (LPS) into the blood, causing inflammatory changes in the body. We confirmed these results in a mouse model of 4NQO-induced esophageal carcinoma in situ and further identified epithelial-mesenchymal transition (EMT) occurrence and TLR4/Myd88/NF-κB pathway activation in mouse esophageal tumors. Additionally, in vitro experiments revealed that LPS from Bacteroides fragile promoted esophageal cancer cell proliferation, migration, and invasion, and induced EMT by activating the TLR4/Myd88/NF-κB pathway. These results reveal that Bacteroides are closely associated with esophageal cancer progression through a higher inflammatory response level and signaling pathway activation that are both common to inflammation and tumors induced by LPS, providing a new biological target for esophageal cancer prevention or treatment., (© 2024. The Author(s).)

Bacteroidetes are prevalent in soil ecosystems and are associated with various eukaryotic hosts, including plants, animals, and humans. The ubiquity and diversity of Bacteroidetes exemplify their impressive versatility in niche adaptation and genomic plasticity. Over the past decade, a wealth of knowledge has been obtained on the metabolic functions of clinically relevant Bacteroidetes, but much less attention has been given to Bacteroidetes living in close association with plants. To improve our understanding of the functional roles of Bacteroidetes for plants and other hosts, we review the current knowledge of their taxonomy and ecology, in particular their roles in nutrient cycling and host fitness. We highlight their environmental distribution, stress resilience, genomic diversity, and functional importance in diverse ecosystems, including, but not limited to, plant-associated microbiomes., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background/Objectives: The phyla Firmicutes and Bacteroidetes are the main constituents of the gut microbiota. An imbalance in the gut microbiota is a sign of dysbiosis, and the Firmicutes-to-Bacteroidetes ratio has been proposed to be a marker of it, especially in the context of obesity. Since Croatia is the country with one of the highest obesity rates in Europe, a pilot observational study was conducted. The aim of the study was to investigate the validity of this potential biomarker in a methodological study using sample processing, DNA sequence analysis and characterization of recruited participants, including various health factors. Methods: A study involving Croatian population was conducted. Participants age, body weight, gender, health history and lifestyle factors were recorded. Gut microbiota composition was analyzed using 16S rRNA sequencing. The F/B ratio was calculated and evaluated in the context of health factors. Statistical analysis was performed to detect the possible association of F/B ratio and excess body weight (kg) and possible impact of certain lifestyle factors. Results: No association between the F/B ratio and excess body weight (kg) was found. Excess body weight was significantly associated with higher age, male gender, and history of appendectomy. No significant health predictors of the F/B ratio were found, but weight gain was positively associated with a higher average F/B ratio. Conclusions: Although this study could not confirm the predictive value of the F/B ratio or any other phyla-related biomarker for excess body weight in the study population, it demonstrated interesting insights into the obesity-associated gut microbiota.

Alterations in the intestinal microbial flora are known to cause various diseases, and many people routinely consume probiotics or prebiotics to balance intestinal microorganisms and the growth of beneficial bacteria. In this study, we selected a peptide from fish (tilapia) skin that induces significant changes in the intestinal microflora of mice and reduces the Firmicutes/Bacteroidetes ratio, which is linked to obesity. We attempted to verify the anti-obesity effect of selected fish collagen peptides in a high-fat-diet-based obese mouse model. As anticipated, the collagen peptide co-administered with a high-fat diet significantly inhibited the increase in the Firmicutes/Bacteroidetes ratio. It increased specific bacterial taxa, including Clostridium_sensu_stricto_1, Faecalibaculum, Bacteroides , and Streptococcus , known for their anti-obesity effects. Consequently, alterations in the gut microbiota resulted in the activation of metabolic pathways, such as polysaccharide degradation and essential amino acid synthesis, which are associated with obesity inhibition. In addition, collagen peptide also effectively reduced all obesity signs caused by a high-fat diet, such as abdominal fat accumulation, high blood glucose levels, and weight gain. Ingestion of collagen peptides derived from fish skin induced significant changes in the intestinal microflora and is a potential auxiliary therapeutic agent to suppress the onset of obesity.

