Biofunctional attributes and storage study of milk fermented by Enterococcus italicus.

Lactic acid bacteria are probiotics in the intestines and have been widely used as natural antioxidants in the food industry. In this study, Enterococcus italicus FM5 with strong antioxidant ability was isolated from fresh milk. The safety evaluation showed that E. italicus FM5 was sensitive to ampicillin, chloramphenicol, erythromycin, vancomycin, rifampicin, and tetracycline, and was not hemolytic. Meanwhile, the whole genome information and biofunctional attributes of this strain were determined and analyzed. Subsequently, E. italicus FM5 was co-cultured with traditional yogurt starters (Streptococcus thermophilus and Lactobacillus bulgaricus) to make fermented milk. The results showed that the addition of E. italicus FM5 could improve the oxygen free radical scavenging ability of the fermented milk, and the scavenging rates of DPPH, ABTS, OH-, and O2- radicals reaching up to 95.54 %, 88.35 %, 93.65 %, and 60.29 %, respectively. Furthermore, the addition of E. italicus FM5 reduced the curd time and improved the water holding capacity of the fermented milk. Besides, the growth of Lb. bulgaricus was significantly promoted when co-cultured with E. italicus FM5, thus the survival cells were increased compared with the traditional fermentation processes. Therefore, this study emphasized the potential to manufacture fermented milk by the co-cultivation of E. italicus with traditional yogurt starters.

The inhibitory effect of swine TAB1 on the replication of pseudorabies virus.

TAK1-binding protein 1 (TAB1) assembles with TAK1 through its C-terminal domain, leading to the self-phosphorylation and activation of TAK1, which plays an important role in the activation of NF-κB and MAPK signaling pathway. Pseudorabies virus (PRV) is the pathogen of Pseudorabies (PR), which belongs to the Alphaherpesvirus subfamily and causes serious economic losses to the global pig industry. However, the impact of swine TAB1 (sTAB1) on PRV infection has not been reported. In this study, evidence from virus DNA copies, virus titer and western blotting confirmed that sTAB1 could inhibit PRV replication and knockout of sTAB1 by CRISPR-Cas9 gene editing system could promote PRV replication. Further mechanistic studies by real-time PCR and luciferase reporter gene assay demonstrated that sTAB1 could enhance the production of inflammatory factors and chemokines, IFN-ß transcription level and IFN-ß promoter activity after PRV infection. In summary, we clarify the underlying mechanism of sTAB1 in inhibiting PRV replication for the first time, which provides a new idea for preventing PRV infection and lays a foundation for PRV vaccine development.

Nine new species of black lichenicolous fungi from the genus Cladophialophora (Chaetothyriales) from two different climatic zones of China.

Lichenicolous fungi are parasites of lichens. Many of these fungi are referred to as "black fungi". A diversity of these black fungi include species that are pathogenic to humans and plants. A majority of black fungi reside in the phylum Ascomycota within the sub-classes Chaetothyriomycetidae and Dothideomycetidae. To explore the diversity of lichenicolous "black fungi" associated with lichens in China, we conducted several field surveys in the Inner Mongolia Autonomous Region and Yunnan Province between 2019 and 2020. We recovered 1,587 fungal isolates from the lichens collected during these surveys. During the preliminary identification of these isolates using the complete internal transcribed spacer (ITS), partial large subunit of nuclear ribosomal RNA gene (LSU), and small subunit of nuclear ribosomal RNA gene (SSU), we identified 15 fungal isolates from the genus Cladophialophora. However, these isolates had low sequence similarities with all known species from the genus. Therefore, we amplified additional gene regions, such as, translation elongation factor (TEF) and partial ß-tubulin gene (TUB), and constructed a multi-gene phylogeny using maximum likelihood, maximum parsimony, and Bayesian inference. In our datasets, we included type sequences where available for all Cladophialophora species. Phylogenetic analyses revealed that none of the 15 isolates belonged to any of the previously described species in the genus. Therefore, using both morphological and molecular data, we classified these 15 isolates as nine new species within the genus Cladophialophora: C. flavoparmeliae, C. guttulate, C. heterodermiae, C. holosericea, C. lichenis, C. moniliformis, C. mongoliae, C. olivacea, and C. yunnanensis. The outcome from this study shows that lichens are an important refugia for black lichenicolous fungi, such as those from Chaetothyriales.

Catalase-peroxidase StKatG is a bacterial manganese oxidase from endophytic Salinicola tamaricis.

