Plastic pollution is an environmental problem that has aroused global concern, and plastics degradation by insect gut microbiota is a new initiative to solve this problem. Despite the important role of insect gut microbiota in the degradation of plastics, little is known about the composition and dynamics of insect gut microbiota. [Objective] To study the effects of feeding plastics on the physiological indices and the composition and dynamics of gut microbiota in the larvae of four insect species. [Methods] Polystyrene (PS), polyethylene (PE), and wheat bran (control) were used as the sole carbon source respectively to feed the larvae of Zophobas atratus Fab., Tenebrio molitor L., Tenebrio obscurus F., and Galleria mellonella L. The dynamics of gut microbiota in the larvae of the four insect species were investigated by fluorescence in situ hybridization, and the correlations between the physiological indices and gut microbiota were analyzed. [Results] All four insect species fed with PS and PE had significantly lower body weight gain and body length increase than those in the control. The survival rates of Z. atratus and T. molitor larvae fed with PS were 25.33% and 11.75%, respectively, higher than those fed with PE. The dominant microbial taxa of the gut microbiota in the four insect species were Firmicutes (relative abundance of 16.98%–54.93%), Betaproteobacteria (5.91%–39.34%), Gammaproteobacteria (4.62%–30.86%) of Proteobacteria, and Euryarchaeota (9.99%–58.05%). [Conclusion] In addition to Firmicutes and Proteobacteria, archaea were also dominant in the gut microbiota in the larvae of the four species fed with PE and PS. The relative abundance of the main microbial taxa in the insect gut varied dynamically over time and was influenced by the types of plastics as well as the insect species. The body weights and body lengths of Z. atratus and T. molitor were correlated with their gut microbiota.

[Objective] Due to the short half-life of mRNA transcript, their anti-degradation ability and ability to recruit ribosomes to initiate translation have significant effects on the gene expression in prokaryotes. However, the multiple functional regions in the 5′ end, of mRNA can affect the half-life and thus the expression of target genes, including the Shine-Dalgarno (SD) sequence and its upstream and downstream translational standby sites (TSSs) and the N-terminal coding sequence (NCS). The own and cross-regional structural differences will affect gene expression. Therefore, it is important to analyze the structure-activity relationship of each functional region. [Methods] We employed primer extension sequencing (SHAPE-seq) to analyze the structural characteristics of seven different 5′mRNAs in Escherichia coli and collected the mRNA abundance and protein level information under their regulation. [Results] Under the regulation of unstructured NCS, the mRNA abundance and protein level were increased by10 and 19 times, respectively. The formation of secondary structure TSS with a stem length of 10 nt increased the mRNA abundance. When the SD sequence was wrapped to form a secondary structure, the efficiency of translation initiation mediated by the SD sequence was affected (protein level downregulation by 10%). The combination of TSS and NCS significantly increased the mRNA abundance and protein level by 11 and 60 folds, respectively. [Conclusion] We characterized the structure of each region of 5′mRNA conducive to prokaryotic gene expression and revealed the structure-activity relationship of each functional region, providing a new regulatory element for target gene expression in industrial microorganisms.

[Objective] To realize the de novo biosynthesis of caffeic acid from glucose by reconstruction of its biosynthetic pathway in Escherichia coli. Fine-tuning gene expression allows us to improve caffeic acid production, which paves a way for the high production of caffeic acid and its derivatives in E. coli. [Methods] The biosynthetic pathway of caffeic acid was reconstructed based on FjTAL and EchpaBC, which encoded the tyrosine ammonia lyase in Flavobacterium johnsoniaeu and the 4-hydroxyphenylacetate 3-hydroxylase complex in E. coli, respectively. The reconstructed pathway was then introduced into commonly used E. coli strains. We improved the expression levels of FjTAL and EchpaBC by screening constitutive promoters, utilizing an intermediate-based biosensor, and increasing the copy number of the key gene. Thus, a total of fourteen recombinant strains were obtained, and the production of caffeic acid and the intermediate p-coumaric acid in these strains was quantified by HPLC. Moreover, the effects of different nitrogen sources and substrate concentrations on the production of caffeic acid were investigated. [Results] We realized de novo biosynthesis of caffeic acid from glucose in E. coli. The use of constitutive promoters other than the commonly used T7 promoter contributed to the yield increase of caffeic acid. When glucose was used as the substrate, the yield of caffeic acid was increased from 1.40 mg/L to 96.40 mg/L. When tyrosine was used as the substrate, the yield of caffeic acid was increased from 1.78 mg/L to 123.31 mg/L. Furthermore, the yield of caffeic acid reached 162.73 mg/L when a p-coumaric acid biosensor instead of a constitutive promoter was used to drive the expression of EchpaBC. Moreover, the yield of caffeic acid was improved to 185.15 mg/L in the case of introducing an extra copy of EchpaBC. [Conclusion] We constructed the strains with high production of caffeic acid by promoter engineering, using an intermediate-base biosensor, and increasing copy number of the key gene. Our study laid a solid foundation for the high production of caffeic acid.