Net growth of microbial populations, that is, changes in abundances over time, can be studied using 16S rRNA fluorescence in situ hybridization (FISH). However, this approach does not differentiate between mortality and cell division rates. We used FISH-based image cytometry in combination with dilution culture experiments to study net growth, cell division, and mortality rates of four bacterial taxa over two distinct phytoplankton blooms: the oligotrophs SAR11 and SAR86, and the copiotrophic phylum Bacteroidetes , and its genus Aurantivirga . Cell volumes, ribosome content, and frequency of dividing cells (FDC) co-varied over time. Among the three, FDC was the most suitable predictor to calculate cell division rates for the selected taxa. The FDC-derived cell division rates for SAR86 of up to 0.8/day and Aurantivirga of up to 1.9/day differed, as expected for oligotrophs and copiotrophs. Surprisingly, SAR11 also reached high cell division rates of up to 1.9/day, even before the onset of phytoplankton blooms. For all four taxonomic groups, the abundance-derived net growth (-0.6 to 0.5/day) was about an order of magnitude lower than the cell division rates. Consequently, mortality rates were comparably high to cell division rates, indicating that about 90% of bacterial production is recycled without apparent time lag within 1 day. Our study shows that determining taxon-specific cell division rates complements omics-based tools and provides unprecedented clues on individual bacterial growth strategies including bottom-up and top-down controls. IMPORTANCE The growth of a microbial population is often calculated from their numerical abundance over time. However, this does not take cell division and mortality rates into account, which are important for deriving ecological processes like bottom-up and top-down control. In this study, we determined growth by numerical abundance and calibrated microscopy-based methods to determine the frequency of dividing cells and subsequently calculate taxon-specific cell division rates in situ . The cell division and mortality rates of two oligotrophic (SAR11 and SAR86) and two copiotrophic ( Bacteroidetes and Aurantivirga ) taxa during two spring phytoplankton blooms showed a tight coupling for all four taxa throughout the blooms without any temporal offset. Unexpectedly, SAR11 showed high cell division rates days before the bloom while cell abundances remained constant, which is indicative of strong top-down control. Microscopy remains the method of choice to understand ecological processes like top-down and bottom-up control on a cellular level., Competing Interests: The authors declare no conflict of interest.

Laccases are multicopper oxidases (MCOs) with a broad application spectrum, particularly in second-generation ethanol biotechnology and the bioremediation of xenobiotics and other highly recalcitrant compounds. Synthetic pesticides are xenobiotics with long environmental persistence, and the search for their effective bioremediation has mobilized the scientific community. Antibiotics, in turn, can pose severe risks for the emergence of multidrug-resistant microorganisms, as their frequent use for medical and veterinary purposes can generate constant selective pressure on the microbiota of urban and agricultural effluents. In the search for more efficient industrial processes, some bacterial laccases stand out for their tolerance to extreme physicochemical conditions and their fast generation cycles. Accordingly, to expand the range of effective approaches for the bioremediation of environmentally important compounds, the prospection of bacterial laccases was carried out from a custom genomic database. The best hit found in the genome of Chitinophaga sp. CB10, a Bacteroidetes isolate obtained from a biomass-degrading bacterial consortium, was subjected to in silico prediction, molecular docking, and molecular dynamics simulation analyses. The putative laccase CB10_180.4889 (Lac_CB10), composed of 728 amino acids, with theoretical molecular mass values of approximately 84 kDa and a pI of 6.51, was predicted to be a new CopA with three cupredoxin domains and four conserved motifs linking MCOs to copper sites that assist in catalytic reactions. Molecular docking studies revealed that Lac_CB10 had a high affinity for the molecules evaluated, and the affinity profiles with multiple catalytic pockets predicted the following order of decreasing thermodynamically favorable values: tetracycline (-8 kcal/mol) > ABTS (-6.9 kcal/mol) > sulfisoxazole (-6.7 kcal/mol) > benzidine (-6.4 kcal/mol) > trimethoprim (-6.1 kcal/mol) > 2,4-dichlorophenol (-5.9 kcal/mol) mol. Finally, the molecular dynamics analysis suggests that Lac_CB10 is more likely to be effective against sulfisoxazole-like compounds, as the sulfisoxazole-Lac_CB10 complex exhibited RMSD values lower than 0.2 nm, and sulfisoxazole remained bound to the binding site for the entire 100 ns evaluation period. These findings corroborate that LacCB10 has a high potential for the bioremediation of this molecule.

