Actinobacteria produce taste and odor in drinking water reservoir: Community composition dynamics, co-occurrence and inactivation models.

Taste and odor (T&O) has become a significant concern for drinking water safety. Actinobacteria are believed to produce T&O during the non-algal bloom period; however, this has not been widely investigated. In this study, the seasonal dynamics of the actinobacterial community structure and inactivation of odor-producing actinobacteria were explored. The results indicated that the diversity and community composition of actinobacteria exhibited significant spatiotemporal distribution. Network analysis and structural equation modeling showed that the actinobacterial community occupied a similar environmental niche, and the major environmental attributes exhibited spatiotemporal dynamics, which affected the actinobacterial community. Furthermore, the two genera of odorous actinobacteria were inactivated in drinking water sources using chlorine. Amycolatopsis spp. have a stronger chlorine resistance ability than Streptomyces spp., indicating that chlorine inactivates actinobacteria by first destroying cell membranes and causing the release of intracellular compounds. Finally, we integrated the observed variability in the inactivation rate of actinobacteria into an expanded Chick-Watson model to estimate its effect on inactivation. These findings will deepen our understanding of the seasonal dynamics of actinobacterial community structure in drinking water reservoirs and provide a foundation for reservoir water quality management strategies.

Nitrogen reduction by aerobic denitrifying fungi isolated from reservoirs using biodegradation materials for electron donor: Capability and adaptability in the lower C/N raw water treatment.

Biological denitrification was considered an efficient and environmentally friendly way to remove the nitrogen in the water body. However, biological denitrification showed poor nitrogen removal performance due to the lack of electron donors in the low C/N water. In this study, three novel aerobic denitrifying fungi (Trichoderma sp., Penicillium sp., and Fusarium sp.) were isolated and enhanced the performance of aerobic denitrification of fungi in low C/N water bodies combined with polylactic acid/polybutylene adipate-co-terephthalate (PLA/PBAT). In this work, the aerobic denitrifying fungi seed were added to denitrifying liquid medium and mixed with PLA/PBAT. The result showed that Trichoderma sp., Penicillium sp., and Fusarium sp. could reduce 89.93 %, 89.20 %, and 87.76 % nitrate. Meanwhile, the nitrate removal efficiency adding PLA/PBAT exceeded 1.40, 1.68, and 1.46 times that of none. The results of material characterization suggested that aerobic denitrifying fungi have different abilities to secrete proteases or lipases to catalyze ester bonds in PLA/PBAT and utilize it as nutrients in denitrification, especially in Penicillium brasiliensis D6. Besides, the electron transport system activity and the intracellular ATP concentration were increased significantly after adding PLA/PBAT, especially in Penicillium brasiliensis D6. Finally, the highest removal efficiency of total nitrogen in landscape water by fungi combined with PLA/PBAT was >80 %. The findings of this work provide new insight into the possibility of nitrogen removal by fungi in low C/N and the recycling of degradable resources.

Spatial and temporal dynamics of actinobacteria in drinking water reservoirs: Novel insights into abundance, community structure, and co-existence model.

The control of taste and odor (T&O) in drinking water reservoirs is the main challenge for water supply. T&O is mainly derived from actinobacteria during non-algal blooms. However, few studies have investigated the actinobacterial community in reservoirs, especially the effects of water quality parameters on actinobacteria. This study analyzed the environmental driving force of the actinobacterial community composition and change in time and space through structural equations and network in drinking water reservoirs. The results showed a high abundance of actinobacteria, up to 2.7 × 104 actinobacteria per 1 L, in the hypolimnion of the Lijiahe reservoir in September, which is one order of magnitude greater than that in the Jinpen reservoir. The two drinking water reservoirs had similar dominant genera, mainly Sporichthya sp., and Mycobacterium sp., and difference in the actinobacterial proportions. However, there was a large difference at the dominant species. Rhodococcus fascians (4.02%) was the dominant species in the Lijiahe reservoir, while Mycobacterium chlorophenolicum (6.64%) was the dominant species in the Jinpen reservoir. Network analysis revealed that the structure of the network in the Lijiahe reservoir was more unstable; thus, it was vulnerable to environmental disturbances. In addition, a low abundance of species may play a critical role in the actinobacterial community structure of aquatic ecosystems. Structural equation modeling analysis suggested that water temperature, dissolved oxygen, and nutrition were the dominant factors affecting the abundance and community of actinobacteria. Overall, these findings broaden the understanding of the distribution and co-existence of actinobacterial communities in drinking water reservoirs and provide valuable clues for the biological controls of T&O and reservoir management.

Nitrogen removal by two strains of aerobic denitrification actinomycetes: Denitrification capacity, carbon source metabolic ability, and raw water treatment.

