Simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata with glucose as carbon source under aerobic conditions.

Previous researches have recognized the vital role of Tetrasphaera elongata in enhanced biological phosphorus removal systems, but the underlying mechanisms remain under-investigated. To address this issue, this study investigated the metabolic characteristics of Tetrasphaera elongata when utilizing glucose as the sole carbon source. Results showed under aerobic conditions, Tetrasphaera elongata exhibited a glucose uptake rate of 136.6 mg/(L·h) and a corresponding phosphorus removal rate of 8.6 mg P/(L·h). Upregulations of genes associated with the glycolytic pathway and oxidative phosphorylation were observed. Noteworthily, the genes encoding the two-component sensor histidine kinase and response regulator transcription factor exhibited a remarkable 28.3 and 27.4-fold increase compared with the group without glucose. Since these genes play a pivotal role in phosphate-specific transport systems, collectively, these findings shed light on a potential mechanism for simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata under aerobic conditions, providing fresh insights into phosphorus removal from wastewaters.

Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in C. parvum biology remain un-elucidated. In this study, a member of the protein family, INS-15 of C. parvum encoded by cgd3_4260, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in Escherichia coli and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the C. parvum invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in E. coli, together with various degraded products. The cgd3_4260 gene had a peak expression at 2 h of in vitro C. parvum culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of C. parvum sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of in vitro growth indicate that INS-15 plays some roles in the invasion or early development of C. parvum.

Cloning and identification of the Frigocyclinone biosynthetic gene cluster from Streptomyces griseus strain NTK 97.

Frigocyclinone is a novel antibiotic with antibacterial and anticancer activities. It is produced by both Antarctica-derived Streptomyces griseus NTK 97 and marine sponge-associated Streptomyces sp. M7_15. Here, we first report the biosynthetic gene cluster of frigocyclinone in the S. griseus NTK 97. The frigocyclinone gene cluster spans a DNA region of 33-kb which consists of 30 open reading frames (ORFs), encoding minimal type II polyketide synthase, aromatase and cyclase, redox tailoring enzymes, sugar biosynthesis-related enzymes, C-glycosyltransferase, a resistance protein, and three regulatory proteins. Based on the bioinformatic analysis, a biosynthetic pathway for frigocyclinone was proposed. Second, to verify the cloned gene cluster, CRISPR-Cpf1 mediated gene disruption was conducted. Mutant with the disruption of beta-ketoacyl synthase encoding gene frig20 fully loses the ability of producing frigocyclinone, while inactivating the glycosyltransferase gene frig1 leads to the production of key intermediate of anti-MRSA anthraquinone tetrangomycin.

LmbU, a Cluster-Situated Regulator for Lincomycin, Consists of a DNA-Binding Domain, an Auto-Inhibitory Domain, and Forms Homodimer.

Few studies were reported about the regulatory mechanism of lincomycin biosynthesis since it was found in 1962. Although we have proved that a cluster-situated regulator (CSR) LmbU (GenBank Accession No. ABX00623.1) positively modulates lincomycin biosynthesis in Streptomyces lincolnensis NRRL 2936, the molecular mechanism of LmbU regulation is still unclear. In this study, we demonstrated that LmbU binds to the target lmbAp by a central DNA-binding domain (DBD), which interacts with the binding sites through the helix-turn-helix (HTH) motif. N-terminal of LmbU includes an auto-inhibitory domain (AID), inhibiting the DNA-binding activity of LmbU. Without the AID, LmbU variant can bind to its own promoter. Interestingly, compared to other LmbU homologs, the homologs within the biosynthetic gene clusters (BGCs) of known antibiotics generally contain N-terminal AIDs, which offer them the abilities to play complex regulatory functions. In addition, cysteine 12 (C12) has been proved to be mainly responsible for LmbU homodimer formation in vitro. In conclusion, LmbU homologs naturally exist in hundreds of actinomycetes, and belong to a new regulatory family, LmbU family. The present study reveals the DBD, AID and dimerization of LmbU, and sheds new light on the regulatory mechanism of LmbU and its homologs.

