Biosynthesis of photostable CdS quantum dots by UV-resistant psychrotolerant bacteria isolated from Union Glacier, Antarctica.

BACKGROUND: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported. RESULTS: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O2 for the formation of reactive oxygen species (ROS) at different levels. CONCLUSIONS: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation.

Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations.

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, ß endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with ß-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.

The mechanism of DEHP degradation by the combined action of biochar and Arthrobacter sp. JQ-1: Mechanisms insight from bacteria viability, degradation efficiency and changes in extracellular environment.

Di(2-ethylhexyl) phthalate (DEHP) has been widely detected in soil, water, and sediment as a priority control pollutant. Immobilized microorganism technology is gradually mature and applied in production. Biochar prepared from agricultural wastes is an excellent immobilized carrier because of its porous structure and abundant functional groups. Environmental acidification was caused by degrading bacteria Arthrobacter sp. JQ-1 (JQ-1) respiration and acidic metabolites during DEHP degradation, which affected the passage life of microorganisms and the removal efficiency of DEHP. The mechanism of DEHP degradation by the combined action of JQ-1 and corn straw biochar (BC) at 600 °C was investigated, and bacterial viability, microenvironmental changes, and kinetic tests were performed in this research. Compared with biodegradation group alone, the degradation rate of DEHP in 1% biochar unloaded and loaded with JQ-1 increased by 18.3% and 30.9%, and its half-life decreased to 23.90 h and 11.95h, a reduction of 31.37 h. The percentage of detected living JQ-1 increased as biochar content increased when loading capacity was less than 1%. In which, (JQ-1-BC2) group was 4.1% higher than (JQ-1-BC1) group. Biochar has the ability to neutralize acidifying environmental pH due to its alkaline functional groups, including lactone group, -OH, -COO-. 1% biochar loaded with JQ-1 increased the pH of the microenvironment by 0.57 and alkaline phosphatase (AKP) activity by 0.0063 U·mL-1, which promoted the reduction of PA. Study suggested that biochar loaded with JQ-1 could simultaneously adsorb and degrade DEHP during the process of DEHP removal. Biochar could be used as a biological stimulant to increase abundance and metabolism, enhance the utilization of DEHP by JQ-1. Biochar (1% (w/v)) loaded with JQ-1 as DEHP removal material showed good performance. Biochar not only as an immobilized carrier, but also as a biostimulant, providing an effective strategy for the collaborative remediation of PAEs contaminated.

Potential and prospects of Actinobacteria in the bioremediation of environmental pollutants: Cellular mechanisms and genetic regulations.

Increasing industrialization and anthropogenic activities have resulted in the release of a wide variety of pollutants into the environment including pesticides, polycyclic aromatic hydrocarbons (PAHs), and heavy metals. These pollutants pose a serious threat to human health as well as to the ecosystem. Thus, the removal of these compounds from the environment is highly important. Mitigation of the environmental pollution caused by these pollutants via bioremediation has become a promising approach nowadays. Actinobacteria are a group of eubacteria mostly known for their ability to produce secondary metabolites. The morphological features such as spore formation, filamentous growth, higher surface area to volume ratio, and cellular mechanisms like EPS secretion, and siderophore production in Actinobacteria render higher resistance and biodegradation ability. In addition, these bacteria possess several oxidoreductase systems (oxyR, catR, furA, etc.) which help in bioremediation. Actinobacteria genera including Arthrobacter, Rhodococcus, Streptomyces, Nocardia, Microbacterium, etc. have shown great potential for the bioremediation of various pollutants. In this review, the bioremediation ability of these bacteria has been discussed in detail. The utilization of various genera of Actinobacteria for the biodegradation of organic pollutants, including pesticides and PAHs, and inorganic pollutants like heavy metals has been described. In addition, the cellular mechanisms in these microbes which help to withstand oxidative stress have been discussed. Finally, this review explores the Actinobacteria mediated strategies and recent technologies such as the utilization of mixed cultures, cell immobilization, plant-microbe interaction, utilization of biosurfactants and nanoparticles, etc., to enhance the bioremediation of various environmental pollutants.

Microbial degradation of organochlorine pesticide: 2,4-Dichlorophenoxyacetic acid by axenic and mixed consortium.

