Efficient production of guanosine in Escherichia coli by combinatorial metabolic engineering.

BACKGROUND: Guanosine is a purine nucleoside that is widely used as a raw material for food additives and pharmaceutical products. Microbial fermentation is the main production method of guanosine. However, the guanosine-producing strains possess multiple metabolic pathway interactions and complex regulatory mechanisms. The lack of strains with efficiently producing-guanosine greatly limited industrial application. RESULTS: We attempted to efficiently produce guanosine in Escherichia coli using systematic metabolic engineering. First, we overexpressed the purine synthesis pathway from Bacillus subtilis and the prs gene, and deleted three genes involved in guanosine catabolism to increase guanosine accumulation. Subsequently, we attenuated purA expression and eliminated feedback and transcription dual inhibition. Then, we modified the metabolic flux of the glycolysis and Entner-Doudoroff (ED) pathways and performed redox cofactors rebalancing. Finally, transporter engineering and enhancing the guanosine synthesis pathway further increased the guanosine titre to 134.9 mg/L. After 72 h of the fed-batch fermentation in shake-flask, the guanosine titre achieved 289.8 mg/L. CONCLUSIONS: Our results reveal that the guanosine synthesis pathway was successfully optimized by combinatorial metabolic engineering, which could be applicable to the efficient synthesis of other nucleoside products.

Biofilm formation of Hafnia paralvei induced by c-di-GMP through facilitating bcsB gene expression promotes spoilage of Yellow River carp (Cyprinus carpio).

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.

Polystyrene microplastics promote intestinal colonization of Aeromonas veronii through inducing intestinal microbiota dysbiosis.

The premise that pathogen colonized microplastics (MPs) can promote the spread of pathogens has been widely recognized, however, their role in the colonization of pathogens in a host intestine has not been fully elucidated. Here, we investigated the effect of polystyrene MPs (PS-MPs) on the colonization levels of Aeromonas veronii, a typical aquatic pathogen, in the loach (Misgurnus anguillicaudatus) intestine. Multiple types of MPs were observed to promote the intestinal colonization of A. veronii, among which PS-MPs exhibited the most significant stimulating effect (67.18% increase in A. veronii colonization). PS-MPs inflicted serious damage to the intestinal tracts of loaches and induced intestinal microbiota dysbiosis. The abundance of certain intestinal bacteria with resistance against A. veronii colonization decreased, with Lactococcus sp. showing the strongest colonization resistance (73.64% decline in A. veronii colonization). Fecal microbiota transplantation was performed, which revealed that PS-MPs induced intestinal microbiota dysbiosis was responsible for the increased colonization of A. veronii in the intestine. It was determined that PS-MPs reshaped the intestinal microbiota community to attenuate the colonization resistance against A. veronii colonization, resulting in an elevated intestinal colonization levels of A. veronii.

Resistance role of Lactobacillus sp. and Lactococcus sp. to copper ions in healthy children's intestinal microorganisms.

Heavy metal exposure is closely associated with gut microbe function and tolerance. However, intestinal microbe responses in children to different copper ion (Cu2+) concentrations have not yet been clarified. Here, in vitro cultivation systems were established for fecal microbe control and Cu2+-treated groups in healthy children. 16S rDNA high-throughput sequencing, meta-transcriptomics and metabolomics were used here to identify toxicity resistance mechanisms at microbiome levels. The results showed that Lactobacillus sp. and Lactococcus sp. exerted protective effects against Cu2+ toxicity, but these effects were limited by Cu2+ concentration. When the Cu2+ concentration was ≥ 4 mg/L, the abundance of Lactobacillus sp. and Lactococcus sp. significantly decreased, and the pathways of antioxidant activity and detoxification processes were enriched at 2 mg/L Cu2+, and beneficial metabolites accumulated. However, at high concentrations of Cu2+ (≥4 mg/L), the abundance of potential pathogen increased, and was accompanied by a downregulation of genes in metabolism and detoxification pathways, which meant that the balance of gut microbiota was disrupted and toxicity resistance decreased. From these observations, we identified some probiotics that are tolerant to heavy metal Cu2+, and warn that only when the concentration limit of Cu2+ in food is 2 mg/L, then a balanced gut microbiota can be guaranteed in children, thereby providing protection for their health.