Manganese (Mn) oxides in iron/manganese plaques are widely distributed in the rhizosphere of wetland plants and contribute significantly to elemental cycling and pollutant removal. Mn oxides are primarily produced by bacterial processes using Mn oxidases. However, the molecular mechanism underlying the formation of rhizosphere Mn oxides is still largely unknown. This study identified a manganese-oxidizing enzyme, the catalase-peroxidase StKatG, from an endophytic bacterium Salinicola tamaricis from the wetland plant. The gene encoding StKatG was cloned and overexpressed in Escherichia coli. The recombinant StKatG displayed different structure and enzymatic properties from the previously reported Mn oxidases. The enzyme activity of StKatG yielded Mn oxides with the mixed-valent state: Mn(II), Mn(III), and Mn(IV). The optimum pH and temperature for StKatG are 7.5 and 50 °C, respectively. Structurally, StKatG is organized into two domains, whereas the reported Mn oxidases are mainly single-domain proteins. Based on the site-directed mutagenesis studies, the presence of aspartic acid (Asp) residues in the loop of StKatG are critical to Mn-oxidizing activity. These findings identified a novel bacterial Mn oxidase and provided insights into the molecular mechanism of Mn oxidation in the plant rhizosphere.

Constructed wetland-microbial fuel cells enhanced with iron carbon fillers for ciprofloxacin wastewater treatment and power generation.

In this study, the following three experimental devices were operated for 70 days for the treatment of ciprofloxacin pollutants in wastewater: constructed wetlands (CW), constructed wetland-microbial fuel cells (EG), and constructed wetland-microbial fuel cells with new iron-carbon fillers (TPFC). The water quality, power generation capacity, microbial community structure, and changes in the resistance gene qnrs were studied. The efficiency of removal of total phosphate in the TPFC (97.1% ± 2.5%) was significantly higher than that in the EG (51.6% ± 4.8%) and the CW (68.1% ± 2.9%). The efficiency of removal of ciprofloxacin was also significantly higher (TPFC: 91.2% ± 3.4%, EG: 82.1% ± 2.3%, and CW: 75.1% ± 5.6%) (P < 0.05). The voltage of TPFC reached 300.16 ± 12.12 mV, which was apparently greater than that of EG (180.36 ± 16.73 mV) (P < 0.05), possibly because of the higher abundance of microorganisms such as Burkholderiaceae, Hydrogenophaga, and Proteobacteria. There were more copies of the resistance gene qnrs (TPFC: 7.74/µL, EG: 5.52/µL, and CW: 2.65/µL), which may be associated with stronger resistance; therefore, the efficiency of removal of ciprofloxacin was higher in the TPFC. TPFCs are a promising way to remove ciprofloxacin in wastewater.

The functional role of fecal microbiota transplantation on Salmonella Enteritidis infection in chicks.

The intestinal microbiota plays important roles in animal health and growth. We investigated the efficacy and mechanisms of fecal microbiota transplantation (FMT) from adult SPF chickens against Salmonella Enteritidis (SE) infection in chicks. We transplanted 160 recipient SPF chicks (1-day-old) that were randomly divided into four groups, Ca (challenge), Cb (non-challenge), Fa (FMT and challenge) and Fb (FMT without challenge). The experiment lasted 40 days. We found that FMT reduced mortality as well as liver inflammatory lesions, promoted weight gain, improved immunity, ameliorated the digestion and absorption ability and inhibited SE colonization in the liver of challenged chicks. 16S rRNA gene high-throughput sequencing indicated that SE challenge caused a significant increase in the relative abundance of Parasutterella in the cecal microbiota of the recipient chicks (P < 0.05). FMT led to the maturation of the intestinal flora of recipients and the relative abundance of the Bacteroides, Rikenellaceae_ RC9_ gut_ group, Prevotellaceae_ UCG_ 001, Prevotellaceae_ Ga6A1_ group and Parabacteroides was significantly increased (P < 0.05). FMT from adult SPF chickens regulated the intestinal microbiota of chicks and increased resistance to SE infection.

Geosmithia Species Associated With Bark Beetles From China, With the Description of Nine New Species.

