Detoxification of tetracycline and synthetic dyes by a newly characterized Lentinula edodes laccase, and safety assessment using proteomic analysis.

Fungal laccase has strong ability in detoxification of many environmental contaminants. A putative laccase gene, LeLac12, from Lentinula edodes was screened by secretome approach. LeLac12 was heterogeneously expressed and purified to characterize its enzymatic properties to evaluate its potential use in bioremediation. This study showed that the extracellular fungal laccase from L. edodes could effectively degrade tetracycline (TET) and the synthetic dye Acid Green 25 (AG). The growth inhibition of Escherichia coli and Bacillus subtilis by TET revealed that the antimicrobial activity was significantly reduced after treatment with the laccase-HBT system. 16 transformation products of TET were identified by UPLC-MS-TOF during the laccase-HBT oxidation process. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that LeLac12 could completely mineralize ring-cleavage products. LeLac12 completely catalyzed 50 mg/L TET within 4 h by adding AG (200 mg/L), while the degradation of AG was above 96% even in the co-contamination system. Proteomic analysis revealed that central carbon metabolism, energy metabolism, and DNA replication/repair were affected by TET treatment and the latter system could contribute to the formation of multidrug-resistant strains. The results demonstrate that LeLac12 is an efficient and environmentally method for the removal of antibiotics and dyes in the complex polluted wastewater.

Quantitative proteomic analysis of the microbial degradation of 3-aminobenzoic acid by Comamonas sp. QT12.

A mab cluster associated with 3-aminobenzoic acid (3AB) degradation was identified in Comamonas sp. QT12. However, the cellular response of Comamonas sp. QT12 to 3-aminobenzoic acid remains unclear. In this study, label-free quantitative proteome analysis based on LC-MS/MS was used to study the protein expression difference of strain QT12 under the condition of using 3AB (3AB) and citric acid/ammonium chloride as substrates (3ABCon). A total of 2068 proteins were identified, of which 239 were significantly up-regulated in 3AB group, 124 were significantly down-regulated in 3AB group, 624 were expressed only in 3AB group, and 216 were expressed only in 3ABCon group in 3AB group. KEGG pathway analysis found that 83 pathways were up-regulated and 49 pathways were down-regulated, In GO analysis, 315 paths were up-regulated and 156 paths were down-regulated. There were 6 genes in the mab cluster that were only detected in the 3AB group.The mab cluster was found to be related to degradation of 3AB. By knockout, it was found that the growth rate of the mutant △orf7 and △orf9 were slowed down. HPLC results showed that the mutant △orf7 and △orf9 could still degrade 3AB, it was found that orf7, orf9 were not key genes about 3AB degradation and they could be replaced by other genes in strain QT12. These findings improve our understanding of the molecular mechanisms underlying the cellular response of 3AB degradation in Comamonas bacterium.

Whole Genome Sequence of an Edible Mushroom Stropharia rugosoannulata (Daqiugaigu).

Stropharia rugosoannulata, also known as Daqiugaigu in China, is a well-known edible mushroom that has been widely cultivated in China in recent years. Many studies have focused on its nutrients, bioactive compounds, and lignin degradation capacity, although there are few molecular and genetic breeding studies due to the lack of genomic information. Here, we present the 47.9 Mb genome sequence of an S. rugosoannulata monokaryotic strain (A15), which has 20 contigs and an N50 of 3.64 Mb, which was obtained by a combination of Illumina and Nanopore sequencing platforms. Further analysis predicted 12,752 protein-coding genes, including 486 CAZyme-encoding genes. Phylogenetic analysis revealed a close evolutionary relationship between S. rugosoannulata and Hypholoma sublateritium, Psilocybe cyanescens, and Galerina marginata based on single-copy orthologous genes. Proteomic analysis revealed different protein expression profiles between the cap and the stipe of the S. rugosoannulata fruiting body. The proteins of the stipe associated with carbon metabolism, energy production, and stress-response-related biological processes had higher abundance, whereas proteins involved in fatty acid synthesis and mRNA splicing showed higher expression in the cap than in the stipe. The genome of S. rugosoannulata will provide valuable genetic resources not only for comparative genomic analyses and evolutionary studies among Basidiomycetes but also for alleviating the bottlenecks that restrict the molecular breeding of this edible mushroom.

Impact of Cold Weather on Setup Errors in Radiotherapy.