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D), an organochlorine herbicide, in the environment has raised public concern as it poses hazard to both humans and the ecosystem. Three potential strains having the capability to degrade 2,4-D were isolated from on site agricultural soil and identified as Arthrobacter sp. SVMIICT25, Sphingomonas sp. SVMIICT11 and Stenotrophomonas sp. SVMIICT13. Over 12 days of incubation, 81-90% of 100 mg/L of 2,4-D degradation was observed at 2% inoculum. A shorter lag phase with 80% of degradation efficiency was observed within 5 days when the inoculum size was increased to 10%. Six microbial consortia were prepared by combining the isolates along with in-house strains, Bacillus sp. and Pseudomonas sp. Consortia R3 (Arthrobacter sp. + Sphingomonas sp.), operated with 10% of inoculum, showed 85-90% degradation within 4 days and 98-100% in 9 days. Further, targeted exo-metabolite analysis confirmed the presence and catabolism of intermediate 2,4-dichlorophenol and 4-chlorophenol compounds.

Mitigation of atrazine-induced oxidative stress on soybean seedlings after co-inoculation with atrazine-degrading bacterium Arthrobacter sp. DNS10 and inorganic phosphorus-solubilizing bacterium Enterobacter sp. P1.

Atrazine toxicity is one of the limiting factors inhibiting sensitive plant growth. Previous studies showed that atrazine-degrading bacteria could alleviate atrazine toxicity. However, there is limited information on how atrazine-degrading bacteria and plant growth-promote bacteria alleviate atrazine toxicity in soybeans. Therefore, the current study aimed to explore the atrazine removal, phosphorus utilization, and the oxidative stress alleviation of atrazine-degrading bacterium Arthrobacter sp. DNS10 and/or inorganic phosphorus-solubilizing bacterium Enterobacter sp. P1 in the reduction of atrazine toxicity in soybean. The results showed that atrazine exposure to soybean seedlings led to significant inhibition in growth, atrazine removal, and phosphorus utilization. However, the co-inoculatied strains significantly increased seedlings biomass, chlorophyll a/b contents, and total phosphorus in leaves accompanied by great reduction of the atrazine-induced antioxidant enzymes activities and malonaldehyde (MDA) contents, as well as atrazine contents in soil and soybeans under atrazine stress. Furthermore, transcriptome analysis highlighted that co-inoculated strains increased the expression levels of genes related to photosynthetic-antenna proteins, carbohydrate metabolism, and fatty acid degradation in leaves. All the results suggest that the co-inoculation mitigates atrazine-induced oxidative stress on soybean by accelerating atrazine removal from soil and phosphorus accumulation in leaves, enhancing the chlorophyll contents, and regulating plant transcriptome. It may be suggested that co-inoculation of atrazine-degrading bacteria and inorganic phosphorus-solubilizing bacteria can be used as a potential method to alleviate atrazine toxicity to the sensitive crops.

Noval bio-organic fertilizer containing Arthrobacter sp. DNS10 alleviates atrazine-induced growth inhibition on soybean by improving atrazine removal and nitrogen accumulation.

Herbicide atrazine restricts nutrient accumulation and thus inhibits the growth of sensitive crops. The application of organic fertilizer is a common measure that contributes to modulating abiotic tolerance of crops and providing nutrients, but its advantages in combination with atrazine degrading microorganisms as bio-organic fertilizer to alleviate atrazine stress on sensitive crops and the associated mechanisms are unknown. We investigated the beneficial effects of organic and bio-organic fertilizer (named DNBF10) containing Arthrobacter sp. DNS10 applications on growth, leaf nitrogen accumulation, root surface structure and root physiological properties of soybean seedlings exposed to 20 mg kg-1 atrazine in soil. Compared with organic fertilizer, bio-organic fertilizer DNBF10 exhibited more reduction in soil atrazine residue and plant atrazine accumulation, as well as alleviation in atrazine-induced root oxidative stress and damaged cells of soybean roots. Transcriptome analysis revealed that DNBF10 application enhanced nitrogen utilization by improving the expression of genes involved in nitrogen metabolism in soybean leaves. Besides, genes expression of cytochrome P450 and ABC transporters involved in atrazine detoxification and transport in soybean leaves were also down-regulated by DNBF10 to diminish phytotoxicity of atrazine to soybean seedlings. These results illustrate the molecular mechanism by which the application of DNBF10 alleviates soybean seedlings growth under atrazine stress, providing a step forward for mitigate the atrazine induced inhibition on soybean seedlings growth through decreasing atrazine residues as well as enhancing damaged root repair and nitrogen accumulation.