Fungi of the genus Geosmithia are frequently associated with bark beetles that feed on phloem on various woody hosts. Most studies on Geosmithia were carried out in North and South America and Europe, with only two species being reported from Taiwan, China. This study aimed to investigate the diversity of Geosmithia species in China. Field surveys in Fujian, Guangdong, Guangxi, Hunan, Jiangsu, Jiangxi, Shandong, Shanghai, and Yunnan yielded a total of 178 Geosmithia isolates from 12 beetle species. The isolates were grouped based on morphology. The internal transcribed spacer, ß-tubulin, and elongation factor 1-α gene regions of the representatives of each group were sequenced. Phylogenetic trees were constructed based on those sequences. In total, 12 species were identified, with three previously described species (Geosmithia xerotolerans, G. putterillii, and G. pallida) and nine new species which are described in this paper as G. luteobrunnea, G. radiata, G. brevistipitata, G. bombycina, G. granulata (Geosmithia sp. 20), G. subfulva, G. pulverea (G. sp. 3 and Geosmithia sp. 23), G. fusca, and G. pumila sp. nov. The dominant species obtained in this study were G. luteobrunnea and G. pulverea. This study systematically studied the Geosmithia species in China and made an important contribution to filling in the gaps in our understanding of global Geosmithia species diversity.

Ophiostomatoid species associated with pine trees (Pinus spp.) infested by Cryphaluspiceae from eastern China, including five new species.

Cryphaluspiceae attacks various economically important conifers. Similar to other bark beetles, Cr.piceae plays a role as a vector for an assortment of fungi and nematodes. Previously, several ophiostomatoid fungi were isolated from Cr.piceae in Poland and Japan. In the present study, we explored the diversity of ophiostomatoid fungi associated with Cr.piceae infesting pines in the Shandong Province of China. We isolated ophiostomatoid fungi from both galleries and beetles collected from our study sites. These fungal isolates were identified using both molecular and morphological data. In this study, we recovered 175 isolates of ophiostomatoid fungi representing seven species. Ophiostomaips was the most frequently isolated species. Molecular and morphological data indicated that five ophiostomatoid fungal species recovered were previously undescribed. Thus, we proposed these five novel species as Ceratocystiopsisyantaiensis, C.weihaiensis, Graphilbumtranslucens, Gr.niveum, and Sporothrixvillosa. These new ophiostomatoid fungi add to the increasing number of fungi known from China, and this evidence suggests that numerous novel taxa are awaiting discovery in other forests of China.

Combination of the endophytic manganese-oxidizing bacterium Pantoea eucrina SS01 and biogenic Mn oxides: An efficient and sustainable complex in degradation and detoxification of malachite green.

Recently, Mn oxides (MnOxs) have been attracting considerable interest in the oxidation of organic pollutants. However, the reduction of MnOx in these reactions leads to the deactivation of the catalyst, which must be frequently regenerated. We evaluated the application of a manganese-oxidizing bacterium (MOB) and MnOx in removing toxic dyes. We studied the co-function of a plant-endophytic MOB, Pantoea eucrina SS01, with its bio-generated MnOx and evaluated the detoxification activity and chemical transformation mechanisms of the complex in malachite green (MG) degradation. We found a synergistic effect between MnOx and the strain. Particularly, strain SS01 could adsorb MG but could not degrade it, whereas the addition of Mn(II) promoted MG degradation by the formation of a complex containing the bacterium and MnOx aggregates (SS01-bio-MnOx), with distinct morphology characteristics. The complex showed a marked sustainability in the degradation of MG into less toxic or non-toxic metabolites. In this process, strain SS01 might have enhanced the regeneration of MnOx, accelerating MG degradation. Our data not only contribute to understanding the mechanism of MG removal by the SS01-bio-MnOx complex, but also provide a scientific basis for the future application of MOB and MnOx.

Serine/threonine Kinases Play Important Roles in Regulating Polyunsaturated Fatty Acid Biosynthesis in Synechocystis sp. PCC6803.

Serine/threonine kinases (STKs) play important roles in prokaryotic cellular functions such as growth, differentiation, and secondary metabolism. When the external environment changes, prokaryotes rely on signal transduction systems, including STKs that quickly sense these changes and alter gene expression to induce the appropriate metabolic changes. In this study, we examined the roles of the STK genes spkD and spkG in fatty acid biosynthesis in the unicellular cyanobacterium Synechocystis sp. PCC6803, using targeted gene knockout. The linoleic acid (C18: 2), γ-linolenic acid (C18: 3n6), α-linolenic acid (C18: 3n3), and stearidonic acid (C18: 4) levels were significantly lower in spkD and spkG gene knockout mutants than in the wild type at a culture temperature of 30°C and a light intensity of 40 µmol⋅m-2⋅s-1. The expression levels of fatty acid desaturases and STK genes differed between the spkD and spkG gene knockout mutants. These observations suggest that spkD and spkG may directly or indirectly affect the fatty acid composition in Synechocystis sp. PCC6803 by regulating the expression of fatty acid desaturases genes. Therefore, the STK genes spkD and spkG play important roles in polyunsaturated fatty acid biosynthesis in Synechocystis sp. PCC6803. These findings could facilitate the development of cyanobacteria germplasm resources that yield high levels of fatty acids. In addition, they provide a theoretical basis for the genetic engineering of cyanobacteria with improved yields of secondary metabolites and increased economic benefits.