Quorum sensing signal autoinducer-2 promotes hydrogen peroxide degradation in water by Gram-positive bacteria.

Hydrogen peroxide is widely used to remedy bacterial and parasitic infections, but its excessive use will cause severe damage to aquatic animals. Moreover, there is no safe, efficient and low-cost method to degrade residual hydrogen peroxide in water. Here we developed a hydrogen peroxide removal mechanism by which autoinducer-2 (AI-2), a quorum sensing signal molecule that can promote the hydrogen peroxide degradation by Gram-positive bacteria. Here, we investigated the promotion effect of AI-2 on hydrogen peroxide degradation by Deinococcus sp. Y35 and the response of the antioxidant system. We further sought to understand the key mechanism underlying the promotion effect of AI-2 on hydrogen peroxide degradation is that, AI-2 contributed to the resistance of strain Y35 to oxidative stress induced by hydrogen peroxide, and altered membrane permeability of strain Y35 that allowed more hydrogen peroxide to enter bacterial cells and be degraded. Additionally, AI-2 can also encourage multiple Gram-positive bacteria to degrade hydrogen peroxide. Accordingly, our study serves as a reference for the regulation mechanism of the signal molecule AI-2 and provides the development of new strategies for hydrogen peroxide degradation.

Autoinducer-2 promotes adherence of Aeromonas veronii through facilitating the expression of MSHA type IV pili genes mediated by c-di-GMP.

IMPORTANCE: Aeromonas veronii can adhere to host cells through different adherence factors including outer-membrane proteins (OMPs), lipopolysaccharide (LPS), and pili, but its adherence mechanisms are still unclear. Here, we evaluated the effect of autoinducer-2 (AI-2) on adherence of A. veronii and its regulation mechanism. After determination of the promotion effect of AI-2 on adherence, we investigated which adherence factor was regulated by AI-2, and the results show that AI-2 only limits the formation of pili. Among the four distinct pili systems, only the mannose-sensitive hemagglutinin (MSHA) type IV pili genes were significantly downregulated after deficiency of AI-2. MshE, an ATPase belonged to MSHA type IV pilin, was confirmed as c-di-GMP receptor, that can bind with c-di-GMP which is positively regulated by AI-2, and the increase of c-di-GMP can promote the expression of MSHA type IV pili genes and adherence of A. veronii. Therefore, this study confirms that c-di-GMP positively regulated by AI-2 binds with MshE, then increases the expression of MSHA pili genes, finally promoting adherence of A. veronii, suggesting a multilevel positive regulatory adhesion mechanism that is responsible for A. veronii adherence.

Cinnamaldehyde Targets the LytTR DNA-Binding Domain of the Response Regulator AgrA to Attenuate Biofilm Formation of Listeria monocytogenes.

The Agr quorum sensing (QS) system is known to contribute to biofilm formation in Listeria monocytogenes. Cinnamaldehyde, a natural food preservative, is considered an inhibitor of Agr-mediated QS in L. monocytogenes. However, the exact mechanism by which cinnamaldehyde acts on Agr remains unclear. In this study, we assessed the effects of cinnamaldehyde on the histidine kinase AgrC and the response regulator AgrA in the Agr system. AgrC kinase activity was not influenced by cinnamaldehyde, and binding between AgrC and cinnamaldehyde was not observed when microscale thermophoresis (MST) was performed, indicating that AgrC was not the target of cinnamaldehyde. AgrA is specifically bound to the agr promoter (P2) to activate the transcription of the Agr system. However, AgrA-P2 binding was prevented by cinnamaldehyde. The interaction between cinnamaldehyde and AgrA was further confirmed with MST. Two conserved amino acids, Asn-178 and Arg-179, located in the LytTR DNA-binding domain of AgrA, were identified as the key sites for cinnamaldehyde-AgrA binding by alanine mutagenesis and MST. Coincidentally, Asn-178 was also involved in the AgrA-P2 interaction. Taken together, these results suggest that cinnamaldehyde acts as a competitive inhibitor of AgrA in AgrA-P2 binding, which leads to suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. IMPORTANCE Listeria monocytogenes can form biofilms on various food contact surfaces, posing a serious threat to food safety. Biofilm formation of L. monocytogenes is positively regulated by the Agr quorum sensing system. Thus, an alternative strategy for controlling L. monocytogenes biofilms is interfering with the Agr system. Cinnamaldehyde is considered an inhibitor of the L. monocytogenes Agr system; however, its exact mechanism of action is still unclear. Here, we found that AgrA (response regulator), rather than AgrC (histidine kinase), was the target of cinnamaldehyde. The conserved Asn-178 in the LytTR DNA-binding domain of AgrA was involved in cinnamaldehyde-AgrA and AgrA-P2 binding. Therefore, the occupation of Asn-178 by cinnamaldehyde suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. Our findings could provide a better understanding of the mechanism by which cinnamaldehyde inhibits L. monocytogenes biofilm formation.