Growth, biofilm formation and atrazine degrading gene (trzN) expression of Arthrobacter sp. DNS10 cultured with montmorillonite, kaolinite and goethite.

The viable and degradation potential of the strains which adhered to soil minerals are essential for eliminating organic pollutants from soil. Herein, the interaction (growth, biofilm formation and survive) of Arthrobacter sp. DNS10, an atrazine degrading strain, with three kinds of typical soil minerals, such as montmorillonite, kaolinite and goethite, as well as the atrazine degradation gene (trzN) expression of the strain in the minerals system were studied. The results showed that montmorillonite had significant promotion effect on the growth of strain DNS10, followed by kaolinite, but goethite significantly inhibited the growth of strain DNS10. In contrast, goethite notably promoted the biofilm formation and there was less biofilm detected in montmorillonite containing system. The percentage of the survival bacteria in the biofilm that formed on montmorillonite, kaolinite and goethite was 53.8%, 40.8% and 28.2%. In addition, there were more reactive oxygen species (ROS) were detected in the cells that exposed to goethite than those of the cells exposed to kaolinite and montmorillonite. These results suggest that the electrostatic repulsion between kaolinite/montmorillonite and strain DNS10 prevents them from contacting each other and facilitates bacterial growth by allowing the strain to obtain more nutrients. Oppositely, the needle-like morphology of goethite might damage the strain DNS10 cell when they were combined by electrostatic attraction, and the goethite induced ROS also aggravate the cytotoxicity of goethite on strain DNS10. In addition, the relative transcription of trzN in the cells contacted with montmorillonite, kaolinite and goethite was 0.94-, 0.27- and 0.20- fold of the no mineral exposure treatment. Briefly, this research suggests that the minerals with different structure and/or physicochemical characteristics might cause various trend for the biofilm formation and degradation potential of the bacteria.

Potential and mechanism for bioremediation of papermaking black liquor by a psychrotrophic lignin-degrading bacterium, Arthrobacter sp. C2.

To meet the challenge of bioremediation of black liquor in pulp and paper mills at low temperatures, a psychrotrophic lignin-degrading bacterium was employed in black liquor treatment for the first time. In this study, Arthrobacter sp. C2 exhibited excellent cold adaptability and lignin degradation ability, with a lignin degradation rate of 65.5% and a mineralization rate of 43.9% for 3 g/L lignin at 15 °C. Bioinformatics analysis and multiple experiments confirmed that cold shock protein 1 (Csp1) was the dominant cold regulator of strain C2, and dye-decolorizing peroxidase (DyP) played a crucial role in lignin degradation. Moreover, structural equation modeling (SEM), mRNA monitoring, and phenotypic variation analysis demonstrated that Csp1 not only mediated cold adaptation but also modulated DyP activity by controlling dyp gene expression, thus driving lignin depolymerization for strain C2 at low temperatures. Furthermore, 96.4% of color, 64.2% of chemical oxygen demand (COD), and 100% of nitrate nitrogen (NO3--N) were removed from papermaking black liquor by strain C2 within 15 days at 15 °C. This study provides insights into the association between the cold regulator and catalytic enzyme of psychrotrophic bacteria and offers a feasible alternative strategy for the bioremediation of papermaking black liquor in cold regions.

Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures.

Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.

Arthrobacter terrae sp. nov., a psychrophilic actinobacterium with multi copies of capA gene isolated from Antarctic soil.

A Gram-staining-positive, non-spore-forming, non-flagellated, ellipsoidal, strain Z1-20 T belonging to the genus Arthrobacter was isolated from a soil sample collected from the Zhongshan station, Antarctic. Phylogenetic analysis of the 16S rRNA gene sequences and phylogenetic analysis revealed that strain Z1-20 T formed a unique single cluster in the genus Arthrobacter and shared high 16S rRNA sequence similarities of 97.1% and 96.9% with A. glacialis HLT2-12-2 T and A. psychrochitiniphilus GP3T, respectively. Values of Digital DNA-DNA hybridization (dDDH) between strain Z1-20 T against A. glacialis HLT2-12-2 T and A. psychrochitiniphilus GP3T were 20.3% and 13.8%, respectively. Average nucleotide identity (ANI) score between strain Z1-20 T against A. glacialis HLT2-12-2 T and A. psychrochitiniphilus GP3T were 72.5% and 72.1%, respectively. Genes for the synthesis of the osmoprotectant glycine betaine and higher copies of capA gene encoding cold shock protein were found in genome of Z1-20 T that may help Z1-20 T in cold-adaptation. Strain Z1-20 T comprised lysine as the diagnostic diamino acid. Based on the results of phylogenetic, phenotypic and chemotaxonomic features, strain Z1-20 T represents a novel species of a novel taxon of genus Arthrobacter, for which the name Arthrobacter terrae gen. nov., sp. nov. is proposed.