The denitrification characteristics of actinomyetes in aquatic ecosystem under aerobic conditions are not well known. Here, two actinomyetes strains M5 and M6 were separated and annotated as Streptomyces sp. Strains M5 and M6 could reduce 95.02% and 96.84 % of total nitrogen, 98.14 % and 97.02 % of total organic carbon under aerobic condition. Nitrogen balance analysis indicated that 78.60 % and 83.01 % of nitrogen was translated into gaseous, with 13.48 % and 10.77 % of nitrogen was assimilated into biomass for strains M5 and M6. The highest removal efficiency of nitrate of strains M5 and M6 in micro-polluted water bodies were 88.61 % and 82.53 %, respectively. Moreover, strains M5 and M6 exhibited remarkable carbon metabolic capacity, especially for esters. Altogether, this study provides a new perspective for understanding the performance of actinomyetes in aerobic denitrification and micro-polluted water reparation.

Characteristics of AccSTIP1 in Apis cerana cerana and its role during oxidative stress responses.

Various environmental stresses, such as heat shock, heavy metals, ultraviolet (UV) radiation and different pesticides, induce a cellular oxidative stress response. The cellular oxidative stress response is usually regulated by heat shock proteins (Hsps) acting as molecular chaperones. Stress-induced phosphoprotein 1 (STIP1), one of the most widely studied co-chaperones, functions as an adaptor that directs Hsp90 to Hsp70-client protein complexes. However, the biological functions of STIP1 remain poorly understood in honeybee (Apis cerana cerana). In this study, AccSTIP1 was identified in Apis cerana cerana. AccSTIP1 transcription was found to be induced by heat (42 °C), HgCl2, H2O2 and different pesticides (emamectin benzoate, thiamethoxam, hexythiazox and paraquat) and inhibited by CdCl2, UV and kresoxim-methyl. Moreover, western blot analysis indicated that the expression profiles of AccSTIP1 were consistent with its transcriptional expression levels. The disc diffusion assay showed that chemically competent transetta (DE3) bacteria expressing a recombinant AccSTIP1 protein displayed the smaller death zones than did control bacteria after exposure to paraquat and HgCl2. The DNA nicking assay suggested that recombinant purified AccSTIP1 protected supercoiled pUC19 plasmid DNA from damage caused by a thiol-dependent mixed-function oxidation (MFO) system. After knocking down AccSTIP1 gene expression via RNA interference (RNAi), the transcript levels of antioxidation-related genes were obviously lower in dsAccSTIP1 honeybees compared with those in the uninjected honeybees. Collectively, these results demonstrated that AccSTIP1 plays an important role in counteracting oxidative stress. This study lays a foundation for revealing the mechanism of AccSTIP1 in the Apis cerana cerana antioxidant system.

Isolation of carboxylesterase (esterase FE4) from Apis cerana cerana and its role in oxidative resistance during adverse environmental stress.

Carboxylesterases (CarEs) play vital roles in metabolising different physiologically important endogenous compounds and in detoxifying various harmful exogenous compounds in insects. Multiple studies of CarEs have focused on pesticide metabolism in insects, while few studies have aimed to identify CarE functions in oxidative resistance, particularly in Apis cerana cerana. In this study, we isolated a carboxylesterase gene, esterase FE4, from Apis cerana cerana and designated it towards an exploration of its roles as an antioxidant and in detoxification. We investigated AcceFE4 expression patterns in response to various stressors. A quantitative real-time PCR analysis revealed that AcceFE4 was up-regulated by H2O2, imidacloprid, and paraquat, and was down-regulated by 4 °C, UV radiation, CdCl2, and HgCl2. Additionally, the protein expression of this gene was down-regulated at 4 °C and up-regulated by H2O2. Disc diffusion assays showed that the AcceFE4 recombinant protein-expressing bacteria had a smaller killing zone than the control group with the paraquat, HgCl2 and cumyl hydroperoxide treatments. Moreover, when the gene was knocked down by RNA interference, we observed that multiple oxidant genes (i.e., AccSOD, AccGST, AccTrx, AccMsrA, and others) were down-regulated in the knockdown samples. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity levels were reduced in the knockdown samples relative to the control group. Finally, we measured the enzyme activity of carboxylesterase and found that the enzyme activity was also reduced in the silent samples. Together, these data suggest that AcceFE4 may be involved in the oxidative resistance response during adverse stress.

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana. The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

Roles of a mitochondrial AccSCO2 gene from Apis cerana cerana in oxidative stress responses.

In eukaryotes, cytochrome c oxidase (COX) is a multimeric protein complex that is the last enzyme in the respiratory electron transport chain of mitochondria. Syntheses of cytochrome c oxidase (SCO) proteins are copper-donor chaperones involved in metalation of the CuA redox center of COX. However, its other precise actions are not yet understood. Here, we report the characterization of AccSCO2 from Apis cerana cerana (Acc). Our data showed that AccSCO2 expression was induced by cold (4°C), CdCl2, HgCl2, ultraviolet (UV) light, and H2O2 and was inhibited by different pesticide treatments. In addition, a disc diffusion assay of recombinant AccSCO2, AccSCO2-R1, and AccSCO2-R2 proteins showed that they played a functional role in protecting cells from oxidative stress involved in copper-dependent manner. Further, following knockdown of AccSCO2 in A. cerana cerana using RNA interference (RNAi), the expression levels of most antioxidant genes (AccGSTD, AccGSTO1, AccGSTS4, AccSOD1, AccSOD2, etc.) were significantly decreased in the AccSCO2-silenced bees compared with the control bees. Moreover, the antioxidant enzymatic activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were all lower in the silenced bees than in the control bees. Finally, the in vivo activity of COX was measured after AccSCO2 knockdown, which revealed a strong reduction in COX activity in the silenced bees. Thus, we hypothesize that AccSCO2 plays important roles in cellular stress responses and anti-oxidative processes, which help to regulate the production of mitochondrial reactive oxygen species and/or the impairment of mitochondrial activity under oxidative stress.

