Gut microbiome of mealworms (Tenebrio molitor Larvae) show similar responses to polystyrene and corn straw diets.

BACKGROUND: Some insects can degrade both natural and synthetic plastic polymers, their host and gut microbes play crucial roles in this process. However, there is still a scientific gap in understanding how the insect adapted to the polystyrene (PS) diet from natural feed. In this study, we analyzed diet consumption, gut microbiota responses, and metabolic pathways of Tenebrio molitor larvae exposed to PS and corn straw (CS). RESULTS: T. molitor larvae were incubated under controlled conditions (25 ± 1 °C, 75 ± 5% humidity) for 30 days by using PS foam with weight-, number-, and size-average molecular weight (Mw, Mn, and Mz) of 120.0, 73.2, and 150.7 kDa as a diet, respectively. The larvae exhibited lower PS consumption (32.5%) than CS (52.0%), and these diets had no adverse effects on their survival. The gut microbiota structures, metabolic pathways, and enzymatic profiles of PS- and CS-fed larvae showed similar responses. The gut microbiota of larvae analysis indicated Serratia sp., Staphylococcus sp., and Rhodococcus sp. were associated with both PS and CS diets. Metatranscriptomic analysis revealed that xenobiotics, aromatic compounds, and fatty acid degradation pathways were enriched in PS- and CS-fed groups; laccase-like multicopper oxidases, cytochrome P450, monooxygenase, superoxidase, and dehydrogenase were involved in lignin and PS degradation. Furthermore, the upregulated gene lac640 in both PS- and CS-fed groups was overexpressed in E. coli and exhibited PS and lignin degradation ability. CONCLUSIONS: The high similarity of gut microbiomes adapted to biodegradation of PS and CS indicated the plastics-degrading ability of the T. molitor larvae originated through an ancient mechanism that degrades the natural lignocellulose. Video Abstract.

Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice.

AIMS: Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC. MAIN METHODS: Standard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing. KEY FINDINGS: Azurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis). SIGNIFICANCE: These results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.

Human supplementation with Pediococcus acidilactici GR-1 decreases heavy metals levels through modifying the gut microbiota and metabolome.

Exposure to heavy metals (HMs) is a threat to human health. Although probiotics can detoxify HMs in animals, their effectiveness and mechanism of action in humans have not been studied well. Therefore, we conducted this randomized, double-blind, controlled trial on 152 occupational workers from the metal industry, an at-risk human population, to explore the effectiveness of probiotic yogurt in reducing HM levels. Participants were randomly assigned to two groups: one consumed probiotic yogurt containing the HM-resistant strain Pediococcus acidilactici GR-1 and the other consumed conventional yogurt for 12 weeks. Analysis of metal contents in the blood revealed that the consumption of probiotic yogurt resulted in a higher and faster decrease in copper (34.45%) and nickel (38.34%) levels in the blood than the consumption of conventional yogurt (16.41% and 27.57%, respectively). Metagenomic and metabolomic studies identified a close correlation between gut microbiota (GM) and host metabolism. Significantly enriched members of Blautia and Bifidobacterium correlated positively with the antioxidant capacities of GM and host. Further murine experiments confirmed the essential role of GM and protective effect of GR-1 on the antioxidative role of the intestine against copper. Thus, the use of probiotic yogurt may be an effective and affordable approach for combating toxic metal exposure through the protection of indigenous GM in humans.ClinicalTrials.gov identifier: ChiCTR2100053222.

Chitooligosaccharides production from shrimp chaff in chitosanase cell surface display system.

Chitin refers to a natural biopolymer, which is economically significant to next-generation biorefineries. In this study, a novel high-yield method with cell surface-display chitosanase (CHI-1) was built to produce chitooligosaccharides (COS) from shrimp chaff through the co-fermentation in the presence of Bacillus subtilis and Acetobacter sp. Under the optimized co-fermentation conditions (5 g/L yeast extracts, 10 g/L KH2PO4, 6% ethanol, 50 g/L glucose), the final deproteinization (DP) and demineralization (DM) efficiency and the chitin yield were achieved as 94, 92 and 18%, respectively. The engineered E. coli BL21-pET23b(+)-NICHI maintained 81% of the initial enzyme activity after 40 days at room temperature. The crude CHI-1 was inactivated after one-day interacting with prepared chitosan. Moreover, E. coli BL21-pET23b(+)-NICHI still maintained excellent hydrolysis ability in 7 days, and the COS yield reached 41%. Accordingly, the proposed method exhibited excellent stability and a high hydrolysis efficiency to produce COS with whole engineered cells.