BACKGROUND: Human milk oligosaccharide (HMO) is a unique component of breastmilk. To date, no study has investigated the correlation between HMO and infant nutritional status particularly through the lens of gut microbiota. Therefore, our study aims to investigate the relationships between 2'-Fucosyllactose (2'-FL) in HMO and Firmicutes/Bacteroidetes (F/B) ratio among stunted infants. METHODS: A case-control study was conducted among 103 mother-infant pairs in Malang City, Indonesia. The quantification of 2'-FL HMO was assessed using High-Performance Liquid Chromatography (HPLC). The F/B ratio was analyzed with real-time poly-chain reaction (RT-PCR). For bivariate analysis, we employed the Spearman correlation and Mann--Whitney tests, while for multivariate analysis, we utilized multiple linear regression. RESULTS: The findings showed that the stunted nutritional status was detected in 49 out of 103 infants. In this group, 40.81% of mothers of infants with a stunted nutritional status had a secretor-positive status, while all mothers of infants with appropriate nutritional status tested positive for the secretor status (100%). However, the association between maternal secretor status and infant nutritional status was not statistically significant (p>0.05). The average levels of 2'-FL HMO in breast milk were lower in the group with stunted infants compared to non-stunted infants (1.21 mg/L vs 1.40 mg/L). The regression analysis revealed a significant association of 2'-FL HMO levels with the presence of Bacteroidetes and value of the F/B ratio (p>0.05). CONCLUSIONS: The breast milk component 2'-FL HMO significantly influences the gut microbiota of stunted infants. Future research aimed at elucidating the mechanisms by which 2'-FL HMO modulates infant gut microbiota should consider not only concentration and specific bacterial taxa but also intake levels [ABSTRACT FROM AUTHOR]

Abstract Gut microbiota plays a crucial role in gastrointestinal tumors. Additionally, gut microbes influence the progression of esophageal cancer. However, the major bacterial genera that affect the invasion and metastasis of esophageal cancer remain unknown, and the underlying mechanisms remain unclear. Here, we investigated the gut flora and metabolites of patients with esophageal squamous cell carcinoma and found abundant Bacteroides and increased secretion and entry of the surface antigen lipopolysaccharide (LPS) into the blood, causing inflammatory changes in the body. We confirmed these results in a mouse model of 4NQO-induced esophageal carcinoma in situ and further identified epithelial–mesenchymal transition (EMT) occurrence and TLR4/Myd88/NF-κB pathway activation in mouse esophageal tumors. Additionally, in vitro experiments revealed that LPS from Bacteroides fragile promoted esophageal cancer cell proliferation, migration, and invasion, and induced EMT by activating the TLR4/Myd88/NF-κB pathway. These results reveal that Bacteroides are closely associated with esophageal cancer progression through a higher inflammatory response level and signaling pathway activation that are both common to inflammation and tumors induced by LPS, providing a new biological target for esophageal cancer prevention or treatment.

Abstract Background Hadal trenches (>6000 m) are the deepest oceanic regions on Earth and depocenters for organic materials. However, how these enigmatic microbial ecosystems are fueled is largely unknown, particularly the proportional importance of complex polysaccharides introduced through deposition from the photic surface waters above. In surface waters, Bacteroidetes are keystone taxa for the cycling of various algal-derived polysaccharides and the flux of carbon through the photic zone. However, their role in the hadal microbial loop is almost unknown. Results Here, culture-dependent and culture-independent methods were used to study the potential of Bacteroidetes to catabolize diverse polysaccharides in Mariana Trench waters. Compared to surface waters, the bathypelagic (1000–4000 m) and hadal (6000–10,500 m) waters harbored distinct Bacteroidetes communities, with Mesoflavibacter being enriched at ≥ 4000 m and Bacteroides and Provotella being enriched at 10,400–10,500 m. Moreover, these deep-sea communities possessed distinct gene pools encoding for carbohydrate active enzymes (CAZymes), suggesting different polysaccharide sources are utilised in these two zones. Compared to surface counterparts, deep-sea Bacteroidetes showed significant enrichment of CAZyme genes frequently organized into polysaccharide utilization loci (PULs) targeting algal/plant cell wall polysaccharides (i.e., hemicellulose and pectin), that were previously considered an ecological trait associated with terrestrial Bacteroidetes only. Using a hadal Mesoflavibacter isolate (MTRN7), functional validation of this unique genetic potential was demonstrated. MTRN7 could utilize pectic arabinans, typically associated with land plants and phototrophic algae, as the carbon source under simulated deep-sea conditions. Interestingly, a PUL we demonstrate is likely horizontally acquired from coastal/land Bacteroidetes was activated during growth on arabinan and experimentally shown to encode enzymes that hydrolyze arabinan at depth. Conclusions Our study implies that hadal Bacteroidetes exploit polysaccharides poorly utilized by surface populations via an expanded CAZyme gene pool. We propose that sinking cell wall debris produced in the photic zone can serve as an important carbon source for hadal heterotrophs and play a role in shaping their communities and metabolism. Video Abstract

