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Sb(III) oxidation by microorganisms plays a key role in the geochemical cycling of antimony and is effective for bioremediation. A previously discovered novel Sb(III)-oxidizing bacteria, Phytobacter sp. X4, was used to elucidate the response patterns of extracellular polypeptides (EPS), antioxidant system, electron transfer and functional genes to Sb(III) under anaerobic conditions. The toxicity of Sb(III) was mitigated by increasing Sb(III) oxidation capacity, and the EPS regulated the content of each component by sensing the concentration of Sb(III). High Sb(III) concentrations induced significant secretion of proteins and polysaccharides of EPS, and polysaccharides were more important. Functional groups including hydroxyl, carboxyl and amino groups on the cell surface adsorbed Sb(III) to block its entry. Hydroxyl radicals and hydrogen peroxide were involved in anaerobic Sb(III) oxidation, as revealed by changes in the antioxidant system and electron spin resonance (EPR) techniques. qPCR confirmed that proteins concerning nitrate and antimony transfer, antimony resistance and antioxidant system were regulated by Sb(III) concentration, and the synergistic cooperation of multiple proteins conferred high antimony resistance to X4. The adaptive antimony resistance mechanism of Phytobacter sp. X4 under anaerobic conditions was revealed, which also provides a reference value for bioremediation method of antimony contamination in anaerobic environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ideonella sakaiensis is a bacterium that can degrade and consume polyethylene terephthalate (PET), a plastic material that was previously considered non-biodegradable. The degradation of PET requires two enzymes, namely poly (ethylene terephthalate) hydrolase (PETase) and mono (2-hydroxyethyl) terephthalate hydrolase (MHETase), which break down PET into terephthalate (TPA) and ethylene glycol (EG), which serve as carbon sources for the bacterium. Previous studies have focused on the enzymatic properties, structure, and mechanism of action of PETase and MHETase. However, the regulation of PETase and MHETase gene expression has not been investigated. This study identified a protein that binds to the MHETase promoter DNA, MHETase gene-regulating protein (MRP) in I. sakaiensis. PET or TPA induced the expression of PETase and MHETase genes. Furthermore, the induction of the MHETase gene was abolished by the deletion of the mrp gene, while the expression of the PETase gene was maintained. In addition, the genes involved in TPA metabolism were not induced in the mrp mutant. Furthermore, the growth of the PET and TPA deteriorated due to mrp mutation. Also, MRP binds to the promoter regions of the MHETase gene and TPA metabolizing genes, but not to the PETase gene promoter. These results suggest that MRP is a transcription factor that activates MHETase and TPA-metabolizing genes., (© 2024 Federation of European Biochemical Societies.)

Polyethylene terephthalate (PET), one of the most widely used plastics in the world, causes serious environmental problems. Recently, scientists have been focused on the enzymatic degradation of PET, an environmentally friendly method that offers an attractive approach to the degradation and recycling of PET. In this work, PET hydrolase from Streptomyces sp. W2061 was biochemically characterized, and the biodegradation of PET was performed using the PET model substrate bis (2-hydroxyethyl terephthalate) (BHET). PET hydrolase has an isoelectric point of 5.84, and a molecular mass of about 50.31 kDa. The optimum pH and temperature were 7.0 and 40°C, respectively. LC-MS analysis of the enzymatic products showed that the PET hydrolase successfully degraded a single ester bond of BHET, leading to the formation of MHET. Furthermore, in silico characterization of the PET hydrolase protein sequence and its predicted three-dimensional structure was designed and compared with the well-characterized IsPETase from Ideonella sakaiensis . The structural analysis showed that the (Gly-x1-Ser-x2-Gly) serine hydrolase motif and the catalytic triad (Ser, Asp, and His) were conserved in all sequences. In addition, we integrated molecular dynamics (MD) simulations to analyze the variation in the structural stability of the PET hydrolase in the absence and presence of BHET. These simulations showed the formation of a stable complex between the PET hydrolase and BHET. To the best of our knowledge, this is the first study on Streptomyces sp. W2061 to investigate the BHET degradation activity of PET hydrolase, which has potential application in the biodegradation of plastics in the environment.

More than 8 billion tons of plastic waste has been generated, posing severe environmental consequences and health risks. Due to prolonged exposure, microplastic particles are found in human blood and other bodily fluids. Despite a lack of toxicity studies regarding microplastics, harmful effects for humans seem plausible and cannot be excluded. As small plastic particles readily translocate from the gut to body fluids, enzyme-based treatment of serum could constitute a promising future avenue to clear synthetic polymers and their corresponding oligomers via their degradation into monomers of lower toxicity than the material they originate from. Still, whereas it is known that the enzymatic depolymerization rate of synthetic polymers varies by orders of magnitude depending on the buffer and media composition, the activity of plastic-degrading enzymes in serum was unknown. Here, we report how an engineered PETase, which we show to be generally trans -selective via induced fit docking, can depolymerize two different microplastic-like substrates of the commodity polymer polyethylene terephthalate (PET) into its non-toxic monomer terephthalic acid (TPA) alongside mono(2-hydroxyethyl)terephthalate (MHET) in human serum at 37 °C. We show that the application of PETase does not influence cell viability in vitro . Our work highlights the potential of applying biocatalysis in biomedicine and represents a first step towards finding a future solution to the problem that microplastics in the bloodstream may pose.