Characterization and bioremediation potential of nickel-resistant endophytic bacteria isolated from the wetland plant Tamarix chinensis.

Wetlands have been proposed as a sink for pollutants such as heavy metals. Wetland plants play a significant role in the phytoremediation of heavy metals. Here, we isolated and characterized three novel nickel (Ni)-resistant endophytic bacteria (NiEB) from the wetland plant Tamarix chinensis. The NiEB were identified as Stenotrophomonas sp. S20, Pseudomonas sp. P21 and Sphingobium sp. S42. All isolates tolerated 50 mg L-1 Ni, with isolates S20 and P21 being more tolerant to Ni at up to 400 mg L-1. Moreover, isolate S42 removed 33.7% of nickel sulfate from the water by forming white precipitates. The three isolates exhibited different plant growth-promoting (PGP) traits related to the production of indole acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Phytotoxicity studies revealed that the growth of the wetland plants in a high Ni concentration (200 mg L-1) recovered after co-incubation with isolate S42. Overall, this study presents the first report of NiEB isolation from wetland plants and provides novel insights into the diverse functions of endophytic bacteria in a plant host with the potential to improve Ni phytoremediation.

Bioremediation of malachite green by cyanobacterium Synechococcus elongatus PCC 7942 engineered with a triphenylmethane reductase gene.

Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.

Diversity evolution of functional bacteria and resistance genes (CzcA) in aerobic activated sludge under Cd(II) stress.

An activated sludge sequencing batch reactor (SBR) was used to treat divalent cadmium (Cd(II)) wastewater for 60 d to investigate the overall treatment performance, evolution of the bacterial community, and abundance of the Cd(II) resistance gene CzcA and shifts in its potential host bacteria. During stable operation with a Cd(II) concentration of 20 mg/L, the average removal efficiencies of Cd(II) and chemical oxygen demand (COD) were more than 85% and that of total phosphorus was greater than 70%, while the total nitrogen (TN) was only about 45%. The protein (PN) content in the extracellular polymeric substances (EPS) increased significantly after Cd(II) addition, while polysaccharides displayed a decreasing trend (p < 0.05), indicating that EPS prefer to release PN to adsorb Cd(II) and protect bacteria from damage. Three-dimensional fluorescence spectral analysis showed that fulvic acid-like substances were the most abundant chemical components of EPS. The addition of Cd(II) adversely affected most denitrifying bacteria (p < 0.05), which is consistent with the low TN removal. In addition, quantitative polymerase chain reaction analysis revealed that CzcA gene abundance decreased as the Cd(II) concentration increased, possibly because expression of the CzcA gene was inhibited by Cd(II) stress. The majority of CzcA gene sequences were carried by Pseudomonas, making it the dominant genus among Cd(II)-resistant bacteria.

Nuclear Import of a Secreted "Candidatus Liberibacter asiaticus" Protein is Temperature Dependent and Contributes to Pathogenicity in Nicotiana benthamiana.

"Candidatus Liberibacter asiaticus" (CLas), one of the causal agents of citrus Huanglongbing (HLB), secretes proteins with functions that are largely unknown. In this study, we demonstrated that CLIBASIA_00460, one of the CLas-encoded Sec-dependent presecretory proteins, might contribute to the phytopathogenicity of CLas. CLIBASIA_00460 was conserved in CLas strains and expressed at a significantly higher level in citrus than in Asian citrus psyllid. Agrobacteria-mediated transient expression in Nicotiana benthamiana epidermal cells showed that the mature CLIBASIA_00460 (m460) without the putative Sec-dependent signal peptide was localized in multiple cellular compartments including nucleus at 25°C, but that nuclear accumulation was greatly decreased as the temperature rose to 32°C. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m460 induced no local symptoms, but tiny necrotic spots were scattered on the systemic leaves. However, NLS-m460, which contains the SV40 nuclear localization sequence (NLS) at the N-terminus to promote nuclear import of m460, caused chlorosis and necrosis in the local leaves and severe necrosis in the systemic leaves. Taken together, these data suggest that CLIBASIA_00460 represented a novel virulence factor of CLas, and that nuclear localization of this protein was temperature dependent and positively correlated with its pathogenicity in planta.

Evolution and resistance of a microbial community exposed to Pb(II) wastewater.

