Promoter analysis of TLR5M and TLR5S revealed NF-κB might be a conserved cis-element in TLR5S promoter of large yellow croaker.

Toll like receptor 5 (TLR5) plays a crucial role in the innate immune response by recognizing bacterial flagellin proteins. In the present study, the genomic and 5'-flanking sequences of LcTLR5M (membrane) and LcTLR5S (soluble) were cloned from the large yellow croaker, Larimichthys crocea. Then, their promoter activities were determined in human embryonic kidney (HEK293T) cells. LcTLR5M contained 4 exons and 3 introns, and LcTLR5S contained 2 exons and 1 intron. Bioinformatic prediction of transcription factor binding sites (TFBSs) indicated that the promoter structures were different between LcTLR5M and LcTLR5S. A dual luciferase assay showed that the deletion mutant -471 to +189 of LcTLR5M promoter possessed the greatest activity with 36 times activity of the control (P < 0.05). For LcTLR5S, two deletion mutants, -1687 to +106 and - 720 to +106, showed high promoter activity. Furthermore, site-directed mutation demonstrated that a -392 to -391 AT/GG substitution in Oct-1 binding site, and a -104 to -103 GG/TT and a -98 to -97 CC/AA substitution in the NF-κB binding site of TLR5S caused a significant decline of luciferase activity (P < 0.05). Moreover, the co-transfection of an NF-κB/p65 over-expression plasmid with wild type TLR5S (-720 to +106) resulted in an extremely significant increase of promoter activity by more than 9 times compared with the NF-kB mutant (P < 0.01). Our findings suggest that the genomic organization and promoter structure may differ between LcTLR5M and LcTLR5S. Oct1 and NF-κB binding sites might be cis-regulatory elements in the LcTLR5S promoter. NF-κB/p65 plays an important role in LcTLR5S promoter activation through binding with the NF-κB binding site.

Formation mechanism of organo-chromium (III) complexes from bioreduction of chromium (VI) by Aeromonas hydrophila.

Chromium is a common heavy metal widely present in aquatic environments. Cost-effective remediation of chromium-contaminated environment can be realized by microbial reduction of Cr(VI) to Cr(III). The genus Aeromonas species is one of such Cr(VI) reducers, whose reduction mechanism remains unrevealed and the main factors governing the Cr(VI) reduction pathways are unknown yet. In this work, the performances and mechanisms of Cr(VI) anaerobic reduction by Aeromonas hydrophila ATCC 7966 were investigated. This strain exhibited excellent Cr(VI) resistance and could utilize a suite of electron donors to support Cr(VI) bioreduction. The Cr(VI) bioreduction processes involved both extracellular (the metal-reducing and respiratory pathway) and intracellular reaction pathways. Adding anthraquinone-2,6-disulfonate or humic acid as a mediator substantially enhanced the Cr(VI) bioreduction. The forms and distribution of the Cr(VI) bioreduction products were affected by the medium composition. Soluble organo-Cr(III) complexes were identified as the main Cr(VI) reduction products when basal salts medium was adopted. Given the environmental ubiquity of the genus Aeromonas, the findings in this work may facilitate a better understanding about the transformation behaviors and fates of Cr(VI) in environments and provide useful clues to tune the bioremediation of chromium-contaminated environments.

Continuous degradation of ciprofloxacin in a manganese redox cycling system driven by Pseudomonas putida MnB-1.