Background: Oxidative stress mediated by reactive oxygen species (ROS) is a common denominator in arsenic toxicity. Arsenic stress in soil affects the water absorption, decrease stomatal conductance, reduction in osmotic, and leaf water potential, which restrict water uptake and osmotic stress in plants. Arsenic-induced osmotic stress triggers the overproduction of ROS, which causes a number of germination, physiological, biochemical, and antioxidant alterations. Antioxidants with potential to reduce ROS levels ameliorate the arsenic-induced lesions. Plant growth promoting rhizobacteria (PGPR) increase the total soluble sugars and proline, which scavenging OH radicals thereby prevent the oxidative damages cause by ROS. The main objective of this study was to evaluate the potential role of Arsenic resistant PGPR in growth of maize by mitigating arsenic stress., Methodology: Arsenic tolerant PGPR strain MD3 (Pseudochrobactrum asaccharolyticum) was used to dismiss the 'As' induced oxidative stress in maize grown at concentrations of 50 and 100 mg/kg. Previously isolated arsenic tolerant bacterial strain MD3 "Pseudochrobactrum asaccharolyticum was used for this experiment. Further, growth promoting potential of MD3 was done by germination and physio-biochemical analysis of maize seeds. Experimental units were arranged in Completely Randomized Design (CRD). A total of 6 sets of treatments viz., control, arsenic treated (50 & 100 mg/kg), bacterial inoculated (MD3), and arsenic stress plus bacterial inoculated with three replicates were used for Petri plates and pot experiments. After treating with this MD3 strain, seeds of corn were grown in pots filled with or without 50 mg/kg and 100 mg/kg sodium arsenate., Results: The plants under arsenic stress (100 mg/kg) decreased the osmotic potential (0.8 MPa) as compared to control indicated the osmotic stress, which caused the reduction in growth, physiological parameters, proline accumulation, alteration in antioxidant enzymes (Superoxide dismutase-SOD, catalase-CAT, peroxidase-POD), increased MDA content, and H 2 O 2 in maize plants. As-tolerant Pseudochrobactrum asaccharolyticum improved the plant growth by reducing the oxidation stress and antioxidant enzymes by proline accumulation. PCA analysis revealed that all six treatments scattered differently across the PC1 and PC2, having 85.51% and 9.72% data variance, respectively. This indicating the efficiency of As-tolerant strains. The heatmap supported the As-tolerant strains were positively correlated with growth parameters and physiological activities of the maize plants., Conclusion: This study concluded that Pseudochrobactrum asaccharolyticum reduced the 'As' toxicity in maize plant through the augmentation of the antioxidant defense system. Thus, MD3 (Pseudochrobactrum asaccharolyticum) strain can be considered as bio-fertilizer., (© 2024. The Author(s).)

Global warming is a leading environmental stress that reduces plant productivity worldwide. Several beneficial microorganisms reduce stress; however, the mechanism by which plant-microbe interactions occur and reduce stress remains to be fully elucidated. The aim of the present study was to elucidate the mutualistic interaction between the plant growth-promoting rhizobacterial strain SH-19 and soybeans of the Pungsannamul variety. The results showed that SH-19 possessed several plant growth-promoting traits, such as the production of indole-3-acetic acid, siderophore, and exopolysaccharide, and had the capacity for phosphate solubilisation. The heat tolerance assay showed that SH-19 could withstand temperatures up to 45 °C. The strain SH-19 was identified as P. megaterium using the 16S ribosomal DNA gene sequence technique. Inoculation of soybeans with SH-19 improved seedling characteristics under high-temperature stress. This may be due to an increase in the endogenous salicylic acid level and a decrease in the abscisic acid level compared with the negative control group. The strain of SH-19 increased the activity of the endogenous antioxidant defense system, resulting in the upregulation of GSH (44.8%), SOD (23.1%), APX (11%), and CAT (52.6%). Furthermore, this study involved the transcription factors GmHSP, GmbZIP1, and GmNCED3. The findings showed upregulation of the two transcription factors GmbZIP1 (17%), GmNCED3 (15%) involved in ABA biosynthesis and induced stomatal regulation, similarly, a downregulation of the expression pattern of GmHSP by 25% was observed. Overall, the results of this study indicate that the strain SH-19 promotes plant growth, reduces high-temperature stress, and improves physiological parameters by regulating endogenous phytohormones, the antioxidant defense system, and genetic expression. The isolated strain (SH-19) could be commercialized as a biofertilizer., (© 2024. The Author(s).)