The present study investigated the utilization of an arabinogalactan from Lycium barbarum (LBP-3) by intestinal Bacteroidetes species. The mixed-culture assay showed 58.4 % LBP-3 was utilized, and Bacteroides caccae and Phocaeicola vulgatus utilized more LBP-3 in single-culture compared to others. During in vitro fermentation of LBP-3, P. vulgatus favored arabinose while B. caccae preferred galactose. Moreover, 9 and 25 oligosaccharides were identified by HPLC-MS n in conditioned media (CM) derived from B. caccae and P. vulgatus, respectively. All of 3 tested Parabacteroides species (P. distasonis, P. goldsteinii, and P. johnsonii) markedly proliferated in CM of B. caccae and P. vulgatus, and proliferations of B. uniformis, B. finegoldii, B. ovatus and B. thetaiotaomicron also increased significantly in CM of B. caccae. The study suggests that the ability of Bacteroidetes species to degrade LBP-3 and sheds light on cooperative interactions of Bacteroides, Phocaeicola, and Parabacteroides species in the presence of LBP-3., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Two heterotrophic bacteroidetes strains were isolated as satellites from autotrophic enrichments inoculated with samples from hypersaline soda lakes in southwestern Siberia. Strain Z-1702 T is an obligate anaerobic fermentative saccharolytic bacterium from an iron-reducing enrichment culture, while Ca. Cyclonatronum proteinivorum Omega T is an obligate aerobic proteolytic microorganism from a cyanobacterial enrichment. Cells of isolated bacteria are characterized by highly variable morphology. Both strains are chloride-independent moderate salt-tolerant obligate alkaliphiles and mesophiles. Strain Z-1702 T ferments glucose, maltose, fructose, mannose, sorbose, galactose, cellobiose, N-acetyl-glucosamine and alpha-glucans, including starch, glycogen, dextrin, and pullulan. Strain Omega T is strictly proteolytic utilizing a range of proteins and peptones. The main polar lipid fatty acid in both strains is iso-C 15:0 , while other major components are various C 16 and C 17 isomers. According to pairwise sequence alignments using BLAST Gracilimonas was the nearest cultured relative to both strains (<90% of 16S rRNA gene sequence identity). Phylogenetic analysis placed strain Z-1702 T and strain Omega T as two different genera in a deep-branching clade of the new family level within the order Balneolales with genus. Based on physiological characteristics and phylogenetic position of strain Z-1702 T it was proposed to represent a novel genus and species Natronogracilivirga saccharolityca gen. nov., sp. nov. (= DSMZ 109061 T =JCM 32930 T =VKM B 3262 T ). Furthermore, phylogenetic and phenotypic parameters of N. saccharolityca and C. proteinivorum gen. nov., sp. nov., strain Omega T (=JCM 31662 T , =UNIQEM U979 T ), make it possible to include them into a new family with a proposed designation Cyclonatronaceae fam. nov.., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