Ciprofloxacin (CIP), as an extensively used antibiotic, has been widely detected at a high level in the environment and has raised environmental pollution concerns. Thus, efficient and cost-effective methods for CIP degradation are highly desired. Biologically produced manganese oxides (BioMnOx) offer a promising perspective for CIP degradation because of their catalytic reactivity and cost-effectiveness. However, the release of Mn(II) from BioMnOx prevents the further oxidation of pollutants. As a consequence, continuous CIP degradation by BioMnOx is not feasible. In this work, a manganese redox cycling system driven by Pseudomonas putida MnB-1 was constructed for continuous degradation of CIP. In such a system CIP was oxidized continuously and rapidly by re-oxidizing the formed Mn(II) to regenerate reactive BioMnOx, which also protected the strain from CIP toxicity. CIP was degraded through N-dealkylation passway. No significant loss of BioMnOx reactivity was observed in three-cycle CIP degradation process, suggesting the stability of this system. An overlooked intracellular BioMnOx, which was involved in CIP degradation, was discovered in P. putida MnB-1. Moreover, the important role of Mn(III) in facilitating CIP removal in this system was also identified. This work provides useful information to better understand the degradation of antibiotic compounds mediated by microbes in environments.

Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation.

Dissimilatory metal reducing bacteria (DMRB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, which are used as external electron acceptors by DMRB for their anaerobic respiration. The EET process has important contribution to environmental remediation mineral cycling, and bioelectrochemical systems. However, the low EET efficiency remains to be one of the major bottlenecks for its practical applications for pollutant degradation. In this work, Shewanella oneidensis MR-1, a model DMRB, was used to examine the feasibility of enhancing the EET and its biodegradation capacity through genetic engineering. A flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in S. oneidensis MR-1. Compared to the control strain, the engineered strain was found to exhibit an improved EET capacity in microbial fuel cells and potentiostat-controlled electrochemical cells, with an increase in maximum current density by approximate 110% and 87%, respectively. The electrochemical impedance spectroscopy (EIS) analysis showed that the current increase correlated with the lower interfacial charge-transfer resistance of the engineered strain. Meanwhile, a three times more rapid removal rate of methyl orange by the engineered strain confirmed the improvement of its EET and biodegradation ability. Our results demonstrate that coupling of improved synthesis of mediators and metal-reducing conduits could be an efficient strategy to enhance EET in S. oneidensis MR-1, which is essential to the applications of DMRB for environmental remediation, wastewater treatment, and bioenergy recovery from wastes.

Cloning and functional characterization of IRAK4 in large yellow croaker (Larimichthys crocea) that associates with MyD88 but impairs NF-κB activation.

As crucial signaling transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling pathway, IL-1R-associated kinase 4 (IRAK4) mediates downstream signaling cascades and plays important roles in innate and adaptive immune responses. In the present study, an IRAK4 orthologue was characterized from large yellow croaker (Larimichthys crocea), named Lc-IRAK4, with a conservative N-terminal death domain and a C-terminal protein kinase domain. The genome of Lc-IRAK4 is structured into eleven exons and ten introns. Expression analysis indicated that Lc-IRAK4 was widely expressed in tested tissues, with the highest level in liver and weakest in muscle. Additionally, in the spleen, liver tissues and blood, it could be induced by poly I:C and LPS stimulation, but not be induced by Vibrio parahemolyticus infection. Fluorescence microscopy assays revealed that Lc-IRAK4 localized in the cytoplasm in HEK 293T cells. It was also determined that Lc-IRAK4 could interact with MyD88, whereas MyD88-mediated NF-κB activation was significantly impaired when co-transfected the two in HEK 293T cells. These findings collectively indicated that although Lc-IRAK4 was evolutionarily conserved in vertebrates, the exact function especially the signaling transduction mediated by IRAK4 in fish immune response was different from that in mammals, which impaired MyD88-mediated NF-κB activation.

Cloning and expression analysis of interferon regulatory factor (IRF) 3 and 7 in large yellow croaker, Larimichthys crocea.