Enrichment Culture but Not Metagenomic Sequencing Identified a Highly Prevalent Phage Infecting Lactiplantibacillus plantarum in Human Feces.

Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human diseases, but its phages in the human gut remain unexplored. Here, we report its first gut phage, Gut-P1, which we systematically screened using metagenomic sequencing, virus-like particle (VLP) sequencing, and enrichment culture from 35 fecal samples. Gut-P1 is virulent, belongs to the Douglaswolinvirus genus, and is highly prevalent in the gut (~11% prevalence); it has a genome of 79,928 bp consisting of 125 protein coding genes and displaying low sequence similarities to public L. plantarum phages. Physiochemical characterization shows that it has a short latent period and adapts to broad ranges of temperatures and pHs. Furthermore, Gut-P1 strongly inhibits the growth of L. plantarum strains at a multiplicity of infection (MOI) of 1e-6. Together, these results indicate that Gut-P1 can greatly impede the application of L. plantarum in humans. Strikingly, Gut-P1 was identified only in the enrichment culture, not in our metagenomic or VLP sequencing data nor in any public human phage databases, indicating the inefficiency of bulk sequencing in recovering low-abundance but highly prevalent phages and pointing to the unexplored hidden diversity of the human gut virome despite recent large-scale sequencing and bioinformatics efforts. IMPORTANCE As Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human gut-related diseases, its bacteriophages may pose a certain threat to their further application and should be identified and characterized more often from the human intestine. Here, we isolated and identified the first gut L. plantarum phage that is prevalent in a Chinese population. This phage, Gut-P1, is virulent and can strongly inhibit the growth of multiple L. plantarum strains at low MOIs. Our results also show that bulk sequencing is inefficient at recovering low-abundance but highly prevalent phages such as Gut-P1, suggesting that the hidden diversity of human enteroviruses has not yet been explored. Our results call for innovative approaches to isolate and identify intestinal phages from the human gut and to rethink our current understanding of the enterovirus, particularly its underestimated diversity and overestimated individual specificity.

Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis.

Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for the commercial production of desirable bioproducts. Sugar transporters are responsible for the import of substrate sugars and the conversion of ethanol and other products. Glucose-facilitated diffusion protein Glf is responsible for facilitating the diffusion of glucose uptake in Z. mobilis. However, another sugar transporter-encoded gene, ZMO0293, is poorly characterized. We employed gene deletion and heterologous expression mediated by the CRISPR/Cas method to investigate the role of ZMO0293. The results showed that deletion of the ZMO0293 gene slowed growth and reduced ethanol production and the activities of key enzymes involved in glucose metabolism in the presence of high concentrations of glucose. Moreover, ZMO0293 deletion caused different transcriptional changes in some genes of the Entner Doudoroff (ED) pathway in the ZM4-ΔZM0293 strain but not in ZM4 cells. The integrated expression of ZMO0293 restored the growth of the glucose uptake-defective strain Escherichia coli BL21(DE3)-ΔptsG. This study reveals the function of the ZMO0293 gene in Z. mobilis in response to high concentrations of glucose and provides a new biological part for synthetic biology.