Enzymatic depolymerization of PET waste emerges as a crucial and sustainable solution for combating environmental pollution. Over the past decade, PET hydrolytic enzymes, such as PETase from Ideonella sakaiensis (IsPETases), leaf compost cutinases (LCC), and lipases, have been subjected to rational mutation to enhance their enzymatic properties. ICCM, one of the best LCC mutants, was selected for overexpression in Escherichia coli BL21(DE3) for in vitro PET degradation. However, overexpressing ICCM presents challenges due to its low productivity. A new stress-inducible T7RNA polymerase-regulating E. coli strain, ASIA hsp , which significantly enhances ICCM production by 72.8 % and achieves higher enzyme solubility than other strains. The optimal cultural condition at 30 °C with high agitation, corresponding to high dissolved oxygen levels, has brought the maximum productivity of ICCM and high PET-hydrolytic activity. The most effective PET biodegradation using crude or pure ICCM occurred at pH 10 and 60 °C. Moreover, ICCM exhibited remarkable thermostability, retaining 60 % activity after a 5-day reaction at 60 °C. Notably, crude ICCM eliminates the need for purification and efficiently degrades PET films., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The enzyme PETase from Ideonella sakaiensis ( Is PETase) strain 201-F6 can catalyze the hydrolysis of polyethylene terephthalate (PET), mainly converting it into mono(2-hydroxyethyl) terephthalic acid (MHET). In this study, we used quantum mechanics/molecular mechanics (QM/MM) simulations to explore the molecular details of the catalytic reaction mechanism of Is PETase in the formation of MHET. The QM region was described with AM1d/PhoT and M06-2 X /6-31+G(d,p) potential. QM/MM simulations unveil the complete enzymatic PET hydrolysis mechanism and identify two possible reaction pathways for acylation and deacylation steps. The barrier obtained at M06-2 X /6-31+G(d,p)/MM potential for the deacylation step corresponds to 20.4 kcal/mol, aligning with the experimental value of 18 kcal/mol. Our findings indicate that deacylation is the rate-limiting step of the process. Furthermore, per-residue interaction energy contributions revealed unfavorable contributions to the transition state of amino acids located at positions 200-230, suggesting potential sites for targeted mutations. These results can contribute to the development of more active and selective enzymes for PET depolymerization.

Three endophytic bacteria, namely BvV, BvP and BvL, were newly isolated from the root nodules of bean, pea and lentil plants respectively cultivated in Mascara the northwest of Algeria, and identified by 16S ribosomal RNA gene sequencing as Brevundimonas naejangsanensis. These strains were able to produce hydrolytic enzymes and hydrogen cyanide. All strains produced a growth-promoting hormone, indole acetic acid, varying in concentration from 83.2 to 171.7 µg/mL. The phosphate solubilizing activity of BvV, BvP and BvL varied from 25.5 to 42.02 µg/mL for tricalcium phosphate. The three antagonistic Brevundimonas spp. showed in vitro the most inhibitory effect on mycelial growth of Fusarium redolens FRC (from 78.33 to 85.55%). Strain BvV, BvP and BvL produced also volatile metabolites which inhibited mycelial FRC growth up to 39.2%. All strains showed significant disease reduction in pot experiments. Chickpea Fusarium yellows severity caused by FRC was reduced significantly from 89.3 to 96.6% in the susceptible cultivar ILC 482 treated with antagonistic B. naejangsanensis. The maximum stimulatory effect on chickpea plants growth was observed by inoculation of strain BvV. This treatment resulted in a 7.40-26.21% increase in shoot height as compared to the control plants. It is concluded that the endophytic bacterial strains of B. naejangsanensis having different plant growth promoting (PGP) activities can be considered as beneficial microbes for sustainable agriculture. To our knowledge, this is the first report to use B. naejangsanensis strains as a new biocontrol agent against F. redolens, a new pathogen of chickpea plants causing Fusarium yellows disease in Algeria., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Environmental pollution caused by plastic waste has become global problem that needs to be considered urgently. In the pursuit of a circular plastic economy, biodegradation provides an attractive strategy for managing plastic wastes, whereas effective plastic-degrading microbes and enzymes are required. In this study, we report that Blastobotrys sp. G-9 isolated from discarded plastic in landfills is capable of depolymerizing polyurethanes (PU) and poly (butylene adipate-co-terephthalate) (PBAT). Strain G-9 degrades up to 60% of PU foam after 21 days of incubation at 28 ℃ by breaking down carbonyl groups via secretory hydrolase as confirmed by structural characterization of plastics and degradation products identification. Within the supernatant of strain G-9, we identify a novel cutinase BaCut1, belonging to the esterase family, that can reproduce the same effect. BaCut1 demonstrates efficient degradation toward commercial polyester plastics PU foam (0.5 mg enzyme/25 mg plastic) and agricultural film PBAT (0.5 mg enzyme/10 mg plastic) with 50% and 18% weight loss at 37 ℃ for 48 h, respectively. BaCut1 hydrolyzes PU into adipic acid as a major end-product with 42.9% recovery via ester bond cleavage, and visible biodegradation is also identified from PBAT, which is a beneficial feature for future recycling economy. Molecular docking, along with products distribution, elucidates a special substrate-binding modes of BaCut1 with plastic substrate analogue. BaCut1-mediated polyester plastic degradation offers an alternative approach for managing PU plastic wastes through possible bio-recycling., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Synthetic plastics, like polyethylene terephthalate (PET), have become an essential part of modern life. Many of these products are remarkably persistent in the environment, and the accumulation in the environment is recognised as a major threat. Therefore, an increasing interest has been focusing on the screening for organisms able to degrade and assimilate the plastic. Ideonella sakaiensis originally isolated from a plastisphere has been reported as a bacterium that was solely thriving on the degradation on PET films. The processes affected by the presence of PET and its monomeric substances terephthalic acid, ethylene glycol, ethyl glycolate, and sodium glyoxylate monohydrate were elucidated by analysis of differential protein expression. The exposure of PET and its monomers induced the MHETase and affect two major pathways: the TCA cycle and the β-oxidation pathway. The increased expression of proteins directly or indirectly involved in these pathways suggests their underlying importance in the degradation of PET by I. sakaiensis since these proteins are mechanistically supporting the enzymes involved in the degradation of PET and its monomers., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites in IsPETase and the efficient post-translational modification in endoplasmic reticulum, IsPETase is heavily glycosylated during secretory expression with P. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of IsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated IsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated IsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of IsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable IsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories., (© 2023. The Author(s).)