Nesterenkonia ebinurensis sp. nov., a Novel Actinobacterium Isolated From Populus euphratica.

During our studies focused on the microorganism diversity and community structure of Populus euphratica at Ebinur lake wetland nature reserve in Xinjiang Uyghur Autonomous Region, PR China, a Gram-positive, aerobic, short rod-shaped bacterium without flagellum, designated as MD2T, was isolated from a piece of resina on Populus euphratica. The isolate grew at temperature of 10-45 °C (optimum 37 °C), pH of 7.0-12.0 (optimum pH 8.0) and NaCl concentration of 1-18% (optimum 3%, w/v). Based on the 16S rRNA gene sequences and the phylogenetic analysis, the strain shared the highest sequence similarities to Nesterenkonia alkaliphila JCM 19766T (96.3%), Nesterenkonia populi KCTC 29119T (95.9%), Nesterenkonia alba CCTCC AB 207011T (95.5%), and was placed within the radiation of Nesterenkonia species in the phylogenetic trees. The draft genome of the isolate was sequenced, which comprised 3,739,891 bp with G + C content of 63 mol%, and was annotated to contain 3614 protein-coding genes, 44 tRNA genes and 5 rRNA genes. Chemotaxonomic analysis indicated that the main respiratory quinones were MK-8 and MK-9, the predominant cellular fatty acids were anteiso-C17:0, anteiso-C15:0 and iso-C16:0, the major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol andphosphatidylinositol. According to the phenotypic, chemotaxonomic and phylogenetic features, strain MD2T is considered to represent a novel species, for which the name Nesterenkonia ebinurensis sp. nov. is proposed. The type strain is MD2T (= KCTC 52999T = MCCC 1K03343T).

Pseudomonas lopnurensis sp. nov., an endophytic bacterium isolated from Populus euphratica at the ancient Ugan river.

A Gram-stain negative, aerobic, rod-shaped, motile by a single polar flagellum, non-spore-forming bacterium, designated strain AL-54T, was isolated from the storage liquid in the stems of Populus euphratica tree at the ancient Ugan River in Xinjiang, PR China. Isolated AL-54T grew optimally at pH 7.0 and temperature 35 °C in the presence of 3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that the isolate belonged to the genus Pseudomonas and was closely related to Pseudomonas songnenensis NEAU-ST5-5 T (97.6%), Pseudomonas zhaodongensis NEAU-ST5-21 T (97.5%), Pseudomonas alcaliphila AL15-21T (97.3%), Pseudomonas toyotomiensis HT-3T (97.3%), Pseudomonas oleovorans subsp. lubricantis RS1T (97.3%), Pseudomonas stutzeri ATCC 17588T (97.3%), Pseudomonas chengduensis CGMCC 2318T (97.2%), and Pseudomonas xanthomarina KMM 1447T (97.1%). Multilocus Sequences Analysis (MLSA) of strain AL-54T based on the three housekeeping genes, rpoB, rpoD and gyrB further confirmed the phylogenetic assignment of the isolates. The G+C content was 64.7 mol%. The DNA-DNA hybridization with P. songnenensis NEAU-ST5-5 T, P. zhaodongensis NEAU-ST5-21T, P. alcaliphila AL15-21T, P. toyotomiensis HT-3T, P. oleovorans subsp. lubricantis RS1T, P. stutzeri ATCC 17588T, P. chengduensis CGMCC 2318T and P. xanthomarina KMM 1447T revealed 44.0%, 44.7%, 60.1%, 48.7%, 49.1%, 60.1%, 58.9% and 60.2% relatedness respectively. The predominant quinone system is ubiquinone-9 (Q-9). The major components of the cellular fatty acids (>10%) were summed feature 8 (comprising C18:1 ω7c /C18:1 ω6c), summed feature 3 (comprising C16:1 ω7c /C16:1 ω6c) and C16:0. The detected major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). On the basis of phenotypic data, chemotaxonomic and phylogenetic properties, strain AL-54T can consider as a novel species within the genus Pseudomonas, for which the name Pseudomonas lopnurensis sp. nov. is proposed. The type strain is AL-54T (= JCM 19136T = CCTCC AB 2013066T = NRRL B-59987T).

The guanidine thiocyanate-high EDTA method for total microbial RNA extraction from severely heavy metal-contaminated soils.