A Study of Soil-Borne Fusarium Wilt in Continuous Cropping Chrysanthemum Cultivar 'Guangyu' in Henan, China.

Cut chrysanthemum, known as a highly favored floral choice globally, experiences a significant decline in production due to continuous cropping. The adverse physiological effects on cut chrysanthemums result from the degradation of a soil's physical and chemical properties, coupled with the proliferation of pathogens. The "Guangyu" cultivar in Xinxiang, Henan Province, China, has been specifically influenced by these effects. First, the precise pathogen accountable for wilt disease was effectively identified and validated in this study. An analysis was then conducted to examine the invasion pattern of the pathogen and the physiological response of chrysanthemum. Finally, the PacBio platform was employed to investigate the dynamic alterations in the microbial community within the soil rhizosphere by comparing the effects of 7 years of monocropping with the first year. Findings indicated that Fusarium solani was the primary causative agent responsible for wilt disease, because it possesses the ability to invade and establish colonies in plant roots, leading to alterations in various physiological parameters of plants. Continuous cropping significantly disturbed the microbial community composition, potentially acting as an additional influential factor in the advancement of wilt.

Phenyllactic acid application to control Listeria monocytogenes biofilms and its growth in milk and spiced beef.

Listeria monocytogenes, as a food-associated pathogen, is able to develop biofilms on different surfaces of food contact, which seriously threatens food safety. Phenyllactic acid (PLA) exhibits excellent inhibitory effects on many bacterial strains including L. monocytogenes. Our study aimed to investigate effects of PLA on L. monocytogenes biofilms and its growth in milk and on spiced beef. Biofilm biomass was measured by the microplate method and biofilm structure was observed by electron microscopy. Growth of L. monocytogenes in food samples was determined by colony counting. Results from the agar dilution method demonstrated that L. monocytogenes 10403S had a PLA minimum inhibitory concentration (MIC) value of 6 mg/ml. Sub-inhibitory concentrations of PLA could inhibit biofilm formation by reducing the secretion of exopolysaccharides and extracellular proteins in L. monocytogenes. PLA at concentrations above 1/2MIC could destroy mature biofilms of L. monocytogenes by decreasing the exopolysaccharides and extracellular proteins in the biofilm framework. Both swimming and swarming motilities of L. monocytogenes were inhibited by PLA. The hemolytic activity of L. monocytogenes was inactivated by PLA. However, the capacity to attach and invade Caco-2 cells was not affected by PLA. The results displayed that PLA had no effect on the expression of genes associated with motility, but reduced the expression level of the hly gene encoding Listeria hemolysin. When added to ultra-high temperature (UHT) whole and pasteurized milk, PLA at 3 mg/ml inhibited L. monocytogenes growth through 14 days of storage at 4 °C. PLA at concentrations ≥3 mg/ml significantly reduced L. monocytogenes counts on spiced beef samples during storage. PLA has potential as an alternative antimicrobial to control L. monocytogenes contamination and its biofilms in food industry.

An Engineered λ Phage Enables Enhanced and Strain-Specific Killing of Enterohemorrhagic Escherichia coli.