The accumulation of macro-, micro- and nano-plastic wastes in the environment is a major global concern, as these materials are resilient to degradation processes. However, microorganisms have evolved their own biological means to metabolize these petroleum-derived polymers, e.g., Ideonella sakaiensis has recently been found to be capable of utilizing polyethylene terephthalate (PET) as its sole carbon source. This study aims to prove its potential capacity to biodegrade two commercial PET materials, obtained from food packaging containers. Plastic pieces of different crystallinity were simultaneously introduced to Ideonella sakaiensis during a seven-week lasting investigation. Loss in weight, appearance of plastics, as well as growth of Ideonella sakaiensis-through quantitative real-time PCR-were determined. Both plastics were found enzymatically attacked in a two-stage degradation process, reaching biodegradation capacities of up to 96%. Interestingly, the transparent, high crystallinity PET was almost fully degraded first, followed by the colored low-crystallinity PET. Results of quantitative real-time PCR-based gene copy numbers were found in line with experimental results, thus underlining its potential of this method to be applied in future studies with Ideonella sakaiensis., (© 2022. The Author(s).)

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETase Mut , one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETase Mut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L -1 purified soluble IsPETase Mut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L -1 purified soluble NusA-IsPETase Mut , which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETase Mut (GroEL/ES) displayed the same catalytic behavior as IsPETase Mut , while the fusion of NusA conferred new enzymatic properties to IsPETase Mut . Although NusA-IsPETase Mut displayed a lower initial hydrolysis capacity than IsPETase Mut , it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETase Mut . Taken together, the latter two catalytic properties of NusA-IsPETase Mut are more likely to contribute to the enhanced product release by NusA-IsPETase Mut PET degradation for two weeks., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The excessive proliferation of Microcystis aeruginosa can lead to ecological damage, economic losses, and threaten animal and human health. For controlling Microcystis blooms, microorganism-based methods have attracted much attention from researchers because of their eco-friendliness and species-specificity. Herein, we first found that a Paucibacter strain exhibits algicidal activity against M. aeruginosa and microcystin degradation capability. The algicidal activity of DH15 (2.1 × 10 4  CFU/ml) against M. aeruginosa (2 × 10 6  cells/ml) was 94.9% within 36 h of exposure. DH15 also degraded microcystin (1.6 mg/L) up to 62.5% after 72 h. We demonstrated that the algicidal activity of DH15 against M. aeruginosa can be mediated by physical attachment and indirect attack: (1) Both washed cells and cell-free supernatant could kill M. aeruginosa efficiently; (2) Treatment with DH15 cell-free supernatants caused oxidative stress, altered the fatty acid profile, and damaged photosynthetic system, carbohydrate, and protein metabolism in M. aeruginosa. The combination of direct and indirect attacks supported that strain DH15 exerts high algicidal activity against M. aeruginosa. The expression of most key genes responsible for photosynthesis, antioxidant activity, microcystin synthesis, and other metabolic pathways in M. aeruginosa was downregulated. Strain DH15, with its microcystin degradation capacity, can overcome the trade-off between controlling Microcystis blooms and increasing microcystin concentration. Our findings suggest that strain DH15 possesses great potential to control outbreaks of Microcystis blooms., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anoxygenic phototrophic bacteria display phenomenal metabolic plasticity leading to distinct phenotypes. Extracellular elevated glucose levels limit photosynthesis in photosynthetic organisms; diversely, cause oxidative stress with ROS generation and "diabetic" like situation in non-photosynthetic organisms. In this study, longer incubations of externally provided glucose (22 mM) inhibited photosynthetic machinery in a phototrophic bacterium, Rubrivivax benzoatilyticus. Data analysis at three time points- exponential, early and late stationary phase, uncovered dynamic protein and metabolite abundance implying metabolic rewiring led non-cultivable state in response to glucose. Protein dynamics datum suggested that proteins related to primary metabolism down-regulated prior to those of secondary metabolism. Numerous proteins for metabolism and energy generation were highly expressed during exponential phase whereas those for membrane transport/translocation and DNA repair accumulated at early and late stationary phase respectively, suggesting a programmed knock-off of phototrophic growth mode and a switch to non-cultivable state. Overall, the omics analyses explicated the metabolic adjustment associated with glucose grown cells of R. benzoatilyticus. Further, our investigation unravelled creation of oxidative stress suggesting physiological stress (oxygen limitation) might be a key player leading to a non-cultivable state in this phototrophic organism. The study, emphasizing microbial glucose intolerance, unlocks the doorway to perceive microorganisms with new perspective. SIGNIFICANCE: Anoxygenic photosynthetic bacteria (APB), thriving under diverse habitat, exhibits magnificent metabolic flexibility. Generally, phototrophy is the preferred growth mode and energy generating route for APB. But, our analyses implicated that the glucose, under phototrophic growth conditions, triggered photobleaching in an APB member, Rubrivivax benzoatilyticus. However, retention of growth along with pigmentation under chemotrophic growth