Nitrogen is an essential element for living organisms on Earth, and it is constantly recycled in the biosphere in different forms. A biohybrid constructed with photocatalytic materials is a new system produced in recent years, which combines photocatalytic materials with electroactive microorganisms, integrating the excellent light trapping performance of photocatalysts and the bioefficient catalytic capability. Therefore, it is of great significance to study how the system plays a role in nitrogen cycling and the related mechanisms. This paper introduces the microbial nitrogen cycling and the nitrogen cycling driven by biohybrids and details several types, advantages and disadvantages, and related mechanisms of electron transfer driven by biohybrids constructed with photocatalytic materials. Finally, this paper makes an outlook on the development prospects in this field from the natures and combinations of photocatalytic materials and microorganisms.

[Objective] To investigate the effects of hydrogen sulfide (H₂S) on the structures of phyllosphere and rhizosphere microbial communities of soybean plants under drought stress. [Methods] High-throughput sequencing of the 16S rRNA gene was combined with bioinformatics analyses (α and β diversity, species composition, co-occurrence networks analysis, etc.) to study the phyllosphere and rhizosphere microbial communities of soybean plants before and after NaHS treatment. [Results] For the soybean plants under normal moisture conditions, the addition of NaHS decreased the diversity and increased the endemic species of phyllosphere microbial community. The addition of NaHS increased the diversity of rhizosphere microbial community of soybean plants under normal moisture conditions but not under severe drought. In addition, exogenous addition of NaHS altered the bacterial co-occurrence network, and the microbial communities in both phyllosphere and rhizosphere were so aggregated that neither rhizobium inoculation nor NaHS addition had significant influences on them. The addition of NaHS mildly affected the relative abundance of operational taxonomic unit (OTU) in the phyllosphere and decreased the relative abundance of OTU in the rhizosphere, which was particularly pronounced under severe drought. Rhizobium inoculation and NaHS addition enriched different microbial taxa in both phyllosphere and rhizosphere. [Conclusion] Under drought stress, H₂S had an insignificant modulating effect on the microbial community structure in the phyllosphere but a pronounced effect on the microbial community structure in the rhizosphere of soybean plants. H₂S reduced the relative abundance of total OTU in the rhizosphere and altered the bacterial co-occurrence network, thus influencing soybean adaptation to drought stress.

[Objective] To investigate the antibiotic resistance profiles, resistance genes, and bla_NDM transmission characteristics of bacterial isolates from six turtle farms in two regions (three in Lanxi (LX) and three in Xiaoshan (XS)) of Zhejiang Province. [Methods] Bacterial strains were identified by 16S rRNA gene sequencing. The Kirby-Bauer method was employed to examine the antibiotic susceptibility of isolated bacteria. PCR was employed to detect resistance genes carried by the isolated bacteria. Conjugation transfer experiments were conducted to obtain bla_NDM-carrying plasmids, and the conjugation frequency was analyzed to assess the transfer capabilities of bacterial donors. Additionally, the modified Carba NP test, plasmid replicon typing, and minimum inhibitory concentration determination were carried out to evaluate the transmission of bla_NDM-carrying plasmids among bacteria. [Results] A total of 244 bacterial strains were isolated, with similar distribution between the two regions and Enterobacteriaceae being dominant. PCR amplification of integrons revealed various resistance genes, including those conferring resistance to aminoglycosides and trimethoprim. We identified numerous resistant bacteria capable of integrating additional exogenous resistance genes. All the isolated strains exhibited resistance to at least one antibiotic, with some displaying resistance to carbapenems. PCR detection of resistance genes indicated the dissemination of carbapenem resistance genes within some farms. This study confirmed the presence of bla_NDM on plasmids capable of inter-bacterial transmission. Moreover, the farms in LX exhibited significantly higher conjugation frequency of bla_NDM-carrying plasmids than the farms in XS. [Conclusion] Antibiotic resistance of bacteria in aquaculture environments is becoming increasingly serious. The carbapenem resistance gene bla_NDM has spread within turtle farms.