Bacteriophages (phages) are ideal alternatives to traditional antimicrobial agents in a world where antimicrobial resistance (AMR) is emerging and spreading at an unprecedented speed. In addition, due to their narrow host ranges, phages are also ideal tools to modulate the gut microbiota in which alterations of specific bacterial strains underlie human diseases, while dysbiosis caused by broad-spectrum antibiotics can be harmful. Here, we engineered a lambda phage (Eλ) to target enterohemorrhagic Escherichia coli (EHEC) that causes a severe, sometimes lethal intestinal infection in humans. We enhanced the killing ability of the Eλ phage by incorporating a CRISPR-Cas3 system into the wild-type λ (wtλ) and the specificity by introducing multiple EHEC-targeting CRISPR spacers while knocking out the lytic gene cro. In vitro experiments showed that the Eλ suppressed the growth of EHEC up to 18 h compared with only 6 h with the wtλ; at the multiplicity of infection (MOI) of 10, the Eλ killed the EHEC cells with ~100% efficiency and did not affect the growth of other laboratory- and human-gut isolated E. coli strains. In addition, the EHEC cells did not develop resistance to the Eλ. Mouse experiments further confirmed the enhanced and strain-specific killing of the Eλ to EHEC, while the overall mouse gut microbiota was not disturbed. Our methods can be used to target other genes that are responsible for antibiotic resistance genes and/or human toxins, engineer other phages, and support in vivo application of the engineered phages. IMPORTANCE Pathogenic strains of Escherichia coli are responsible for 0.8 million deaths per year and together ranked the first among all pathogenic species. Here, we obtained, for the first time, an engineered phage, Eλ, that could specifically and efficiently eliminate EHEC, one of the most common and often lethal pathogens that can spread from person to person. We verified the superior performance of the Eλ over the wild-type phage with in vitro and in vivo experiments and showed that the Eλ could suppress EHEC growth to nondetectable levels, fully rescue the EHEC-infected mice, and rescore disturbed mouse gut microbiota. Our results also indicated that the EHEC did not develop resistance to the Eλ, which has been the biggest challenge in phage therapy. We believe our methods can be used to target other pathogenic strains of E. coli and support in vivo application of the engineered phages.

Single-Cell Sequencing Identifies the Heterogeneity of CD8+ T Cells and Novel Biomarker Genes in Hepatocellular Carcinoma.

CD8+ T cells are required for the establishment of antitumor immunity, and their substantial infiltration is associated with a good prognosis. However, CD8+ T cell subsets in the tumor microenvironment may play distinct roles in tumor progression, prognosis, and immunotherapy. In this study, we used the scRNA-seq data of hepatocellular carcinoma (HCC) to reveal the heterogeneity of different CD8+ T cell subsets. The scRNA-seq data set GSE149614 was obtained from the GEO database, and the transcriptome and sample phenotypic data of TCGA-LIHC were obtained from the TCGA database. CD8+ T cell subtypes and metabolic gene sets were obtained from published reports. The data processing and analysis of CD8+ T cell groups was performed by R language. The PPI network was constructed to obtain the hub genes, and the KM survival curve of the hub genes was further plotted to determine the hub genes with differences in survival. CD8+ T cells in HCC were divided into 7 subsets, and the cytotoxic CD8 T cells 4 subset showed considerable differences between the TP53-mutant and nonmutant groups, as well as between different degrees of cirrhosis, HCC grades, stages, ages, and body weights. Cytotoxic CD8 T cells 4 differential genes were analyzed by TCGA-LIHC data and single-cell sequencing data set. 10 hub genes were found: FGA, ApoA1, ApoH, AHSG, FGB, HP, TTR, TF, HPX, and APOC3. Different subsets of CD8+ T cells were found to contribute to heterogeneous prognosis and pathway activity in HCC. Alterations in the cytotoxic and immune checkpoint gene expression during CD8+ T cell differentiation were also identified. We found that cytotoxic CD8 T cells 4 is closely associated with survival and prognosis of HCC and identified four differential genes that can be used as biological markers for survival, prognosis, and clinically relevant characteristics of HCC. Results of this study could help finding targets for immunotherapy of HCC and aid in the accelerated development of immunotherapy for HCC.

Dynamic simulation and optimization of integrated clean energy water systems.

Nuclear hybrid energy systems (NHES) are a viable option to provide clean power by combining renewable energy sources such as wind and solar. This study analyzes two types of NHES that use small modular reactors (SMR) and wind turbines to produce clean energy and water. The first system uses freeze desalination (FD) and the second system uses reverse osmosis (RO) to produce clean water. Both systems are optimized using net present value at two case locations. The FD system can better meet the energy demand using the stored thermal energy to boost the power during peak hours, which allows less capital investment on its design compared to the RO system. However, the results from the two cases reveal that the RO system can be more economic when water price is more than $1.50/m3. A sensitivity analysis also identified the critical system parameters on the net present value of the systems.