mode supports that glucose gradually knocked off the phototrophic growth mode of R. benzoatilyticus and switched to an alternate energy driving route or less energy demanding non-cultivabile state. Thus, the change in lifestyle i.e. photoheterotrophic growth instead of chemotrophic perhaps, might be the prime culprit and key player in inducing the said state of non-cultivability, akin to diabetes. The study, shedding light on the plausible regulation of cultivability, unveils the programmed regulated switching between different growth modes of the organism and illuminates the importance of glucose intolerance by microorganisms. Through this investigation, we appeal that the studies on 'glucose intolerance in microorganisms' also need due attention that will perhaps change our outlook to perceive micro-organisms in relation to their physiological life style., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Biodegradation of polyethylene terephthalate (PET) is one of fundamental ways to solve plastic pollution. As various microbial hydrolases have an extra domain unlike PETase from Ideonella sakaiensis (IsPETase), research on the role of these extra domain in PET hydrolysis is crucial for the identification and selection of a novel PET hydrolase. Here, we report that a PET hydrolase from Burkholderiales bacterium RIFCSPLOWO2_02_FULL_57_36 (BbPETase) with an additional N-terminal domain (BbPETase AND ) shows a similar hydrolysis activity toward microcrystalline PET and a higher thermal stability than IsPETase. Based on detailed structural comparisons between BbPETase and IsPETase, we generated the BbPETase S335N/T338I/M363I/N365G variant with an enhanced PET-degrading activity and thermal stability. We further revealed that BbPETase AND contributes to the thermal stability of the enzyme through close contact with the core domain, but the domain might hinder the adhesion of enzyme to PET substrate. We suggest that BbPETase is an enzyme in the evolution of efficient PET degradation and molecular insight into a novel PET hydrolase provides a novel strategy for the development of biodegradation of PET., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the β1-β2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the β7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems., (© 2021 Wiley Periodicals LLC.)

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo , we have developed a gene disruption system in I. sakaiensis . The pT18 mobsacB -based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene ( pyrF ) from the genome of the wild-type strain, producing the Δ pyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ pyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ petase and Δ mhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δ petase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis , which was then applied for constructing Δ petase and Δ mhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis , while PETase and MHETase play essential roles in its PET assimilation.

The two novel bacterial strains designated 1Y17 T and 4Y10 T from aquaculture water were characterized using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains 1Y17 T and 4Y10 T belonged to the genus Inhella and were close to Inhella crocodyli CCP-18 T , Inhella inkyongensis IMCC1713 T and Inhella fonticola TNR-25 T . Strains 1Y17 T and 4Y10 T shared 98.6% identity with each other and less than 99.0% identity with their relatives above. The phylogenomic analysis indicated that the two strains formed two independent branches distinct from their relatives. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the two strains were 21.3 and 80.9% below the two thresholds of 70% dDDH and 95-96% ANI for species definition; those between the two novel strains and their relatives were far below thresholds for species definition, implying that they represent two novel genospecies. The predominant fatty acids of the two strains were summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and C 16:0 ; the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; the major quinone and polyamine were Q-8 and putrescine. Their genomic DNA G + C contents were 69.3 and 65.0%. The two novel strains can produce poly-β-hydroxybutyrate, matching with the presence of the three synthetic related genes of the phaC-phaA-phaB in their genomes. Based on the genotypic and phenotypic characteristics such as aesculin and gelatin hydrolysis, strains 1Y17 T and 4Y10 T are concluded to represent two novel species of the genus Inhella, for which the names Inhella proteolytica sp. nov. (type strain 1Y17 T  = GDMCC 1.1830 T  = KACC 21948 T ) and Inhella gelatinilytica sp. (type strain 4Y10 T  = GDMCC 1.1829 T  = KCTC 82338 T ) are proposed., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mitsuaria sp. TWR114 is a biocontrol agent against tomato bacterial wilt (TBW). We aimed to gain genomic insights relevant to the biocontrol mechanisms and colonization ability of this strain. The draft genome size was found to be 5,632,523 bp, with a GC content of 69.5%, assembled into 1144 scaffolds. Genome annotation predicted a total of 4675 protein coding sequences (CDSs), 914 pseudogenes, 49 transfer RNAs, 3 noncoding RNAs, and 2 ribosomal RNAs. Genome analysis identified multiple CDSs associated with various pathways for the metabolism and transport of amino acids and carbohydrates, motility and chemotactic capacities, protection against stresses (oxidative, antibiotic, and phage), production of secondary metabolites, peptidases, quorum-quenching enzymes, and indole-3-acetic acid, as well as protein secretion systems and their related appendages. The genome resource will extend our understanding of the genomic features related to TWR114's biocontrol and colonization abilities and facilitate its development as a new biopesticide against TBW., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression., (© 2021. The Author(s).)