ABSTRACT: The present study was conducted to investigate the stimbiotic mechanism of xylo-oligosaccharide (XOS) in degrading the complex polysaccharides by the caecal bacteria of the chicken, by applying a proteomic approach. A total of 800 as-hatched Ross 308 broiler chicks were equally divided into 4 experimental pens (200 chicks per pen) at a commercial poultry barn, allocating 2 pens per treatment. Birds were fed ad libitum with 2 dietary treatments; CON (without XOS) and XOS (with 0.1g XOS/kg diet) from d 0 to 35. From each pen, 60 Individual birds were weighed weekly whereas caecal content was obtained from 5 birds cervically dislocated on d 35. The caecal bacteria were lysed and their proteins were quantified using label-free quantitative proteomic mass spectrometry. The results showed that XOS significantly increased (P < 0.05) bird weight on d 7, 14, 21, and 28, and body weight gain on d 7, 14, 21, and 35 compared to CON. However, no difference (P > 0.05) in body weight gain was observed from d 0 to 35 between CON and XOS. The proteomic analysis of caecal bacteria revealed that 29 proteins were expressed differently between the CON and the XOS group. Out of 29, 20 proteins were significantly increased in the XOS group compared to CON and 9 of those proteins belonged to the starch-utilizing system (Sus)-like system of the gram-negative Bacteroidetes. Bacteroides thetaiotaomicron (Bt) is a significant constituent of the human gut microbiota, known for its remarkable ability to hydrolyze most glycosidic bonds of polysaccharides. This microorganism possesses a 5-protein complex in its outer membrane, named the starch utilization system (Sus), responsible for adhering to, breaking down, and transporting starch into the cell. Sus serves as an exemplar system for numerous polysaccharide utilization loci that target glycans found in Bt and other members of the Bacteroidetes phylum. The proteins of the Sus-like system are involved in the degradation of complex polysaccharides and transportation of the oligosaccharides into the periplasm of the caecal bacteria where they are further broken down into smaller units. These smaller units are then transported into the cytoplasm of the cell where they are utilized in metabolic pathways leading to potential generation of short-chain fatty acids, thus improving the nutritive value of residual feed. In conclusion, XOS supplementation upregulates the expression of the proteins of the Sus-like system indicating its role as a stimbiotic.

A Gram-stain-negative bacterium, designated strain LY-5  T , was isolated from an activated sludge sample collected from a river in Yancheng city, Jiangsu province, China. Cells of strain LY-5  T , were strictly aerobic, non-motile and rod-shaped. Growth occurred at 15-37 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum 7.0) and at 0-3% (w/v) NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene and genome sequences indicated that strain LY-5  T formed a distinct phyletic branch within the family Chitinophagaceae, with closest relatives being members of the genera Phnomibacter, Aurantisolimonas, and Chitinophaga, sharing 88.5-90.3% sequence similarity. Moreover, the average amino acid identity (AAI) between strain LY-5  T and its closed phylogenetic neighbors was below 70%, indicating it belongs to a novel genus. The predominant cellular fatty acids of LY-5  T were iso-C 15:0 , iso-C 15:1 G and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c), and the only isoprenoid quinone was menaquinone-7 (MK-7). The major polar lipids identified in strain LY-5  T comprised phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids. The genome size of strain LY-5  T was 4.8 Mbp with a G + C content of 38.9%. Based on the evidence presented in this study, strain LY-5  T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Polluticaenibacter yanchengensis gen. nov., sp. nov. (Type strain LY-5  T  = CCTCC AB 2023260  T  = KCTC 102218  T ) is proposed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Obtaining body condition is an important life history challenge that directly impacts individual fitness and is particularly important for hibernating animals, whose maintenance of adequate body fat and mass is essential for survival. It is well-documented that host-associated microorganisms play a vital role in animal physiology and behavior. Recent work demonstrates that gut microbes are associated with fat accumulation and obesity, particularly the phyla Firmicutes and Bacteroidetes. The focus of most microbiome studies has been on human health or involved lab-reared animals used as a model system. However, these microbes likely are important for individual fitness in wild populations and provide potential mechanistic insights into the adaptability and survival of wildlife. Here we tested whether symbiotic microorganisms within the phyla of Firmicutes and Bacteroidetes were associated with summer mass gain in an exceptionally well-studied wild population of yellow-bellied marmots (Marmota flaviventer) by analyzing 207 fecal samples collected over 5 summer active seasons. Results showed that marmots with higher mass gain rates had a greater relative abundance of Firmicutes. In contrast, a higher relative abundance of Bacteroidetes was associated with lower mass gain rates, but only for marmots living in harsher environments. Similar patterns were found at the family level where Ruminococcaceae, a member of Firmicutes, was associated with higher mass gain rates, and Muribaculaceae, a member of Bacteroidetes, was associated with lower mass gain rates in harsher environments. Although correlative, these results highlight the potential importance of symbiotic gut microbiota to mass gain in the wild—a trait associated with survival and fitness in many taxonomic groups. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the stimbiotic mechanism of xylo-oligosaccharide (XOS) in degrading the complex polysaccharides by the caecal bacteria of the chicken, by applying a proteomic approach. A total of 800 as-hatched Ross 308 broiler chicks were equally divided into 4 experimental pens (200 chicks per pen) at a commercial poultry barn, allocating 2 pens per treatment. Birds were fed ad libitum with 2 dietary treatments; CON (without XOS) and XOS (with 0.1g XOS/kg diet) from d 0 to 35. From each pen, 60 Individual birds were weighed weekly whereas caecal content was obtained from 5 birds cervically dislocated on d 35. The caecal bacteria were lysed and their proteins were quantified using label-free quantitative proteomic mass spectrometry. The results showed that XOS significantly increased (P < 0.05) bird weight on d 7, 14, 21, and 28, and body weight gain on d 7, 14, 21, and 35 compared to CON. However, no difference (P > 0.05) in body weight gain was observed from d 0 to 35 between CON and XOS. The proteomic analysis of caecal bacteria revealed that 29 proteins were expressed differently between the CON and the XOS group. Out of 29, 20 proteins were significantly increased in the XOS group compared to CON and 9 of those proteins belonged to the starch-utilizing system (Sus)-like system of the gram-negative Bacteroidetes. Bacteroides thetaiotaomicron (Bt) is a significant constituent of the human gut microbiota, known for its remarkable ability to hydrolyze most glycosidic bonds of polysaccharides. This microorganism possesses a 5-protein complex in its outer membrane, named the starch utilization system (Sus), responsible for adhering to, breaking down, and transporting starch into the cell. Sus serves as an exemplar system for numerous polysaccharide utilization loci that target glycans found in Bt and other members of the Bacteroidetes phylum. The proteins of the Sus-like system are involved in the degradation of complex polysaccharides and transportation of the oligosaccharides into the periplasm of the caecal bacteria where they are further broken down into smaller units. These smaller units are then transported into the cytoplasm of the cell where they are utilized in metabolic pathways leading to potential generation of short-chain fatty acids, thus improving the nutritive value of residual feed. In conclusion, XOS supplementation upregulates the expression of the proteins of the Sus-like system indicating its role as a stimbiotic., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a helical loop on the cell surface. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell and revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction. Several electron microscopic analyses revealed the presence of a possible multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. These results provide insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along tracks that may form a multi-rail system underneath the outer membrane. The insights may give clues as to how the SprB filaments get their driving force., (© 2023. The Author(s).)