Prediction of Hepatocellular Carcinoma Prognosis and Immune Cell Infiltration Using Gene Signature Associated with Inflammatory Response.

It has been demonstrated that the inflammatory response influences cancer development and can be used as a prognostic biomarker in various tumors. However, the relevance of genes associated with inflammatory responses in hepatocellular carcinoma (HCC) remains unknown. The Cancer Genome Atlas (TCGA) database was analyzed using weighted gene coexpression network analysis (WGCNA) and differential analysis to discover essential inflammatory response-related genes (IFRGs). Cox regression studies, both univariate and multivariate, were employed to develop a prognostic IFRGs signature. Additionally, Gene Set Enrichment Analysis (GSEA) was used to deduce the biological function of the IFRGs signature. Finally, we estimated immune cell infiltration using a single sample GSEA (ssGSEA) and x-cell. Our results revealed that, among the major HCC IFRGs, two (DNASE1L3 and KLKB1) were employed to create a predictive IFRG signature. The IFRG signature could correctly predict overall survival (O.S) as per Kaplan-Meier time-dependent roc curves analysis. It was also linked to pathological tumor stage and T stage and might be used as a prognostic predictor in HCC. GSEA analysis concluded that the IFRG signature might influence the immune response in HCC. Immunological cell infiltration and immune checkpoint molecule expression differed in the high-risk and low-risk groups. As a result of our findings, DNASILE may play a role in the tumor microenvironment. However, more research is necessary to confirm the role of DNASE1L3 and KLKB1.

Transcriptome analysis of potential flocculation-related genes in Streptomyces sp. hsn06 with flocculation activity on Chlorella vulgaris biomass.

Chlorella vulgaris is a biomass energy provider with promising potential to help alleviate the energy crisis. Streptomyces sp. hsn06, as an actinomycete, can harvest C. vulgaris biomass safely and efficiently through flocculation activity, and proteins contribute greatly to the flocculation effect. However, potential flocculation protein-related genes are unclear. The mycelia of strain hsn06 after culture with glucose as the sole carbon source exhibited significantly higher flocculation activity as well as higher protein contents than those cultured with starch as the carbon source. To further explore the flocculation mechanism, the mycelia of strain hsn06 with distinct flocculation activities after culture with different carbon sources were examined by transcriptome analysis. We found that 403 genes were differentially up-regulated in mycelia cultured with glucose, compared to those cultured with starch as the carbon source. Five significantly differentially expressed protein-related genes were determined and confirmed by qRT-PCR, which indicated that three of the selected genes were potential flocculation-related genes. These results advance our understanding of potential flocculation-related genes during the harvesting of microalgal biomass.

Role of the VirSR-VirAB system in biofilm formation of Listeria monocytogenes EGD-e.

The ability of Listeria monocytogenes, an important foodborne pathogen, to form biofilms in food processing environments leads to increased opportunity for contamination of food products, which is a major concern for food safety. In this study, the role of a complex system composed of the VirSR two-component signal transduction system (TCS) and the ATP-binding cassette (ABC) transporter VirAB in biofilm formation of L. monocytogenes EGD-e was investigated. Biofilm formation was measured using the microplate assay with crystal violet staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and attachment and swarming motility were compared between strain EGD-e and its isogenic deletion mutants. Additionally, the relative expression levels of genes associated with the early steps of biofilm development in the wild-type and mutant strains were also determined by RT-qPCR. Results from microplate assay, CLSM and SEM showed that VirR is not required for biofilm formation in L. monocytogenes EGD-e. A central finding of this study is that both VirAB and VirS are essential for biofilm formation and they could function as a whole in biofilm formation of L. monocytogenes EGD-e. The results also demonstrated that both VirAB and VirS are involved in attachment, but they are not associated with swarming motility. Results from RT-qPCR showed that flaA, motA and motB were downregulated in the mutant strains ΔvirAB and ΔvirS, which could be the possible reason for reduced attachment and biofilm formation in these mutants. This study provides a better understanding of the mechanisms involved in biofilm formation of L. monocytogenes, leading to improved processes to control this biofilm-forming foodborne pathogen.