Objective: To investigate the influence of cold weather on setup errors of patients with chest and pelvic disease in radiotherapy. Methods: The image-guided data of the patients were collected from the Radiotherapy Center of Cancer Hospital Affiliated to Guangxi Medical University from October 2020 to February 2021. During this period, the cold weather days were December 15, 16, and 17, 2020, and January 7 and 8, 2021. For body fixation in radiotherapy, an integrated plate and a thermoplastic mold were employed in 18 patients with chest disease, while an integrated plate and a vacuum pad were applied in 19 patients with pelvic disease. All patients underwent cone beam computed tomography (CBCT) scans in the first five treatments and once a week thereafter. The obtained data were registered to the planning CT image to get the setup errors of the patient in the translational direction including X, Y, and Z axes and rotational direction including R X , R Y , and R Z . Then, the Mann-Whitney U test was performed. The expansion boundary values of the chest and pelvis were calculated according to the formula M PTV=2.5∑+0.7δ. Results: A total of 286 eligible results of CBCT scans were collected. There were 138 chest CBCT scans, including 26 taken in cold weather and 112 in usual weather, and 148 pelvic CBCT scans, including 33 taken in cold weather and 115 in usual weather. The X-, Y-, and Z-axis translational setup errors of patients with chest disease in the cold weather group were 0.16 (0.06, 0.32) cm, 0.25 (0.17, 0.52) cm, and 0.35 (0.21, 0.47) cm, respectively, and those in the usual weather group were 0.14 (0.08, 0.29) cm, 0.23 (0.13, 0.37) cm, and 0.18 (0.1, 0.35) cm, respectively. The results indicated that there was a statistical difference in the Z-axis translational error between the cold weather group and the usual weather group (U = 935.5; p=0.005 < 0.05), while there was no statistical difference in the rotational error between the two groups. The external boundary values of X, Y, and Z axes in the cold weather group were 0.57 cm, 0.92 cm, and 0.99 cm, respectively, and those in the usual weather group were 0.57 cm, 0.78 cm, and 0.68 cm, respectively. There was no significant difference in the translational and rotational errors of patients with pelvic disease between the cold weather group and the usual weather group (p < 0.05). The external boundary values of X, Y, and Z axes were 0.63 cm, 0.79 cm, and 0.68 cm in the cold weather group and 0.61 cm, 0.79 cm, and 0.61 cm in the usual weather group, respectively. Conclusion: The setup error of patients undergoing radiotherapy with their bodies fixed by an integrated plate and a thermoplastic mold was greater in cold weather than in usual weather, especially in the ventrodorsal direction.

Label-Free Quantitative Proteomic Analysis of the Global Response to Indole-3-Acetic Acid in Newly Isolated Pseudomonas sp. Strain LY1.

Indole-3-acetic acid (IAA), known as a common plant hormone, is one of the most distributed indole derivatives in the environment, but the degradation mechanism and cellular response network to IAA degradation are still not very clear. The objective of this study was to elucidate the molecular mechanisms of IAA degradation at the protein level by a newly isolated strain Pseudomonas sp. LY1. Label-free quantitative proteomic analysis of strain LY1 cultivated with IAA or citrate/NH4Cl was applied. A total of 2,604 proteins were identified, and 227 proteins have differential abundances in the presence of IAA, including 97 highly abundant proteins and 130 less abundant proteins. Based on the proteomic analysis an IAA degrading (iad) gene cluster in strain LY1 containing IAA transformation genes (organized as iadHABICDEFG), genes of the ß-ketoadipate pathway for catechol and protocatechuate degradation (catBCA and pcaABCDEF) were identified. The iadA, iadB, and iadE-disrupted mutants lost the ability to grow on IAA, which confirmed the role of the iad cluster in IAA degradation. Degradation intermediates were analyzed by HPLC, LC-MS, and GC-MS analysis. Proteomic analysis and identified products suggested that multiple degradation pathways existed in strain LY1. IAA was initially transformed to dioxindole-3-acetic acid, which was further transformed to isatin. Isatin was then transformed to isatinic acid or catechol. An in-depth data analysis suggested oxidative stress in strain LY1 during IAA degradation, and the abundance of a series of proteins was upregulated to respond to the stress, including reaction oxygen species (ROS) scavenging, protein repair, fatty acid synthesis, RNA protection, signal transduction, chemotaxis, and several membrane transporters. The findings firstly explained the adaptation mechanism of bacteria to IAA degradation.

Cloning and heterologous expression of a novel halo/alkali-stable multi-domain xylanase (XylM18) from a marine bacterium Marinimicrobium sp. strain LS-A18.