Tomato (Solanum lycopersicum L.) is an important vegetable cultivated around the world. Under field conditions, tomato can be negatively affected by water scarcity in arid and semiarid regions. The application of native plant growth-promoting rhizobacteria (PGPR) isolated from arid environments has been proposed as an inoculant to mitigate abiotic stresses in plants. In this study, we evaluated rhizobacteria from Cistanthe longiscapa (syn Calandrinia litoralis and Calandrinia longiscapa), a representative native plant of flowering desert (FD) events (Atacama Desert, Chile), to determine their ability to reduce water scarcity stress on tomato seedlings. The isolated bacterial strains were characterized with respect to their PGPR traits, including P solubilization, 1-aminocyclopropane-1-carboxylate deaminase activity, and tryptophan-induced auxin and exopolysaccharide production. Three PGPR consortia were formulated with isolated Bacillus strains and then applied to tomato seeds, and then, the seedlings were exposed to different levels of water limitations. In general, tomato seeds and seedlings inoculated with the PGPR consortia presented significantly (P ≤ 0.05) greater plant growth (48 to 60 cm of height and 171 to 214 g of weight) and recovery rates (88 to 100%) compared with those without inoculation (37 to 51 cm of height; 146 to 197 g of fresh weight; 54 to 92% of recovery) after exposure to a lack of irrigation over different time intervals (24, 72 and 120 h) before transplantation. Our results revealed the effectiveness of the formulated PGPR consortia from FD to improve the performance of inoculated seeds and seedlings subjected to water scarcity; thus, the use of these consortia can represent an alternative approach for farmers facing drought events and water scarcity associated with climate change in semiarid and arid regions worldwide.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales ), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insoluble substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The analysis provided values of k cat and K M , as well as an apparent specificity constant in the conventional units of M -1 s -1 . These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high k cat rather than a low K M . Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, whereas the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites., (© 2020 Wiley-VCH GmbH.)

Poly(ethylene terephthalate) (PET) is one of the most commonly used plastics worldwide and its accumulation in the environment is a global problem. PETase from Ideonella sakaiensis 201-F6 was reported to exhibit higher hydrolytic activity and specificity for PET than other enzymes at ambient temperature. Enzymatic degradation of PET using PETase provides an attractive approach for plastic degradation and recycling. In this work, extracellular PETase was achieved by Escherichia coli BL21 using a Sec-dependent translocation signal peptide, pelB, for secretion. Furthermore, engineering of the pelB through random mutagenesis and screening was performed to improve the secretion efficiency of PETase. Evolved pelB enabled higher PETase secretion by up to 1.7-fold. The improved secretion of PETase led to more efficient hydrolysis of the PET model compound, bis (2-hydroxyethyl) terephthalic acid (BHET), PET powder, and PET film. Our study presents the first example of the increasing secretion of PETase by an engineered signal peptide, providing a promising approach to obtain extracellular PETase for efficient enzymatic degradation of PET.

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand., (© 2021 Elsevier Inc. All rights reserved.)

Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2 , but only the two FB strains possess the putative Fe oxidase genes mtoAB The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation. IMPORTANCE As AMD moves through the environment, it impacts aquatic ecosystems, but at the same time, these ecosystems can naturally attenuate contaminated waters via acid neutralization and catalyzing metal precipitation. This is the case in the former Ronneburg uranium-mining district, where AMD impacts creek sediments. We isolated and characterized two iron-oxidizing Thiomonas species that are mildly acidophilic to neutrophilic and that have two genetic pathways for iron oxidation. These Thiomonas species are well positioned to naturally attenuate AMD as it discharges across the landscape., (Copyright © 2020 American Society for Microbiology.)

Antimicrobial resistance is a serious threat to human health worldwide, prompting research efforts on a massive scale in search of novel antibiotics to fill an urgent need for a remedy. Teixobactin, a macrocyclic depsipeptide natural product, isolated from uncultured bacteria ( Eleftheria terrae) , displayed potent activity against several Gram-positive pathogenic bacteria. The distinct pharmacological profile and interesting structural features of teixobactin with nonstandard amino acid (three d-amino acids and l- allo -enduracididine) residues attracted several research groups to work on this target molecule in search of novel antibiotics with new mechanism. Herein, we present a comprehensive and critical perspective on immense possibilities offered by teixobactin in the domain of drug discovery. Efforts made by various research groups since its isolation are discussed, highlighting the molecule's considerable potential with special emphasis on replacement of amino acids. Critical analysis of synthetic efforts, SAR studies, and the way forward are provided hereunder.