A novel organophosphate hydrolase from Arthrobacter sp. HM01: Characterization and applications.

Bioremediation systems coupled to efficient microbial enzymes have emerged as an attractive approach for the in-situ removal of hazardous organophosphates (OPs) pesticides from the polluted environment. However, the role of engineered enzymes in OPs-degradation is rarely studied. In this study, the potential OPs-hydrolase (opdH) gene (Arthrobacter sp. HM01) was isolated, cloned, expressed, and purified. The recombinant organophosphate hydrolase (ropdH) was âˆ¼29 kDa; which catalyzed a broad-range of OPs-pesticides in organic-solvent (∼99 % in 30 min), and was found to increase the catalytic efficiency by 10-folds over the native enzyme (kcat/Km: 107 M-1s-1). The degraded metabolites were analyzed using HPLC/GCMS. Through site-directed mutagenesis, it was confirmed that, conserved metal-bridged residue (Lys-127), plays a crucial role in OPs-degradation, which shows âˆ¼18-folds decline in OPs-degradation. Furthermore, the catalytic activity and its stability has been enhanced by >2.0-fold through biochemical optimization. Thus, the study suggests that ropdH has all the required properties for OPs bioremediation.

Genome sequences of Arthrobacter spp. that use a modified sulfoglycolytic Embden-Meyerhof-Parnas pathway.

Sulfoglycolysis pathways enable the breakdown of the sulfosugar sulfoquinovose and environmental recycling of its carbon and sulfur content. The prototypical sulfoglycolytic pathway is a variant of the classical Embden-Meyerhof-Parnas (EMP) pathway that results in formation of 2,3-dihydroxypropanesulfonate and was first described in gram-negative Escherichia coli. We used enrichment cultures to discover new sulfoglycolytic bacteria from Australian soil samples. Two gram-positive Arthrobacter spp. were isolated that produced sulfolactate as the metabolic end-product. Genome sequences identified a modified sulfoglycolytic EMP gene cluster, conserved across a range of other Actinobacteria, that retained the core sulfoglycolysis genes encoding metabolic enzymes but featured the replacement of the gene encoding sulfolactaldehyde (SLA) reductase with SLA dehydrogenase, and the absence of sulfoquinovosidase and sulfoquinovose mutarotase genes. Excretion of sulfolactate by these Arthrobacter spp. is consistent with an aerobic saprophytic lifestyle. This work broadens our knowledge of the sulfo-EMP pathway to include soil bacteria.

Degradation of antibacterial 1-octylpyrrolidin-2-one by bacterial pairs isolated from river water and soil.

The study of bacterial degradation of 1-octylpyrrolidin-2-one (NOP) by river water and soil bacteria was the main aim of the research. Although the compound demonstrated bacteriostatic as well as bactericidal effects against Gram-positive and certain Gram-negative bacteria at concentrations ranging from 100 to 1000 mg L-1, its concentration of 100 mg L-1 was successfully degraded by microbial communities of both river water and alluvial soil; removal efficiencies reached 87.2 and 88.4% of dissolved organic carbon, respectively. Isolation of the strains responsible for the process showed that bacterial degradation was initiated by the octane-utilising bacteria of the genus Phenylobacterium, which used four carbon atoms of the NOP octyl chain and oxidised terminal carbon atom of the remaining chain. The structure of the intermediate produced by phenylobacteria was elucidated following the results obtained from the detailed electrospray mass spectrometry (ESI-MS) analysis; these experiments showed that it is a 4-(2-oxopyrrolidin-1-yl)butanoic acid. This intermediate was further degraded by other bacterial members of appropriate microbial communities, namely Bordetella petrii and Arthrobacter sp. Further tests proved that these bacteria were able to assimilate the nitrogen atom of the lactam ring and thus complete the degradation process.