Structure and function of microbial community associated with phenol co-substrate in degradation of benzo[a]pyrene in coking wastewater.

Coking wastewater (CWW) contains high contents of phenols and other toxic and refractory compounds including polycyclic aromatic hydrocarbons (PAHs) with the most carcinogenic benzo[a]pyrene (BaP) among them. The mechanism of PAHs/BaP degradation in activated sludge of CWW treatment with phenol as co-substrate was studied. For characterizing the structure and functions of microbial community associated with BaP degradation with phenol as co-substrate, high-throughput MiSeq sequencing was used to examine the 16S rRNA genes of microbiology, revealing noticeable shifts in CWW activated sludge bacterial populations. Major genera involved in anaerobic degradation were Tissierella_Soehngenia, Diaphorobacter and Geobacter, whereas in aerobic degradation Rhodanobacter, Dyella and Thauera prevailed. BaP degradation with phenol as co-substrate induced bacterial diversification in CWW activated sludge in opposite trends when anaerobic and aerobic conditions were applied. In order to predict the microbial community functional profiling, a bioinformatics software package of phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was run to find that some dominant genera enriched in the BaP pathway may own the ability to degrade PAHs/BaP. Further experiments should focus on testing the dominant genera in BaP degradation at different oxygen levels.

Expression dynamics of gonadotropin-releasing hormone-I and its mutual regulation with luteinizing hormone in chicken ovary and follicles.

Gonadotropin-releasing hormone-I (GnRH-I) has been identified in the ovaries of vertebrate species, and this decapeptide is a key regulator of reproductive functions. However, its biological action and regulatory mechanism in the chicken ovary remain to be characterized. In this study, the expression of GnRH-I gene in chicken hypothalamus and ovaries at different developmental stages and different sizes of follicles was investigated, and the effect of GnRH-I mRNA on chicken follicular cells was analyzed in vitro. The results showed that the expression of GnRH-I was dramatically decreased in the hen ovary compared to that in the hypothalamus after sexual maturation. In the mature ovarian follicles, GnRH-I mRNA levels were significantly higher in theca cells than that in granulosa cells. Overexpression of GnRH-I decreased the expression of luteinizing hormone receptor (LHR) mRNA in theca cells from preovulatory follicles but had no effect on granulosa cells. Treatment of theca cells with different concentrations of luteinizing hormone (LH) significantly increased GnRH-I mRNA expression at low doses (50 ng/ml) but significantly decreased it at higher doses (200 ng/ml). Furthermore, GnRH-I inhibited LH-induced LHR expression at the lower dose of LH (50 ng/ml). These findings provide strong evidence indicating that GnRH-I is an important regulator in the chicken ovary.

Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128.

Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.

[Evaluation of Advanced Nitrogen Removal from Coking Wastewater Using Sulfide Iron-containing Sludge as a Denitrification Electron Donor].

In general, it is difficult to reach the total nitrogen discharge standard in the effluent after municipal and industrial wastewater treatment. The problems hindering advanced denitrification include an unstable C/N ratio in the influent wastewater, increased hydraulic loading with increasing reflux ratio, reduced reaction kinetics, high energy consumption, and secondary pollution and high sludge yield resulting from addition of organic carbon sources. Therefore, deep denitrification with the advantages of energy savings and easy operation is urgently needed. To address these issues, chemical iron sulfide sludge, collected after the pretreatment of sulfur-containing industrial wastewater, was used as a solid-phase electron donor to perform advanced denitrification using autotrophic denitrifiers. In this study, the secondary biological effluent of coking wastewater was the influent for denitrification and the performance of denitrification, transformation of sulfide and iron in the sludge, and microbial community changes were investigated. The optimal reaction conditions and effect range of the technology for deep denitrification of wastewater were then calculated. When the concentrations of NO3--N and NO2--N in the influent were (74.54±0.57) and (1.11±0.19) mg·L-1, respectively, the corresponding concentrations in the effluent were reduced to (2.78±1.08) and (2.87±0.71) mg·L-1, respectively, with a hydraulic retention time (HRT) of 18 h. The removal rate of TON (NO3--N+NO2--N) was as high as 90.0%, of which the reduction rate of NO3--N and the accumulation rate of NO2--N were 12.06 and 7.74 mmol·(L·d)-1, respectively. This study showed that the use of chemical sulfide iron sludge as an electron donor for deep denitrification is of practical importance, as it could simplify the subsequent treatment of sulfur- and iron-rich chemical sludge, finally reaching the goal of resource utilization.