The interferon regulatory factor (IRF)3 and IRF7 are considered to play essential roles in innate immune system's antiviral responses. In this report, the full-length cDNA and genomic structure and immune response characterizations of IRF3 and IRF7 were investigated in large yellow croaker, Larimichthys crocea. The full-length cDNA of L. crocea (Lc)IRF3 was of 2204 bp, including a 5'-terminal untranslated region (UTR) of 41 bp, a 3'-terminal UTR of 774 bp and an open reading frame (ORF) of 1389 bp encoding a polypeptide of 462 amino acids residues. The full-length cDNA of LcIRF7 was of 1979 bp, including a 5'-terminal UTR of 47 bp, a 3'-terminal UTR of 636 bp and an ORF of 1296 bp encoding a polypeptide of 431 amino acids. The putative amino acid sequence of both LcIRF3 and LcIRF7 contained a typical IRF domain at the N-terminal and an IRF3 domain at the C-terminal. Furthermore, we obtained 4517 nucleotides (nt) LcIRF3 genome sequence based on the full-length cDNA, which contained 11 exons and 10 introns. The full-length genome sequence of LcIRF7 was of 3991 nucleotides, including 9 exons and 8 introns. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of LcIRF3 and LcIRF7 with the most predominant expression of LcIRF3 and LcIRF7 in the liver and in the gill, respectively. The expression levels of LcIRF3 and LcIRF7 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were tested in blood, spleen and liver. The results showed that the highest relative expression of LcIRF3 was in the liver at 24 h after poly I:C injection with 90 times greater than that of the non-injection group (p < 0.05). Moreover, LcIRF3 transcription increased significantly at most time point in blood and spleen tissue after poly I:C stimulation compared with that of the control group. After LPS injection, the peak value of LcIRF7 was in the liver with 207 times (at 3 h) as much as that in the control group (p < 0.05). In addition, LcIRF7 expression was significantly induced by poly I:C injection in spleen. Both LcIRF3 and LcIRF7 transcripts did not show significant change after V. parahaemolyticus stimulation. These results indicated that IRF3 and IRF7 might play an important role in large yellow croaker's defense against viral and bacterial infection.

Characterization of Toll-like receptor 3 gene in large yellow croaker, Pseudosciaena crocea.

Toll-like receptor 3 (TLR3) plays an important role in innate immune responses. In this report, the full-length cDNA sequence and genomic structure of Pseudosciaena crocea TLR3 (PcTLR3) were identified and characterized. The full-length cDNA of PcTLR3 was of 3384 bp, including a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 589 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The full-length genome sequence of PcTLR3 was composed of 5721 nucleotides, including five exons and four introns. The putative PcTLR3 protein contained a signal peptide sequence, 16 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR3 in most tissues, with the predominant expression in liver, then intestine, and the weakest expression in blood cells. The expression of PcTLR3 after injection with poly inosinic:cytidylic (I:C) and Vibrio parahemolyticus was tested in spleen, blood cells and liver. The results indicated that PcTLR3 transcripts could be induced in the three tissues by injection with poly I:C. The highest expression was in the blood cells with 43.5 times (at 6h) greater expression than in the control (p<0.05). In addition, after V. parahemolyticus challenge, a moderate up-regulation and down-regulation of PcTLR3 was found in blood cells and liver, respectively. Our results suggested that PcTLR3 might play an important role in fish's defense against both viral and bacterial infection.

Molecular cloning and expression of IRF1 in large yellow croaker, Pseudosciaena crocea.

The interferon regulatory factor (IRF) family is known to be crucial in mediating the host defense against pathogen infection by binding to characteristic elements in promoters within interferon (IFN) genes and IFN-inducible genes. In this report, the full-length cDNA of IRF1 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1667 bp, including a 5'-terminal untranslated region (UTR) of 142 bp, a 3'-terminal UTR of 674 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acids. The putative amino acid sequence contained a typical IRF domain at the N-terminal. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of IRF1 in most detected tissues, with the predominant expression in the gill and spleen and the weakest expression in the brain. The expression of IRF1 after challenged with LPS and poly I:C was tested in blood, spleen and liver, which showed that IRF1 changed obviously with the most significantly up-regulated expression was 37 times (at 6 h) after injection with poly I:C in the blood and 13 times (at 3 h) after injection with LPS in the liver compared with the control values (p < 0.01). These results indicated that as a crucial factor in regulating the IFN and IFN-inducible elements in mammals, IRF1 might play an important role in large yellow croaker defense against the pathogen infection.