[Objective] This study aims to validate the functions of two ferritin-encoding genes: bacterioferritin (Bfr)-encoding gene (atu2771) and DNA-binding protein from starved cells (Dps)-encoding gene (atu2477), in Agrobacterium fabrum, to determine the open reading frame (ORF) of the Bfr-encoding gene, to investigate the effects of terminal fusion, heme group, and key residues on the function and self-assembly of A. fabrum Bfr, and to explore the potential applications of the two ferritin nano-cages. [Methods] We re-introduced the two ferritin-encoding genes into the ferritin-deficient mutants of A. fabrum respectively via plasmids to verify if the re-introduction could complement the ferritins of the ferritin-deficient mutants and thus validate the functions of the two genes. Native PAGE was employed to separate the ferritins in the crude extract of A. fabrum and potassium ferrocyanide (an iron-specific staining reagent) was used to stain the ferritins. Various peptides or protein were fused to the termini of two ferritins to test if the terminal fusion would affect the functions and self-assembly of the two ferritins. Site-directed mutation was then employed to test the effects of the key residue and heme group on the function and self-assembly of Bfr. [Results] Iron-specific staining on the ferritins separated by native PAGE showed that the Bfr-encoding gene expressed Bfr in all the tested A. fabrum strains, whereas the Dps-encoding gene expressed Dps in none of the tested A. fabrum strains. Complementary experiment with two different Bfr-encoding ORFs (encoding 161 residues and 169 residues) showed that Bfr in the wild type was encoded by the ORF encoding 161 residues. The results demonstrated that terminal fusions with different peptides or protein influenced but did not abolish the function and self-assembly of Bfr. The substitution of Met₆₀, which was predicted to chelate the iron of heme, indicated that heme affected the function and self-assembly of Bfr but was not indispensable. [Conclusion] A. fabrum utilizes Bfr to store iron. The ORF of the Bfr-encoding gene utilizes UUG (a rare start code) as its start code and encodes a Bfr composed of 161 residues. The Dps-encoding gene of A. fabrum expressed in none of the tested conditions. The structures of both Bfr and Dps of A. fabrum are stable enough to withstand the terminal fusion with various peptides or protein, suggesting that both Bfr and Dps nano-cages demonstrate great promise for biotechnological applications.

[Objective] To establish a methodology assessing the existence and active state of biofilms on mudflat. [Methods] We sampled the biofilms on mudflat and the surface sediments in the nearby regions, and compared the dominant algal species and their abundance between the biofilms and their nearby regions by 18S rRNA gene sequencing. Furthermore, we employed chlorophyll a (Chl-a) assay and flow cytometry to compare the concentration of Chl-a and the amount of Chl-a-containing cells between the biofilms and their nearby regions. [Results] The mudflat biofilms mainly harbored Diatomea, Dinoflagellata, Ochrophyta, Chlorophyta, Cryptophyceae, and Phragmoplastophyta, the relative abundance of which, however, varied significantly in different seasons or geographic locations. There was a significant difference in the concentration of Chl-a between biofilms and their nearby regions. The amount of Chl-a-containing cells in biofilms was significantly higher than that in the nearby regions. A method for assessing biofilms on mudflat was established based on the relative abundance of algae, the concentration of Chl-a, and the amount of Chl-a-containing cells. In brief, a sample is classified as a biofilm in the case of the relative abundance of algae higher than 40%, the Chl-a concentration higher than 500 mg/m³, and the amount of Chl-a-containing cells more than 500 cells/μL. Otherwise, the sample is classified as inexistence of biofilm. Additionally, the amount of Chl-a-containing cells more than 1 500 cells/μL indicates that the biofilm is in the vigorous growth phase, and that between 500 cells/μL and 1 500 cells/μL suggests that the biofilm is in the colonization or recession phase. [Conclusion] We compared the dominant algal species and their relative abundance, the concentration of Chl-a, and the amount of Chl-a-containing cells between biofilms on mudflat and their nearby regions, and established a methodology assessing the existence and active state of biofilms on mudflat based on the above indicators. The findings enriched the knowledge of biofilms on mudflat and provided a theoretical basis for understanding the microbial carbon sequestration capacity of biofilms on mudflat.