Molecular analyses relying on RNA, as a direct way to unravel active microbes and their functional genes, have received increasing attention from environmental researchers recently. However, extracting sufficient and high-quality total microbial RNA from seriously heavy metal-contaminated soils is still a challenge. In this study, the guanidine thiocyanate-high EDTA (GTHE) method was established and optimized for recovering high quantity and quality of RNA from long-term heavy metal-contaminated soils. Due to the low microbial biomass in the soils, we combined multiple strong denaturants and intense mechanical lysis to break cells for increasing RNA yields. To minimize RNAase and heavy metals interference on RNA integrity, the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 ml g-1 soil and 10 to 100 mM, respectively. This optimized GTHE method was applied to seven severely contaminated soils, and the RNA recovery efficiencies were 2.80 ~ 59.41 µg g-1 soil. The total microbial RNA of non-Cr(VI) (NT) and Cr(VI)-treated (CT) samples was utilized for molecular analyses. The result of qRT-PCR demonstrated that the expressions of two tested genes, chrA and yieF, were respectively upregulated 4.12- and 62.43-fold after Cr(VI) treatment. The total microbial RNA extracted from NT and CT samples, respectively, reached to 26.70 µg and 30.75 µg, which were much higher than the required amount (5 µg) for metatranscriptomic library construction. Besides, ratios of mRNA read were more than 86%, which indicated the high-quality libraries constructed for metatranscriptomic analysis. In summary, the GTHE method is useful to study microbes of contaminated habitats.

Exploring novel Cr(VI) remediation genes for Cr(VI)-contaminated industrial wastewater treatment by comparative metatranscriptomics and metagenomics.

Microbial remediation is a promising method to treat Cr(VI) in industrial wastewater. The remediation efficiency and stress-resistance ability of Cr(VI) remediation genes in microbes are the limiting factors for their application in industrial wastewater treatment. To screen novel highly efficient Cr(VI) remediation genes, comparative metatranscriptomic and metagenomic analyses were performed on long-term Cr(VI)-contaminated riparian soil with/without additional Cr(VI) treatment. The most suitable Cr(VI) treatment time was determined to be 30 min according to the high quality RNA yield and fold changes in gene expression. Six novel genes, which had complete open reading frames (ORFs) in metagenomic libraries, were identified from unculturable microbes. In the phenotypic functional assay, all novel genes enhanced the Cr(VI) resistance/reduction ability of E. coli. In the industrial wastewater treatment, E-mcr and E-gsr presented at least 50% Cr(VI) removal efficiencies in the presence of 200-600 µM of Cr(VI), without a decrease in efficiency over 17 days. The stress resistance assay showed that gsr increased the growth rate of E. coli by at least 30% under different extreme conditions, and thus, gsr was identified as a general stress-response gene. In the Cr valence distribution assay, E-mcr presented ~40 µM higher extracellular Cr (III) compared to E-yieF. Additionally, transmission electron microscopy (TEM) of E-mcr showed bulk black agglomerates on the cell surface. Thus, mcr was identified as a membrane chromate reductase gene. This research provides a new idea for studying novel highly efficient contaminant remediation genes from unculturable microbes.

Pseudomonas tarimensis sp. nov., an endophytic bacteria isolated from Populus euphratica.

An endophytic bacterium, MA-69T, was isolated from the storage liquid in the stems of Populuseuphratica trees at the ancient Ugan River in Xinjiang, PR China. Strain MA-69T was found to be short rod-shaped, Gram-stain-negative, non-spore-forming, aerobic and motile by means of a monopolar flagellum. According to phylogenetic analysis based on 16S rRNA gene sequences, strain MA-69T was assigned to the genus Pseudomonas with highest 16S rRNA gene sequence similarity of 97.5 % to Pseudomonas azotifigens JCM 12708T, followed by Pseudomonas matsuisoli JCM 30078T (97.5 %), Pseudomonas balearica DSM 6083T (97.1 %), Azotobacter salinestris ATCC 49674T (96.1 %) and Pseudomonas indica DSM 14015T (95.9 %). Analysis of strain MA-69T based on the three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the isolate to be distinctly delineated from species of the genus Pseudomonas. The DNA G+C content of strain MA-69T was 64.1 mol%. DNA-DNA hybridization with Pseudomonas azotifigens JCM 12708T, Pseudomonas matsuisoli JCM 30078T and Pseudomonas balearica DSM 6083T revealed 62.9, 60.1 and 49.0 % relatedness, respectively. The major fatty acids in strain MA-69T were summed feature 3 (25.7 %), summed feature 8 (24.0 %), C19 : 0cyclo ω8c (19.9 %), C16 : 0 (14.6 %) and C12 : 0 (6.3 %). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Q-9 was the major quinone in strain MA-69T. Based on phenotypic, chemotaxonomic and phylogenetic properties, strain MA-69T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas tarimensis sp. nov. is proposed. The type strain is MA-69T (=CCTCC AB 2013065T=KCTC 42447T).