Ideonella sakaiensis produces an enzyme, PETase, that is capable of hydrolyzing polyethylene terephthalate (PET) plastic. We demonstrate that although I. sakaiensis can grow on amorphous plastic, it does not grow on highly crystalline plastic under otherwise identical conditions. Both amorphous film and amorphous plastic obtained from commercial food containers support the growth of the bacteria, whereas highly crystalline film and the highly crystalline body of a plastic water bottle do not support growth. Highly crystalline PET can be melted and rapidly cooled to make amorphous plastic which then supports bacterial growth, whereas the same plastic can be melted and slowly cooled to make crystalline plastic which does not support growth. We further subject a plastic water bottle to a top-to-bottom analysis, finding that only amorphous sections are degraded, namely the finish (threading), the topmost portion of the shoulder which connects to the finish, and the area immediately surrounding the centre of the base. Finally, we use these results to estimate that the percentage of non-degradable plastic in plastic water bottles ranges from 52% to 82% (depending on size), demonstrating that most of the plastic found in PET water bottles will not be degraded by I. sakaiensis., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.

Regulation of the expression of the gene for chlorite dismutase (cld), located on the chlorate reduction composite transposon of the chlorate reducer Ideonella dechloratans, was studied. A 200 bp upstream sequence of the cld gene, and mutated and truncated versions thereof, was used in a reporter system in Escherichia coli. It was found that a sequence within this upstream region, which is nearly identical to the canonical FNR-binding sequence of E. coli, is necessary for anaerobic induction of the reporter gene. Anaerobic induction was regained in an FNR-deficient strain of E. coli when supplemented either with the fnr gene from E. coli or with a candidate fnr gene cloned from I. dechloratans. In vivo transcription of the suggested fnr gene of I. dechloratans was demonstrated by qRT-PCR. Based on these results, the cld promoter of I. dechloratans is suggested to be a class II-activated promoter regulated by an FNR-type protein of I. dechloratans. No fnr-type genes have been found on the chlorate reduction composite transposon of I. dechloratans, making anaerobic upregulation of the cld gene after a gene transfer event dependent on the presence of an fnr-type gene in the recipient., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Recent reports link Sutterella with gastrointestinal diseases, the most intriguing being therapeutic failure in ulcerative colitis (UC). Sutterella does not appear to induce substantial inflammation; rather, it has a capacity to degrade IgA. This activity, however, is not conserved, presenting a key target to deciphering the influence of Sutterella on the host., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Melanins are chemically diverse ubiquitous pigments found across the life forms synthesized via different biochemical pathways mainly from L-tyrosine or acetyl CoA. Though few reports suggest the possibility of tryptophan-based melanin synthesis, however, such tryptophan-based melanin and its biosynthesis remained a biochemical riddle. Here we report tryptophan-based melanin production by bacterium, Rubrivivax benzoatilyticus JA2. Aerobic cultures of strain JA2 produced brown pigment when grown on L-tryptophan-containing media. Purified pigment showed typical physico-chemical properties of melanin. Further, extensive spectroscopic studies revealed that pigment is an amorphous, indole-type polymer with stable free radical centers. Further, hydrolysis of the brown pigment revealed the presence of indole moiety, confirming the indolic nature of the pigment. Demonstration of in vitro and in vivo pigment synthesis directly from L-tryptophan or hydroxytryptophan confirms tryptophan-based melanin synthesis in strain JA2. Interestingly, canonical melanin biosynthetic inhibitors did not affect the pigment synthesis indicating possible non-canonical tryptophan-based melanin biosynthesis in strain JA2. Further, the exometabolite profiling and precursor feeding studies suggests that L-tryptophan converted to hydroxytryptophan/hydroxyindoles and their subsequent polymerization lead to the formation of melanin. The current study sheds light on biosynthetic diversity of melanins and L-tryptophan can be a potential precursor for melanin synthesis in life forms.

Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substance(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity, transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be more profound to life-forms than previously thought., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Burkholderiales are an emerging source of bioactive secondary metabolites and have the potential to be a robust chassis for metabolites from Gram-negative bacteria. However, only a few constitutive promoters can be utilized in Burkholderiales. Herein, we described the screening of strong constitutive promoters from Burkholderiales strain DSM 7029, and 37 promoters identified from transcriptome sequencing were cloned and characterized using a firefly luciferase reporter and were further verified by qPCR analysis. These promoters were then used to drive a complex 56-kb epothilone BGC from myxobacterium and a 23-kb rhizomide BGC from Paraburkholderia rhizoxinica , and the successful production of epothilone and rhizomide was observed in DSM 7029, with improved yields compared to the production achieved by previously used promoters. Additionally, these promoters are also functional in other Burkholderiales species. Thus, these promoters are highly useful for optimizing yields of important metabolites in Burkholderiales and for mining cryptic biosynthetic pathways in DSM 7029.