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel&#95;I&#95;48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel&#95;I&#95;48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression.

Background: The gut microbiome, which is the collection of microorganisms living in the gastrointestinal tract, has been shown to play a significant role in the development of metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM). Studies have found that the ratio of Firmicutes to Bacteroidetes (F/B) is higher in obese individuals compared to lean individuals and tends to decrease with weight loss. However, the relationship between the F/B ratio and T2DM in Korean individuals, with or without obesity, is not fully understood. Objective: The objective of this study is to compare the F/B ratios and metabolic profiles of lean and obese Korean individuals with T2DM. Methods: In this study, 36 individuals with type 2 diabetes mellitus (T2DM) were recruited and classified into four groups (I, II, III, and IV) based on their body mass index (BMI). Group I had a BMI of less than 23.0, group II had a BMI between 23.0 and 24.9, group III had a BMI between 25.0 and 29.9, and group IV had a BMI of 30 kg/m2 or greater. Fecal samples were collected from all participants and sent to Chunlab Inc. (located in Seoul, Republic of Korea) for analysis. The changes in the major microbial phyla within the samples were investigated using quantitative real-time PCR. The collected data were then statistically analyzed using the SPSS program. Results: The levels of triglycerides and alanine transaminase in group I were significantly lower than in the other three groups. The amount of Actinobacteria in group IV was the highest among all four groups. The ratio of Firmicutes to Bacteroidetes increased as BMI increased, and this ratio was positively correlated with AST activity. Conclusions: Our study showed that there is a correlation between the degree of obesity in individuals with diabetes and their gut microbiome. Additionally, the ratio of Firmicutes to Bacteroidetes (F/B ratio) may play a role in the metabolic effects of fatty liver disease, as it may contribute to obesity.

Ixotrophy is a contact-dependent predatory strategy of filamentous bacteria in aquatic environments for which the molecular mechanism remains unknown. We show that predator-prey contact can be established by gliding motility or extracellular assemblages we call "grappling hooks." Cryo-electron microscopy identified the grappling hooks as heptamers of a type IX secretion system substrate. After close predator-prey contact is established, cryo-electron tomography and functional assays showed that puncturing by a type VI secretion system mediated killing. Single-cell analyses with stable isotope-labeled prey revealed that prey components are taken up by the attacker. Depending on nutrient availability, insertion sequence elements toggle the activity of ixotrophy. A marine metagenomic time series shows coupled dynamics of ixotrophic bacteria and prey. We found that the mechanism of ixotrophy involves multiple cellular machineries, is conserved, and may shape microbial populations in the environment.