Co-metabolic biodegradation of 4-bromophenol in a mixture of pollutants system by Arthrobacter chlorophenolicus A6.

Brominated phenols are listed as priority pollutants together with nitrophenol and chlorophenol are the key components of paper pulp wastewater. However, the biodegradation of bromophenol in a mixed substrate system is very scanty. In the present investigation, simultaneous biodegradation kinetics of three substituted phenols 4-bromophenol (4-BP), 4-nitrophenol (4-NP), and 4-chlorophenol (4-CP) were investigated using Arthrobacter chlorophenolicus A6. A 23 full factorial design was applied with varying 4-BP and 4-CP from 75-125 mg/L and 4-NP from 50-100 mg/L. Almost complete degradation of this mixture of substituted phenols was achieved at initial concentration combinations of 125, 125, and 100 mg/L of 4-CP, 4-BP, and 4-NP, respectively, in 68 h. Statistical analysis of the results revealed that, among the three variables, 4-NP had the most prominent influence on the degradation of both 4-CP and 4-BP, while the concentration of 4-CP had a strong negative interaction effect on the biodegradation of 4-NP. Irrespective of the concentration levels of these three substrates, 4-NP was preferentially biodegraded over 4-CP and 4-BP. Furthermore, 4-BP biodegradation rates were found to be higher than those of 4-CP, followed by 4-NP. Besides, the variation of the biomass yield coefficient of the culture was investigated at different initial concentration combinations of these substituted phenols. Although the actinomycetes consumed 4-NP at a faster rate, the biomass yield was very poor. This revealed that the microbial cells were more stressed when grown on 4-NP compared to 4-BP and 4-CP. Overall, this study revealed the potential of A. chlorophenolicus A6 for the degradation of 4-BP in mixed substrate systems.

Siderophores provoke extracellular superoxide production by Arthrobacter strains during carbon sources-level fluctuation.

Superoxide and other reactive oxygen species (ROS) shape microbial communities and drive the transformation of metals and inorganic/organic matter. Taxonomically diverse bacteria and phytoplankton produce extracellular superoxide during laboratory cultivation. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (A. QXT-31) secreted a type of siderophore [deferoxamine (DFO)], which provoked extracellular superoxide production by A. QXT-31 during carbon sources-level fluctuation. Several other siderophores also demonstrated similar effects to A. QXT-31. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbon sources metabolism by A. QXT-31) ETC and superoxide production. Considering that most aerobes secrete siderophore(s) and undergo carbon sources-level fluctuation, the superoxide-generation pathway is likely a common pathway by which aerobes produce extracellular superoxide in the environment, thus influencing the microbial community and cycling of elements. Our results pointed that the ubiquitous siderophore might be the potential driving force for the microbial generation of superoxide and other ROS and revealed the important role of iron physiology in microbial ROS generation.

Rationally engineered chitin deacetylase from Arthrobacter sp. AW19M34-1 with improved catalytic activity toward crystalline chitin.

Chitin and its derivatives have anticoagulant, antimicrobial, and antioxidant properties, but the poor solubility of chitin limits its application in different fields. In this study, site-directed mutagenesis was performed to enhance the deacetylation activity of chitin deacetylases CDA from Arthrobacter (ArCE4). The mutant Mut-2-8 with Y172E/E200S/Y201W showed a 2.84- fold and 1.39-fold increase in catalytic efficiency (kcat/Km) for the deacetylation of (GluNAc)5 and α-chitin, respectively. These results demonstrated that the mutations significantly improved the activation of ArCE4 on crystalline chitin. The molecular docking study confirmed that the enhancement of catalytic efficiency is due to the extra two hydrogen bonds and one acetyl group. In summary, the activity of Mut-2-8 to insoluble chitin was significantly improved by reactional design, which is beneficial to resolve the issues of the expensive cost of the enzymes and low efficiency. Mut-2-8 exhibits potential applications in the chitosan industry.

A Cold-Shock Protein from the South Pole-Dwelling Soil Bacterium Arthrobacter sp. Confers Cold Tolerance to Rice.