This study investigated the treatment performance of activated sludge on Pb(II)-containing wastewater, including contaminant removal efficiency, extracellular polymeric substances, pbrT gene content and the microbial community. The average removal efficiencies of ammonia nitrogen, chemical oxygen demand, total phosphorus, total nitrogen and Pb(II) were 40% ±â€¯4%, 91% ±â€¯3%, 95% ±â€¯3%, 51% ±â€¯5% and 92% ±â€¯9% during the stable operation stage, respectively. Moreover, the extracellular polymeric substance -protein contents increased significantly from day 0 to day 60 (p < 0.05). The most abundant fluorescent component in extracellular polymeric substances was a humic acid-like substance, and its fluorescence intensity increased significantly from day 0 to day 60 (p < 0.05). Adsorption of negatively charged organic functional groups in extracellular polymeric substances was identified as a major component of the removal of Pb(II). Most of the denitrifying bacteria associated with nitrogen removal showed an increasing trend during the acclimation stage, which may have resulted in high total nitrogen removal efficiency. In addition, pbrT uptake protein was found to be responsible for the uptake of Pb(II) into cells. The abundance of the pbrT gene showed a downward trend (p < 0.05) after adding Pb(II), probably because expression of the pbrT gene was inhibited under Pb(II) stress. Sphingopyxis containing the pbrT gene was the dominant resistance genus, and its relative abundance increased significantly (p < 0.05) from day 0 to day 60. This study provided a theoretical basis for the treatment of Pb(II)-containing wastewater.

Chemical composition of extracellular polymeric substances and evolution of microbial community in activated sludge exposed to ibuprofen.

Ibuprofen (IBU) containing wastewater with a concentration of 1-5 mg/L was treated in an activated sludge sequencing batch reactor (SBR), for 60 days, in order to investigate the overall performance of the SBR, the parameter variations during a typical cycle, the chemical composition and content of extracellular polymeric substances (EPS) and the evolution of microbial community. The average removal efficiencies of COD, NH4+-N and TN were >85%, while >40% of the IBU was removed and the removal efficiencies of TP fluctuated around ~ 75%. The EPS content increased significantly with IBU addition (p < 0.01). Fulvic acid-like substances in the chemical composition of EPS increased during the stable operation phase. Proteobacteria associated with nitrogen removal was the dominant phylum, which can also resist IBU stress. For the denitrifying bacteria, the OTUs of both Rhodobacter and Pseudomonas increased from day 1-30 and reduced on day 60 (p < 0.01), which was opposite to the results observed for Rhodocyclaceae (phosphorus-accumulating bacteria). The OTUs of Acidovorax showed an increasing trend (p < 0.01), whereas the OTUs for Nitrospira (nitrite oxidizers) and Nitrosomonas (ammonia oxidizers) decreased significantly (p < 0.05).

Biodegradation of malachite green by an endophytic bacterium Klebsiella aerogenes S27 involving a novel oxidoreductase.

Endophytic microorganisms can metabolize organic contaminants and assist in plant growth, thus facilitating the phytoremediation of polluted environments. An endophytic bacterium capable of decoloring malachite green (MG) was isolated from the leaves of the wetland plant Suaeda salsa and was identified as Klebsiella aerogenes S27. Complete decolorization of MG (100 mg/l) was achieved in 8 h at 30 °C and pH 7.0. Ultraviolet-visible spectroscopy and Fourier-transform infrared spectroscopy analyses indicated the degradation of MG by the isolate. The enzymic assays of the strain showed the triphenylmethane reductase (TMR) activity. A gene encoding putative TMR-like protein (named as KaTMR) was cloned and heterologously expressed in Escherichia coli. KaTMR showed only 42.6-43.3% identities in amino acids compared with well-studied TMRs, and it phylogenetically formed a new branch in the family of TMRs. The degraded metabolites by recombinant KaTMR were detected by liquid chromatography-mass spectrometry, showing differences from the products of reported TMRs. The biotransformation pathway of MG was proposed. Phytotoxicity studies revealed the less-toxic nature of the degraded metabolites compared to the dye. This study presented the first report of an endophyte on the degradation and detoxification of triphenylmethane dye via a novel oxidoreductase, thus facilitating the study of the plant-endophyte symbiosis in the bioremediation processes.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

OBJECTIVE: Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. RESULTS: Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. CONCLUSIONS: The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Complete Genome Sequence of the Heavy-Metal-Tolerant Endophytic Type Strain of Salinicola tamaricis.

The first complete genome sequence of a recently described Salinicola tamaricis species was determined for the strain F01T (=CCTCC AB 2015304T =KCTC 42855T). The strain was isolated from the leaves of wetland plant Tamarix chinensis Lour and shows a high tolerance to heavy metals, such as manganese, nickel, lead, and copper ions.