Correction to: The mineralization of oxalic acid and bio-treated coking wastewater by catalytic ozonation using nickel oxide.

In Table 1, "SO2-4" and "NO-3" should be corrected to "[Formula: see text]" and "[Formula: see text]", respectively. The original article was corrected.

The mineralization of oxalic acid and bio-treated coking wastewater by catalytic ozonation using nickel oxide.

Coking wastewater after biological treatment still possesses potential environmental risk and should be mineralized further. This work focused on the mineralization of bio-treated coking wastewater using catalytic ozonation by NiO. First, oxalic acid, the typical by-product of advanced oxidation process (AOPs), was used to test the catalytic performance of NiOs, prepared by modified hydrothermal methods upon addition of different surfactants. This demonstrated that NiO upon addition of hexadecyltrimethylammonium (CTAB) had the best catalytic activity, due to its high concentration surface hydroxyl density and strong stability. Moreover, the best NiO was applied for the catalytic ozonation of bio-treated coking wastewater. Under our experimental conditions, the total organic carbon (TOC) removal reached 100% after 420 min. In addition, the spectroscopic analysis suggested that compounds with conjugated structures could be significantly removed by both ozonation and catalytic ozonation. Some of these substances were transformed into by-products with aliphatic C-C and O=C-O groups such as organic acids that can inhibit further mineralization.

[Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A2+(A1-A2)/(1+(x/x0)p) (A1=1.28; A2=-0.066; x0=12560.75; p=0.74) with a correlation coefficient (R2) of 0.997. The lowest detectable limit was 0.65 µg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

Distribution and migration of heavy metals in soil and crops affected by acid mine drainage: Public health implications in Guangdong Province, China.

Acid mine drainages (AMD) contain high concentrations of heavy metals, and their discharges into streams and rivers constitute serious environmental problems. This article examines the effects of AMD on soil, plant and human health at Dabaoshan mine in Guangdong Province, China. Although the large scale mining was stopped in 2011, the heavy metal pollution in soil continues to endanger crops and human health in that region. The objectives of this study were to elucidate distribution and migration of Cd, Cu, Zn, As and Pb and associated health implications to local inhabitants. We collected and analyzed 74 crop samples including 28 sugarcane, 30 vegetables, 16 paddy rice and the corresponding soil samples, used correlation and linear relationship for transformation process analysis, and applied carcinogenic and non-carcinogenic risk for hazard evaluation. Results showed that the local soils were heavily polluted with Cd, Cu and As (especially for Cd) and the mean Igeo value was as high as 3.77. Cadmium, Cu, and Zn in rice and vegetables were comparable with those found four years ago, while As and Pb in edible parts were 2 to 5 times lower than before. The root uptake of Cd and Zn contributed mainly to their high concentrations in crops due to high exchangeable fraction of soil, while leafy vegetables accumulated elevated As and Pb contents mainly due to the atmospheric deposition. Metal concentrations in sugarcane roots were higher than those in rice and vegetable roots. The risk assessment for crops consumption showed that the hazard quotients values were of 21 to 25 times higher than the threshold level for vegetables and rice, indicating a potential non-carcinogenic risk to the consumers. The estimated mean total cancer risk value of 0.0516 more than 100 times exceeded the USEPA accepted risk level of 1×10(-4), indicating unsuitability of the soil for cultivating the food crops. Therefore, the local agricultural and the land-use policies need to be reevaluated.