Ultrastructural changes of Saccharomyces cerevisiae in response to ethanol stress.

In the fermentative process using Saccharomyces cerevisiae to produce bioethanol, the performance of cells is often compromised by the accumulation of ethanol. However, the mechanism of how S. cerevisiae responds against ethanol stress remains elusive. In the current study, S. cerevisiae cells were cultured in YPD (yeast extract - peptone - dextrose) medium containing various concentrations of ethanol (0%, 2.5%, 5%, 7.5%, 10%, and 15% (v/v)). Compared with the control group without ethanol, the mean cell volume of S. cerevisiae decreased significantly in the presence of 7.5% and 10% ethanol after incubation for 16 h (P < 0.05), and in the presence of 15% ethanol at all 3 sampling time points (1, 8, and 16 h) (P < 0.05). The exposure of S. cerevisiae cells to ethanol also led to an increase in malonyldialdehyde content (P < 0.05) and a decrease in sulfhydryl group content (P < 0.05). Moreover, the observations through transmission electron microscopy enabled us to relate ultrastructural changes elicited by ethanol with the cellular stress physiology. Under ethanol stress, the integrity of the cell membrane was compromised. The swelling or distortion of mitochondria together with the occurrence of a single and large vacuole was correlated with the addition of ethanol. These results suggested that the cell membrane is one of the targets of ethanol, and the degeneration of mitochondria promoted the accumulation of intracellular reactive oxygen species.

Biosafety evaluation of bacteriophages for treatment of diarrhea due to intestinal pathogen Escherichia coli 3-2 infection of chickens.

Intestinal Escherichia coli caused diarrhea in chicken makes serious damage directly to the chicken culture industry. Bacteriophage therapy is able to control the diarrhea in chickens effectively. In this study, the biosafety of bacteriophages was evaluated for treating intestinal pathogenic E. coli, which induced diarrhea in chickens. Ten bacteriophages were isolated from feces of chickens with diarrhea using the ill-chicken intestinal pathogenic E. coli 3-2 as target organism. Three bacteriophages propagated on E. coli 3-2 with relative big and clear plaques were selected and used together for toxicity experiment and evaluating the effect of therapy on chicken weight gain. In 3 weeks of trial, no mice given with or without mixed bacteriophages died, and the weight of mice of the experimental group did not show significant difference to the control group after 3 weeks infection. Besides remarkable decreasing the death rate of chickens with diarrhea, treatment of mixed bacteriophages also promoted the weight gain and saved the diet consumption as the utilize rate of diet increased 11% compared with the control group. These observations indicated that a mixture of three bacteriophages would be biosafe for rapid and effective preventing pathogenic E. coli infections.

Metabolic responses to ethanol in Saccharomyces cerevisiae using a gas chromatography tandem mass spectrometry-based metabolomics approach.

During the fermentation process, Saccharomyces cerevisiae cells are often inhibited by the accumulated ethanol, and the mechanism of the S. cerevisiae response to ethanol is not fully understood. In the current study, a systematic analytical approach was used to investigate the changes in the S. cerevisiae cell metabolome that were elicited by treatment with various concentrations of ethanol. Gas chromatography-mass spectrometry and a multivariate analysis were employed to investigate the ethanol-associated intracellular biochemical changes in S. cerevisiae. The intracellular metabolite profiles that were found upon treatment of the cells with different concentrations of ethanol were unique and could be distinguished with the aid of principal component analysis. Furthermore, partial least-squares-discriminant analysis revealed a group classification and pairwise discrimination between the control without ethanol and ethanol treated groups, and 29 differential metabolites with variable importance in the projection value greater than 1 were identified, which was also confirmed by the subsequent hierarchical cluster analysis. The metabolic relevance of these compounds in the response of S. cerevisiae to ethanol stress was investigated. Under ethanol stress, the glycolysis was inhibited and the use of carbon sources for fermentation was diminished, which might account for the growth inhibition of S. cerevisiae cells. It was suggested that S. cerevisiae cells change the levels of fatty acids, e.g., hexadecanoic, octadecanoic and palmitelaidic acids, to maintain the integrity of their plasma membrane through decreasing membrane fluidity in the medium containing ethanol. Moreover, the increased levels of some amino acids idemtified in the cells of ethanol-treated experimental group might also confer ethanol tolerance to S. cerevisiae. These results reveal that the metabolomics strategy is a powerful tool to gain insight into the molecular mechanism of a microorganism's cellular response to environmental stress factors.