Life stages and morphological variations of Limnocythere inopinata (Crustacea, Ostracoda) from Lake Jiang-Co (northern Tibet): a bioculture experiment.

Limnocythere inopinata (Baird, 1843) is a Holarctic species, abundant in a number of Recent and fossil ostracod assemblages, and has many important taxonomic and (paleo)ecological applications. However, the life cycle and morphological characteristics of the living L. inopinata are still unclear. A bioculture experiment was designed to study life stages and morphological variations from stage A-8 to adult in this species. The living animals were collected from Lake Jiang-Co, in the northern Tibetan Plateau. Results reveal that this species possesses a special growth pattern with the maximum size increase occurring at the transition from the instars A-5 to A-4. The growth pattern deviates from Brooks' rule and one population from Lake Dali, eastern Mongolian Plateau. This suggests that the life history of L. inopinata may be influenced by environmental factors. Some morphological differences between Lake Jiang-Co and European populations of L. inopinata are also uncovered. Therefore, a detailed morphological description of this population is provided, but refrain from erecting a new species at the present stage because those differences appear to be inconsistent.

Whole Transcriptome Analysis Provides Insights Into the Molecular Mechanisms of Chlamydospore-Like Cell Formation in Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus naturally isolated from hardwoods and widely used in environmental pollution control because it produces extracellular peroxidases. It forms chlamydospores during nitrogen starvation, which naturally occurs in the habitat of P. chrysosporium. Chlamydospores protect fungi against many stresses; the molecular basis underlying chlamydospore formation in basidiomycetes is poorly explored. Chlamydospores in P. chrysosporium have a different cell wall compared with hyphae, as confirmed by cell wall digestion and microscopy. Furthermore, this study investigated the transcriptome of P. chrysosporium in different life stages, including conidium, hypha, and chlamydospore formation, through RNA sequencing. A total of 2215 differentially expressed genes were identified during these processes. The expression patterns of genes involved in several molecular events critical for chlamydospore formation, including starch and sucrose metabolism, phosphatase and kinase, and transcription factors, were determined. This study serves as a basis for further investigating the function of chlamydospore formation in the biotechnologically relevant fungus P. chrysosporium.

The sug operon involves in resistance to quaternary ammonium compounds in Listeria monocytogenes EGD-e.

In recent years, an increasing number of Listeria monocytogenes strains with resistance to quaternary ammonium compounds (QACs) have been reported. However, the genetic basis for QACs resistance in L. monocytogenes remains poorly understood. In the present study, we have characterized the operon lmo0852/lmo0853/lmo0854 (designated sugR/sugE1/sugE2) that contributes to QACs' resistance in L. monocytogenes EGD-e. We constructed the gene deletion mutants and the complemented strains, determined minimum inhibitory concentrations (MICs) of these strains against antimicrobial agents, assessed the transcription levels of target genes by RT-qPCR, and measured the promoter activity by using ß-galactosidase assays. We also investigated the interaction between the promoter DNA and the putative regulatory protein by electrophoretic mobility shift assay (EMSA). The sug operon consists of a putative TetR family regulator encoded by sugR and two small multidrug resistance (SMR) efflux pumps encoded by sugE1 and sugE2. Our results showed that either SugE1 or SugE2 is sufficient for QACs' resistance, indicating their function overlapping in QACs' resistance. Interestingly, lacking one sugE gene could lead to a significant increase in transcription of the other sugE gene in the presence of benzalkonium chloride (BC). Additionally, SugR negatively regulates the transcription of the sug locus by binding to the operon promoter. Given that QACs are commonly used in food industry, the findings from this study will help us to have a better understanding of the adaptations of L. monocytogenes to this type of disinfectant. Key points •Either SugE1 or SugE2 was sufficient for QACs resistance. •The functions of two sug genes overlap and compensate each other in QACs resistance. •SugR is the transcriptional suppressor of sugE1 and sugE2. •SugR regulates the sug locus by binding to the operon promoter.