Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.

For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.

Transcriptomic and Proteomic Analysis of Gardnerella vaginalis Responding to Acidic pH and Hydrogen Peroxide Stress.

Gardnerella vaginalis is the main pathogen that causes bacterial vaginosis. In the healthy vaginal microecological environment of a woman, the lactobacilli produce lactate and hydrogen peroxide to inhibit the growth of pathogens such as G. vaginalis. The lack of lactobacilli results in a high pH and low hydrogen peroxide in the vagina which facilitate G. vaginalis growth, leading to the imbalance of the vaginal microecology. In this study, lactate and hydrogen peroxide were added to a G. vaginalis culture medium to simulate the co-culture of the lactobacilli and G. vaginalis, and then the genes related to the stress response of G. vaginalis were identified using transcriptomics and proteomics. It was indicated that, among all the upregulated genes, most of them encoded transporters associated with the efflux of harmful substances, and the majority of the downregulated genes were related to the biofilm formation and epithelial cell adhesion. This study may help find new drug targets for G. vaginalis for the development of novel therapies for bacterial vaginosis.

A fast RT-qPCR system significantly shortens the time for SARS-CoV-2 nucleic acid test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global development. Rapid and accurate diagnosis is critical for containing the pandemic and treating patients in time. As the gold standard for SARS-CoV-2 diagnosis, the qualitative reverse transcription-PCR (RT-qPCR) test has long been criticized for its long detection time. In this study, we optimized the primers and probes targeting SARS-CoV-2 ORF1ab and N gene designed by the Chinese Center for Disease Control and Preventions (CDC) to increase their Tm values to meet the optimal elongation temperature of Taq DNA polymerase, thus greatly shortened the elongation time. The higher elongation temperature in turn narrowed the temperature range of the reaction and saved more time. In addition, by shortening the distance between the fluorophore at the 5' end and the quencher in the middle we got a probe with higher signal-to-noise ratio. Finally, by using all these measures and optimized RT-qPCR program we successfully reduced the time (nucleic acid extraction step is not included) for nucleic acid test from 74 min to 26 min.

SARS-CoV-2 RdRp uses NDPs as a substrate and is able to incorporate NHC into RNA from diphosphate form molnupiravir.

The coronavirus disease 2019 has been ravaging throughout the world for three years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 employs the viral RNA dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Systematic characterization of RdRp will undoubtedly aid in the development of antiviral drugs targeting RdRp. Here, our research reveals that RdRp can recognize and utilize nucleoside diphosphates as a substrate to synthesize RNA with an efficiency of about two thirds of using nucleoside triphosphates as a substrate. Nucleoside diphosphates incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate ß-d-N4-hydroxycytidine into RNA while using diphosphate form molnupiravir as a substrate. This incorporation results in genome mutation and virus death. It is also observed that diphosphate form molnupiravir is a better substrate for RdRp than the triphosphate form molnupiravir, presenting a new strategy for drug design.

Antibiotic resistance and pathogenicity assessment of various Gardnerella sp. strains in local China.

Gardnerella overgrowth is the primary cause of bacterial vaginosis (BV), a common vaginal infection with incidences as high as 23-29% worldwide. Here, we studied the pathogenicity, drug resistance, and prevalence of varying Gardnerella spp. We isolated 20 Gardnerella strains from vaginal samples of 31 women in local China. Ten strains were then selected via phylogenetic analysis of cpn60 and vly gene sequences to carry out genome sequencing and comparative genomic analysis. Biofilm-formation, sialidase, and antibiotic resistance activities of the strains were characterized. All strains showed striking heterogeneity in genomic structure, biofilm formation and drug resistance. Two of the ten strains, JNFY3 and JNFY15, were classified as Gardnerella swidsinskii and Gardnerella piotii, respectively, according to their phenotypic characteristics and genome sequences. In particular, seven out of the ten strains exhibited super resistance (≥ 128 µg/mL) to metronidazole, which is the first line of treatment for BV in China. Based on the biochemical and genomic results of the strains, we proposed a treatment protocol of prevalent Gardnerella strains in local China, which provides the basis for accurate diagnosis and therapy.

Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations.

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded. The three nucleotides closest to the 3' end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein. This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.

DexA70, the Truncated Form of a Self-Produced Dextranase, Effectively Disrupts Streptococcus mutans Biofilm.

Billions of people suffer from dental caries every year in spite of the effort to reduce the prevalence over the past few decades. Streptococcus mutans is the leading member of a specific group of cariogenic bacteria that cause dental caries. S. mutans forms biofilm, which is highly resistant to harsh environment, host immunity, and antimicrobial treatments. In this study, we found that S. mutans biofilm is highly resistant to both antimicrobial agents and lysozyme. DexA70, the truncated form of DexA (amino acids 100-732), a dextranase in S. mutans, prevents S. mutans biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. DexA70 treatment markedly enhances biofilm sensitivity to antimicrobial agents and lysozyme, indicating its great potential in combating biofilm-related dental caries.

YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner.

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.

Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide.

Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD+ during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (adhE) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in Clostridium ljungdahlii A metabolic experiment using 13C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases.IMPORTANCE Ethanol production from carbon monoxide (CO) as a carbon and energy source by Clostridium ljungdahlii and "Clostridium autoethanogenum" is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of C. ljungdahlii in the future.

Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode.

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Synergistic Cellulose Hydrolysis Dominated by a Multi-Modular Processive Endoglucanase from Clostridium cellulosi.

Recalcitrance of biomass feedstock remains a challenge for microbial conversion of lignocellulose into biofuel and biochemicals. Clostridium cellulosi, one thermophilic bacterial strain dominated in compost, could hydrolyze lignocellulose at elevated temperature by secreting more than 38 glycoside hydrolases belong to 15 different families. Though one multi-modular endoglucanase CcCel9A has been identified from C. cellulosi CS-4-4, mechanism of synergistic degradation of cellulose by various cellulases from strain CS-4-4 remains elusive. In this study, CcCel9A, CcCel9B, and CcCel48A were characterized as processive endoglucanase, non-processive endoglucanase, and exoglucanase, respectively. To understand how they cooperate with each other, we estimated the approximate concentration ratio on the zymogram and optimized it using purified enzymes in vitro. Synergism between individual glycoside hydrolase during cellulose hydrolysis in the mixture was observed. CcCel9A and CcCel48A could degrade cellulose chain from non-reducing ends and reducing ends, respectively, to cello-oligosaccharide. CcCel9B could cut cellulose chain randomly and cello-oligosaccharides with varied length were released. In addition, a ß-glucosidase BlgA from Caldicellulosiruptor sp. F32 which could cleave cello-oligosaccharides including G2-G6 to glucose was added to the enzyme mixture to remove the product inhibition of its partners. The combination and ratios of these cellulases were optimized based on the release rate of glucose. Hydrolysis of corn stalk was conducted by a four-component cocktail (CcCel9A:CcCel9B:CcCel48A:BlgA = 25:25:10:18), and only glucose was detected as main production by using high-performance anion-exchange chromatography. Processive endoglucanase CcCel9A, dominated in secretome of C. cellulosi, showed good potential in developing cellulase cocktail due to its exquisite cooperation with various cellulases.

Depiction of carbohydrate-active enzyme diversity in Caldicellulosiruptor sp. F32 at the genome level reveals insights into distinct polysaccharide degradation features.