Halophilic bacteria are good bioresources for halotolerant alkaline enzymes. A multi-domain high-molecular-weight endo-ß-1,4-xylanase gene, xylM18, was cloned from a halophilic marine bacterium Marinimicrobium sp. LS-A18. XylM18 is different from any of the functionally reported xylanases. It has a glycosyl hydrolase (GH) 43 domain, a GH10 domain, and two serine-rich linkers, representing a novel family. The gene, encoding 1022 residues, was cloned and heterologously expressed in Escherichia coli BL21(DE3) cells. Purified XylM18 was proved to be a xylanase. It showed diminished activity without salt and showed activity with a broad NaCl range from 0.2 to 25% (w/v). NaCl can increase the optimal temperature from 30 °C (0% NaCl) to 50 °C (10% NaCl). The purified XylM18 was active between pH 6.0 and 10.0 and was optimally active at pH 7.0. The xylanase activities were basically unchanged at a NaCl concentration range from 10 to 20% or pH from 7 to 10 after 24 h incubation. The apparent Km and Vmax values of XylM18 for xylan were 2.76 mg/mL and 60.0 U/mg, respectively. The GH10 domain of this enzyme, XylM18-GH10, was expressed and characterized. XylM18-GH10 also showed xylanase activity and maintained halo-stable property. The apparent Km and Vmax values of XylM18-GH10 for xylan were 1.60 mg/mL and 130.1 U/mg, respectively. Other domains of XylM18 showed no xylanase activity. In summary, XylM18 is a halo-tolerant and alkali-stable endoxylanase which is a suitable candidate for xylan biodegradation in high-salt and alkali conditions. To our knowledge, this is the first report of a multidomain high-molecular-weight xylanase.

Isolation of a 3-hydroxypyridine degrading bacterium, Agrobacterium sp. DW-1, and its proposed degradation pathway.

A 3-hydroxypyridine degrading bacterium, designated strain DW-1, was isolated from petroleum contaminated soil in Liao River China. 16S rRNA-based phylogenetic analysis indicates that strain DW-1 belongs to genus Agrobacterium. The optimal cultivation temperature and pH for strain DW-1 with 3-hydroxypyridine were 30 °C and 8.0, respectively. Under optimal conditions, strain DW-1 could completely degrade up to 1500 mg/L of 3-hydroxypyridine in 66 h. The 3-hydroxypyridine degradation pathway of strain DW-1 was suggested by HPLC and LC-MS analysis. The first reaction of 3-hydroxypyridine degradation in strain DW-1 was α-hydroxylation so that the major metabolite 2,5-dihydroxypyridine was produced, and then 2,5-dihydroxypyridine was transformed by a Fe2+-dependent dioxygenase to form N-formylmaleamic acid. N-Formylmaleamic acid will be transformed to maleic acid and fumaric acid through maleamic acid. This is the first report of the 3-hydroxypyridine degradation pathway and the utilization of 3-hydroxypyridine by a Agrobacterium sp. It may be potentially used for the bioremediation of environments polluted with 3-hydroxypyridine.

Microbial Degradation of Nicotinamide by a Strain Alcaligenes sp. P156.

A novel Alcaligenes sp. strain P156, which can utilize nicotinamide as its sole source of carbon, nitrogen and energy, was enriched and isolated from soil in a solid waste treatment plant. Aerobic growth and degradation with nicotinamide were characterized. Seven nicotinamide degradation-related genes were obtained by sequence alignment from the genome sequence of strain P156. Four genes, designated naaA, naaD, naaE and naaF, were cloned and heterologously expressed. Nicotinamide degradation is initiated by deamination to form nicotinic acid catalyzed by the nicotinamidase NaaA, which shares highest amino acid sequence identity (27.2%) with nicotinamidase from Arabidopsis thaliana. Nicotinic acid is converted to 6-hydroxynicotinic acid, which is further oxidized to 2,5-dihydroxypyridine (2,5-DHP). 2,5-DHP is then transformed to a ring-cleavage product, N-formylmaleamic acid, by an Fe2+ dependent dioxygenase NaaD. N-formylmaleamic acid is transformed to fumaric acid through maleamic acid and maleic acid by NaaE and NaaF, respectively. To our knowledge, this is the first report of the complete microbial degradation of nicotinamide in bacteria. Nicotinamide is considered as a model compound for the study of microbial degradation of pyridinic compounds, and the nicotinamide degrading related genes in strain P156 were distributed differently from the reported similar gene clusters. Therefore, this study contribute to the knowledge on the degradation of pyridinic compounds.

A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74 µM and 6.49 ± 0.17 s-1, respectively, and those for NADH were 189.85 ± 55.70 µM and 7.41 ± 1.39 s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.

Novel Gene Encoding 5-Aminosalicylate 1,2-Dioxygenase from Comamonas sp. Strain QT12 and Catalytic Properties of the Purified Enzyme.

The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. FeII is required for the catalytic activity of the purified enzyme. The apparent Km and Vmax values of MabB for 5ASA are 52.0 ± 5.6 µM and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (∼70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.