Novel insights into anoxic/aerobic(1)/aerobic(2) biological fluidized-bed system for coke wastewater treatment by fluorescence excitation-emission matrix spectra coupled with parallel factor analysis.

Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) was applied to investigate the contaminant removal efficiency and fluorescent characteristic variations in a full scale coke wastewater (CWW) treatment plant with a novel anoxic/aerobic(1)/aerobic(2) (A/O(1)/O(2)) process, which combined with internal-loop fluidized-bed reactor. Routine monitoring results indicated that primary contaminants in CWW, such as phenols and free cyanide, were removed efficiently in A/O(1)/O(2) process (removal efficiency reached 99% and 95%, respectively). Three-dimensional excitation-emission matrix fluorescence spectroscopy and PARAFAC identified three fluorescent components, including two humic-like fluorescence components (C1 and C3) and one protein-like component (C2). Principal component analysis revealed that C1 and C2 correlated with COD (correlation coefficient (r)=0.782, p<0.01 and r=0.921, p<0.01), respectively) and phenols (r=0.796, p<0.01 and r=0.914, p<0.01, respectively), suggesting that C1 and C2 might be associated with the predominating aromatic contaminants in CWW. C3 correlated with mixed liquor suspended solids (r=0.863, p<0.01) in fluidized-bed reactors, suggesting that it might represent the biological dissolved organic matter. In A/O(1)/O(2) process, the fluorescence intensities of C1 and C2 consecutively decreased, indicating the degradation of aromatic contaminants. Correspondingly, the fluorescence intensity of C3 increased in aerobic(1) stage, suggesting an increase of biological dissolved organic matter.

Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment.

The use of a recombinant bacterial vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live bacterial vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) vector and found one, P(viuB), with characteristics suitable for our use. We fused P(viuB) with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-ß-d-thiogalactopyranoside)-inducible P(T7)-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in bacterial vector vaccine applications.

QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda.

The inter-kingdom communication with the mammalian hosts mediated by autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE), and transduced by two-component systems QseBC has recently been described. As a fish pathogen and opportunistic pathogen for human beings, Edwardsiella tarda develops surface structures such as flagellar and fimbriae to cause different hemagglutination phenotypes and serotypes and initiate pathogen-host recognition and invasion process. E. tarda survives within macrophages in fish using type III secretion system (TTSS). Here, the genes of E. tarda two-component system, qseB and qseC, were found to be co-transcribed. Phylogenetic analysis indicated that evolution of QseC strongly correlated to different host niches. Compared with the wild type and their complemented strains, ΔqseB and ΔqseC mutants exhibited significant impaired flagellar motilities. Mammalian Epi was able to stimuli the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in ΔqseB but repressed in ΔqseC. Disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements EseB and EsaC, and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence-related surface structures (flagellum, fimbriae and secretion system) and that hormone might stimulate the virulence of the pathogen in fish.

Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish.

Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA-EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.

Characterization of Edwardsiella tarda rpoS: effect on serum resistance, chondroitinase activity, biofilm formation, and autoinducer synthetases expression.

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant rpoS and the complemental strain rpoS (+). Compared with the wild type and rpoS (+), rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.

PCR-RFLP to detect codon 248 mutation in exon 7 of p53 tumor suppressor gene.

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of p53 tumor suppressor gene. MspI restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or single nucleotide polymorphisms in individual genomes.

Cloning and characterization of the ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10.

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.

Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1.

Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1. angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP. In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V. anguillarum serotype O1 strain MVM425. A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant. The angE-encoded protein was overexpressed in E. coli and purified by a three-step procedure. Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5'-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture. Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V. anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.