Thermophilic bacterium Caldicellulosiruptor sp. F32 can utilize cellulose-, hemicellulose-containing biomass, including unpretreated wheat straw. We have conducted a bioinformatics analysis of the carbohydrate-active enzyme (CAZyme) in the genome of Caldicellulosiruptor sp. F32, which reveals a broad substrate range of the strain. Among 2285 predicted open reading frames (ORFs), 73 (3.2%) CAZyme encoding genes, including 44 glycoside hydrolases (GHs) distributing in 22 GH families, 6 carbohydrate esterases (CEs), 3 polysaccharide lyases (PLs), 21 glycosyl transferases (GTs), and 25 carbohydrate-binding modules (CBMs) were found. An in-depth bioinformatics analysis of CAZyme families that target cellulose, hemicellulose, chitin, pectin, starch, and ß-1,3-1,4-glucan degradation were performed to highlight specialized polysaccharide degrading abilities of strain F32. A great number of orthologous multimodular CAZymes of Caldicellulosiruptor sp. F32 were found in other strains of genus Caldicellulosiruptor. While, a portion of the CAZymes of Caldicellulosiruptor sp. F32 showed sequence identity with proteins from strains of genus Clostridium. A thermostable ß-glucosidase BlgA synergistically facilitated the enzymatic degradation of Avicel by endo-1,4-ß-glucanase CelB, which indicated that the synchronous action of synergism between CAZymes enhanced the lignocellulose degradation by Caldicellulosiruptor sp. F32.

Desertibacter xinjiangensis sp. nov., isolated from the soil of a Euphrates poplar forest, and emended description of the genus Desertibacter.

A pale pink and strictly aerobic bacterium, designated strain M71(T), was isolated from the soil of a Euphrates poplar forest in Xingjiang, PR China. Cells of the strain were Gram-reaction-negative, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 10-37 °C (optimum 30 °C), at pH 6.0-9.0 (optimum pH 7.0-8.0) and with 0-2.0% NaCl (w/v, optimum 0%). Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain M71(T) belongs to the genus Desertibacter in the family Rhodospirillaceae. The 16S rRNA gene sequence of this strain showed 96.2% sequence similarity with the type strain of Desertibacter roseus 2262(T). The respiratory quinone was Q-10 and the predominant cellular fatty acids were C(18:1)ω7c (53.2%), C(16:1)ω5c (11.0%), summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c, 10.2%) and C(16:0) (8.5%). The DNA G+C content was 71.2 mol% (HPLC). The strain contained phosphatidylcholine and phosphatidylethanolamine as the predominant polar lipids. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain M71(T) is considered to represent a novel species of the genus Desertibacter, for which the name Desertibacter xinjiangensis sp. nov. is proposed. The type strain is M71(T) ( =CCTCC AB 209291(T) =CIP 110127(T)).

Dysgonomonas macrotermitis sp. nov., isolated from the hindgut of a fungus-growing termite.

A Gram-stain-negative, facultatively anaerobic, non-motile and coccoid- to short-rod-shaped bacterium, designated strain Dys-CH1(T), was isolated from the hindgut of a fungus-growing termite Macrotermes barneyi. The optimal pH and cultivation temperature of strain Dys-CH1(T) were pH 7.2-7.6 and 35-37 °C, respectively. Sequence analysis of 16S rRNA gene showed that Dys-CH1(T) shared 94.6 % and 90.9 % similarity with Dysgonomonas capnocytophagoides JCM 16697(T) and Dysgonomonas gadei CCUG 42882(T), respectively. Strain Dys-CH1(T) was found to be different from other species of the genus Dysgonomonas with validly published names with respect to taxonomically important traits, including habitat, biochemical tests, DNA G+C content, bile resistance, fatty-acid composition and susceptibility to antimicrobial agents. On the basis of these characteristics, strain Dys-CH1(T) represents a novel species of the genus Dysgonomonas for which the name Dysgonomonas macrotermitis sp. nov. is proposed. The type strain is Dys-CH1(T) ( = JCM 19375(T) = DSM 27370(T)).

Synergism of glycoside hydrolase secretomes from two thermophilic bacteria cocultivated on lignocellulose.

Two cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genus Clostridium and showed 98.87% and 98.86% identity with Clostridium stercorarium subsp. leptospartum ATCC 35414(T) and Clostridium cellulosi AS 1.1777(T), respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the ß-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the ß-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs).