[Objective] To develop probiotics for gynecological inflammation, we isolated and screened out Lactobacillus with antimicrobial and probiotic properties from the vagina of healthy women. [Methods] The plate streaking method was employed to isolate the Lactobacillus strains from vaginal samples, and the isolates were then identified based on morphological and 16S rRNA gene sequencing evidence. The growth and adhesion of the five strains were characterized. The Oxford cup method with Escherichia coli and Staphylococcus aureus as indicator strains was employed to assess the antimicrobial activities of the strains. The microtiter plate method was used to measure the inhibitory effects of the strains on Candida albicans. The antimicrobial components were explored by organic acid elimination and hydrogen peroxide elimination methods. [Results] Five strains of Lactobacillus were isolated, including three strains (Q2.1, BHC04, and Q8.5) of Lactobacillus crispatus and two strains (Q6.3 and BHG05) of Lactobacillus gasseri. All the five strains of Lactobacillus had strong growth and high acid production. Strains Q2.1, BHC04, and BHG05 had a short delay period and reached a plateau growth stage after 20 h, and strains BHG05, Q6.3, and Q8.5 had high acid production, with the culture medium finally reaching pH 3.80–4.03. The adhesion capacity (hydrophobicity, self-agglutination rate, and co-agglutination rate with pathogens) of L. crispatus Q2.1, BHC04, and Q8.5 was significantly higher than that of the positive control strain, L. delbrueckii DM8909. The inhibitory effects of the five strains on E. coli, S. aureus, and C. albicans were stronger than those of the positive control (nisin). Strains BHC04, BHG05, and Q8.5 showcased stronger inhibitory effects on E. coli than DM8909. The inhibitory effect of BHG05 on C. albicans was significantly stronger than that of DM8909, with the inhibition rate reaching up to (73.14±0.14)%. The inhibitory effects of BHC04 and Q8.5 on C. albicans were not significantly different from that of DM8909, with the inhibition rates reaching up to (72.80±0.30)% and (72.93±0.10)%, respectively. According to the results above, we selected BHC04 and BHG05 as high quality strains with antimicrobial potential. The five strains produced organic acids and hydrogen peroxide to exert antimicrobial effects. [Conclusion] Two strains of Lactobacillus with antimicrobial effects and excellent probiotic properties were screened out. They can be used as candidate strains of antimicrobial probiotics for the prevention and treatment of gynecological inflammation caused by E. coli, S. aureus, and C. albicans.

[Objective] To select the elite strains among the microorganisms isolated from the rhizosphere of Brassica oleracea var. capitata and evaluate the secondary metabolism potential of the strains, laying a solid foundation for mining the microbial resources in the rhizosphere of this plant. [Methods] The roots of B. oleracea var. capitata were collected from Xiannüshan Town in Wulong District of Chongqing. The conventional methods were then used to isolate microbial strains, and one strain of Serratia marcescens was selected for genome sequencing (PacBio RS Ⅱ and Illumina HiSeq) and bioinformatic analysis. The antiSMASH was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. Red/ET recombination was employed to capture the BGC for prodigiosin. [Results] One prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The genome of this strain contained 11 putative BGCs (1–11) for secondary metabolites, of which 9 BGCs displayed low similarities to the BGCs encoding known compounds. This result suggested that the strain had great potential of producing novel secondary metabolites. Prodigiosin was identified by HPLC and high-resolution mass spectrometry (HRMS). Heterologous expression of BGC4 resulted in the production of prodigiosin in Escherichia coli. Four common promoters were used to drive the expression of BGC in E. coli, and the highest prodigiosin production was observed with the rpoS promoter. [Conclusion] A prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The BGC for prodigiosin was captured from the chromosome of this strain and expressed successfully in the surrogate host E. coli BAP1.