Background/Objectives : The phyla Firmicutes and Bacteroidetes are the main constituents of the gut microbiota. An imbalance in the gut microbiota is a sign of dysbiosis, and the Firmicutes-to-Bacteroidetes ratio has been proposed to be a marker of it, especially in the context of obesity. Since Croatia is the country with one of the highest obesity rates in Europe, a pilot observational study was conducted. The aim of the study was to investigate the validity of this potential biomarker in a methodological study using sample processing, DNA sequence analysis and characterization of recruited participants, including various health factors. Methods : A study involving Croatian population was conducted. Participants age, body weight, gender, health history and lifestyle factors were recorded. Gut microbiota composition was analyzed using 16S rRNA sequencing. The F/B ratio was calculated and evaluated in the context of health factors. Statistical analysis was performed to detect the possible association of F/B ratio and excess body weight (kg) and possible impact of certain lifestyle factors. Results : No association between the F/B ratio and excess body weight (kg) was found. Excess body weight was significantly associated with higher age, male gender, and history of appendectomy. No significant health predictors of the F/B ratio were found, but weight gain was positively associated with a higher average F/B ratio. Conclusions : Although this study could not confirm the predictive value of the F/B ratio or any other phyla-related biomarker for excess body weight in the study population, it demonstrated interesting insights into the obesity-associated gut microbiota.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4 + T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4 + T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 β-sandwich (β1–β7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus (‘motif C-t.’) and the loop connecting strands β3 and β4 (‘motif Lβ3β4’) as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lβ3β4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

ABSTRACT Bacteria have to persist under low iron conditions in order to adapt to the nutritional immunity of a host. Since the knowledge of iron stimulon of Bacteroidetes is sparse, we examined oral (Porphyromonas gingivalis and Prevotella intermedia) and gut (Bacteroides thataiotaomicron) representatives for their ability to adapt to iron deplete and iron replete conditions. Our transcriptomics and comparative genomics analysis show that many iron-regulated mechanisms are conserved within the phylum. They include genes upregulated in low iron, as follows: fldA (flavodoxin), hmu (hemin uptake operon), and loci encoding ABC transporters. Downregulated genes were frd (ferredoxin), rbr (rubrerythrin), sdh (succinate dehydrogenase/fumarate reductase), vor (oxoglutarate oxidoreductase/dehydrogenase), and pfor (pyruvate:ferredoxin/flavodoxin oxidoreductase). Some genus-specific mechanisms, such as the sus of B. thetaiotaomicron coding for carbohydrate metabolism and the xusABC coding for xenosiderophore utilization were also identified. While all bacteria tested in our study had the nrfAH operon coding for nitrite reduction and were able to reduce nitrite levels present in culture media, the expression of the operon was iron dependent only in B. thetaiotaomicron. It is noteworthy that we identified a significant overlap between regulated genes found in our study and the B. thetaiotaomicron colitis study (W. Zhu, M. G. Winter, L. Spiga, E. R. Hughes et al., Cell Host Microbe 27:376–388, 2020, http://dx.doi.org/10.1016/j.chom.2020.01.010). Many of those commonly regulated genes were also iron regulated in the oral bacterial genera. Overall, this work points to iron being the master regulator enabling bacterial persistence in the host and paves the way for a more generalized investigation of the molecular mechanisms of iron homeostasis in Bacteroidetes. IMPORTANCE Bacteroidetes are an important group of anaerobic bacteria abundant both in the oral and gut microbiomes. Although iron is a required nutrient for most living organisms, the molecular mechanisms of adaptation to the changing levels of iron are not well known in this group of bacteria. We defined the iron stimulon of Bacteroidetes by examination of the transcriptomic response of Porphyromonas gingivalis and Prevotella intermedia (both belong to the oral microbiome) and Bacteroidetes thetaiotaomicron (belongs to the gut microbiome). Our results indicate that many of the iron-regulated operons are shared among the three genera. Furthermore, using bioinformatics analysis, we identified a significant overlap between our in vitro studies and transcriptomic data derived from a colitis study, thus underscoring the biological significance of our work. Defining the iron-dependent stimulon of Bacteroidetes can help to identify the molecular mechanisms of iron-dependent regulation as well as better understand the persistence of the anaerobes in the human host.