Altererythrobacter xinjiangensis sp. nov., isolated from desert sand, and emended description of the genus Altererythrobacter.

A Gram-negative, rod-shaped, non-motile, strictly aerobic bacterium, strain S3-63(T), was isolated from desert sand of Xinjiang, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S3-63(T) had highest similarity to type strains of the genus Altererythrobacter, i.e. Altererythrobacter marinus H32(T) (97.2% similarity), Altererythrobacter marensis MSW-14(T) (95.9%), Altererythrobacter aestuarii KCTC 22735(T) (95.5%), Altererythrobacter epoxidivorans JCS350(T) (95.1%), Altererythrobacter namhicola KCTC 22736(T) (95.1%), Altererythrobacter luteolus SW-109(T) (95.0%) and Altererythrobacter indicus LMG 23789(T) (93.5%). Growth occurred at 20-37 °C (optimum 30 °C), at pH 7.0-9.0 (optimum pH 8.0) and in 0-3% (w/v) NaCl (optimum 1%). The major respiratory quinone was ubiquinone-10 and the predominant cellular fatty acids were C(18:1)ω7c (50.8%), summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c; 12.6%), C(16:0) (12.3%), C(14:0) 2-OH (7.3%) and C(17:1)ω6c (4.5%). The DNA G+C content was 64.6 mol%. Therefore, the phylogenetic, physiological and chemotaxonomic data demonstrated that strain S3-63(T) represents a novel species of the genus Altererythrobacter, for which the name Altererythrobacter xinjiangensis sp. nov. is proposed; the type strain is S3-63(T) (=CCTCC AB 207166(T)=CIP 110125(T)). An emended description of the genus Altererythrobacter is provided.

Isolation and characterization of a chromium-resistant bacterium Serratia sp. Cr-10 from a chromate-contaminated site.

A novel bacterium, Cr-10, was isolated from a chromium-contaminated site and capable of removing toxic chromium species from solution by reducing hexavalent chromium to an insoluble precipitate. Sequence analysis of 16S rRNA gene of strain Cr-10 showed that it was most closely related to Serratia rubidaea JCM 1240(T) (97.68%). Physiological and chemotaxonomic data also supported that strain Cr-10 was identified as Serratia sp., a genus which was never specially reported chromate-resistant before. Serratia sp., Cr-10 was tolerant to a concentration of 1,500 mg Cr(VI) L(-1), which was the highest level reported until now. The optimum pH and temperature for reduction of Cr(VI) by Serratia sp. Cr-10 were found to be 7.0 and 37 °C, respectively. The Cr(VI) reduction was significantly influenced by additional carbon sources, and among them fructose and lactose offered maximum reduction, with a rate of 0.28 and 0.25 mg Cr(VI) L(-1) h(-1), respectively. The cell-free extracts and filtrate of the culture were able to reduce Cr(VI) while concentration of total chromium remained stable in the process, indicating that the enzyme-catalyzed mechanism was applied in Cr(VI) reduction by the isolate. Additionally, it was found that there was hardly any chromium on the cell surface of the strain, further supporting that reduction, rather than bioadsorption, plays a major role in the Cr(VI) removal.

Pedobacter xinjiangensis sp. nov., from desert, Xinjiang.

A Gram-negative, rod-shaped, gliding, aerobic bacterium, designated 12157(T), was isolated from the desert of Xinjiang, China and subjected to a polyphasic taxonomic study. The strain 12157(T) grew optimally at pH 7.0 and 30 degrees Celsius. MK-7 was the predominant respiratory menaquinone. The DNA G+C content was 42.0 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolate was mostly related to the members of the genus Pedobacter, with similarities ranging from 90.0 % to 93.7 %. Phylogenetic evidence and the results of phenotypic, genotypic and chemotaxonomic analysis support the establishment of a novel species, Pedobacter xinjiangensis sp. nov., with strain 12157(T) (=CCTCC AB 208092(T)=NRRL B-51338(T)) as the type strain.