Low temperature is a critical environmental factor restricting the physiology of organisms across kingdoms. In prokaryotes, cold shock induces the expression of various genes and proteins involved in cellular processes. Here, a cold-shock protein (ArCspA) from the South Pole-dwelling soil bacterium Arthrobacter sp. A2-5 was introduced into rice, a monocot model plant species. Four-week-old 35S:ArCspA transgenic rice plants grown in a cold chamber at 4 °C survived for 6 days. Cold stress significantly decreased the chlorophyll content in WT plants after 4 days compared with that in 35S:ArCspA transgenic plants. RNA-seq analysis was performed on WT and 35S:ArCspA transgenic rice with/without cold stress. GO terms such as "response to stress (GO:0006950)", "response to cold (GO:0009409)", and "response to heat (GO:0009408)" were significantly enriched among the upregulated genes in the 35S:ArCspA transgenic rice under normal conditions, even without cold-stress treatment. The expression of five cold stress-related genes, Rab16B (Os11g0454200), Rab21 (Os11g0454300), LEA22 (Os01g0702500), ABI5 (Os01 g0859300), and MAPK5 (Os03g0285800), was significantly upregulated in the transgenic rice compared with the WT rice. These results indicate that the ArCspA gene might be involved in the induction of cold-responsive genes and provide cold tolerance.

Enhanced ATP and antioxidant levels for cAMP biosynthesis by Arthrobacter sp. CCTCC 2013431 with polyphosphate addition.

OBJECTIVES: When citrate and pyruvate were utilized to strengthen ATP generation for high cAMP production, oxidative stress became more severe in cells resulting in lower cell viability and cAMP formation at the late fermentation phase. To further improve cAMP biosynthesis, the effects of polyphosphate on cAMP fermentation performance together with intracellular ATP and oxidation levels were investigated under high oxidative stress condition and then high efficient cAMP fermentation process based on polyphosphate and salvage synthesis was developed and studied. RESULTS: With 2 g/L-broth sodium hexametaphosphate added at 24 h was determined as the optimal condition for cAMP production by Arthrobacter sp. CCTCC 2013431 in shake flasks. Under high oxidative stress condition caused by adding 15 mg/L-broth menadione, cAMP contents and cell viability were improved greatly due to hexametaphosphate addition and also exceeded those of control (without hexametaphosphate and menadione added) when fermentations were conducted in a 7 L bioreactor. Meanwhile, ATP levels and antioxidant capacity were improved obviously by hexametaphosphate as well. Moreover, a fermentation process with hexametaphosphate and hypoxanthine coupling added was developed by which cAMP concentration reached 7.25 g/L with an increment of 87.1% when compared with only hypoxanthine added batch and the high ROS contents generated from salvage synthesis were reduced significantly. CONCLUSION: Polyphosphate could improve intracellular ATP levels and antioxidant capacity significantly under high oxidative stress condition resulting in enhanced cell viability and cAMP fermentation production no matter by de novo synthesis or salvage synthesis.

Enhanced endogenous amino acids and energy metabolism level for cAMP biosynthesis by Arthrobacter sp. CCTCC 2013431 with citrate as cosubstrate.

OBJECTIVES: In our previous study, citrate was used as auxiliary energy substance for improving cAMP fermentation performance, however, the regulation mechanism of citrate on improved cAMP contents was not clear. To elucidate the regulation mechanism, cAMP fermentations with/without citrate addition were conducted in a 7 L fermentor using Arthrobacter sp. CCTCC 2013431 and assays on key enzymes activities, energy metabolism level, amino acids contents and peroxidation level were performed. RESULTS: With 3 g/L-broth sodium citrate added, cAMP concentration and conversion yield from glucose reached 4.34 g/L and 0.076 g/g which were improved by 30.7% and 29.8%, respectively, when compared with those of control. Citrate changed carbon flux distribution among different routes and more carbon flux was directed into pentose phosphate pathway beneficial to cAMP synthesis. Meanwhile, energy metabolism together with precursor amino acids levels were improved significantly owing to strengthened metabolic intensity of tricarboxylate cycle by exogenous citrate utilization which provided energy and substance basis for cAMP production. Moreover, higher glutamate synthesis and oxidative stress caused by citrate addition consumed excessive NADPH derived from pentose phosphate pathway by which feedback suppression for pentose phosphate pathway was relieved efficiently. CONCLUSION: Citrate promoted cAMP fermentation production by Arthrobacter sp. CCTCC 2013431 due to enhanced precursor amino acids, energy metabolism level and relieved feedback suppression for pentose phosphate pathway.