The cyanotoxin cylindrospermopsin was discovered during a drinking water-related outbreak of human poisoning in 1979. Knowledge about the degradation of cylindrospermopsin in waterbodies is limited. So far, only few cylindrospermopsin-removing bacteria have been described. Manganese-oxidizing bacteria remove a variety of organic compounds. However, this has not been assessed for cyanotoxins yet. We investigated cylindrospermopsin removal by manganese-oxidizing bacteria, isolated from natural and technical systems. Cylindrospermopsin removal was evaluated under different conditions. We analysed the correlation between the amount of oxidized manganese and the cylindrospermopsin removal, as well as the removal of cylindrospermopsin by sterile biogenic oxides. Removal rates in the range of 0.4-37.0 μg L -1 day -1 were observed. When MnCO 3 was in the media Pseudomonas sp. OF001 removed ∼100% of cylindrospermopsin in 3 days, Comamonadaceae bacterium A210 removed ∼100% within 14 days, and Ideonella sp. A288 and A226 removed 65% and 80% within 28 days, respectively. In the absence of Mn 2+ , strain A288 did not remove cylindrospermopsin, while the other strains removed 5-16%. The amount of manganese oxidized by the strains during the experiment did not correlate with the amount of cylindrospermopsin removed. However, the mere oxidation of Mn 2+ was indispensable for cylindrospermopsin removal. Cylindrospermopsin removal ranging from 0 to 24% by sterile biogenic oxides was observed. Considering the efficient removal of cylindrospermopsin by the tested strains, manganese-oxidizing bacteria might play an important role in cylindrospermopsin removal in the environment. Besides, manganese-oxidizing bacteria could be promising candidates for biotechnological applications for cylindrospermopsin removal in water treatment plants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microbial transformation of arsenic species and their interaction with the carbon cycle play a major role in the mobility of this toxic metalloid in the environment. The influence of simple or complex organic substrates on arsenic bio-oxidation was studied using two bacterial strains: one - the arsenivorans strain of Thiomonas delicata - is able to use AsIII as sole energy source; the other, Herminiimonas arsenicoxydans, is not. Experiments were performed at two AsIII concentrations (75 and 2 mg/L). At 75 mg/L As, for both strains, expression of aioA gene decreased when yeast extract concentration was raised from 0.2 to 1 g/L. At 2 mg/L As, the presence of either yeast extract or simple (succinate or acetate) organic substrates in the medium during bacterial growth decreased the AsIII-oxidation rate by both strains. When added specifically during oxidation test, yeast extract but not simple organic substrates seems to have a negative effect on AsIII oxidation. Taken together, results confirm the negative influence of simple or complex organic substrates on the kinetics of microbial AsIII oxidation and suggest that this effect results from different mechanisms depending on the type of organic substrate. Further, for the first time, the influence of a complex organic substrate, yeast extract, on aioA gene expression has been evidenced., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of Editors-in-Chief and first Author. The article duplicates significant parts of a paper that had already appeared in Fish & Shellfish Immunology, Volume 93 (2019) 726-731, https://doi.org/10.1016/j.fsi.2019.06.052. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process. The first author informed the journal that the article was published without the knowledge of the co-authors., (Copyright © 2019. Published by Elsevier Ltd.)

Background: The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment-a significant portion thereof in the world's oceans. In 2016, Ideonella sakaiensis, a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I. sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste., Results: Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions., Conclusion: We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P. tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.

The demand for agricultural crops continues to escalate with an increasing population. To meet this demand, marginal land can be used as a sustainable source for increased plant productivity. However, moisture stress not only affects crop growth and productivity but also induces plants' susceptibility to various diseases. The positive role of plant growth hormone, salicylic acid (SA), on the defence systems of plants has been well documented. With this in mind, a combination of plant growth promoting rhizobacteria (PGPR) and SA was used to evaluate its performance on wheat grown under rainfed conditions (average moisture 10-14%). The selected bacterial strains were characterized for proline production, indole-3-acetic acid (IAA), hydrogen cyanide (HCN), ammonia (NH3), and exopolysaccharides (EPS). Wheat seeds of two genotypes, Inqilab-91 (drought tolerant) and Shahkar-2013 (drought sensitive), which differed in terms of their sensitivity to drought stress, were soaked for three hours prior to sowing in 24-hour old cultures of the bacterial strains Planomicrobium chinense strain P1 (accession no. MF616408) and Bacillus cereus strain P2 (accession no. MF616406). SA was applied (150 mg/L), as a foliar spray on one-month-old wheat seedlings. A significant reduction in the physiological parameters was noted in the plants grown in rainfed conditions but the PGPR and SA treatment effectively ameliorated the adverse effects of moisture stress. The wheat plants treated with PGPR and SA showed significant increases in leaf protein and sugar contents and maintained higher chlorophyll content, chlorophyll fluorescence (fv/fm) and performance index (PI) under rainfed conditions. Leaf proline content, lipid peroxidation, and antioxidant enzyme activity were higher in the non-inoculated plants grown in rainfed conditions but significantly reduced in the inoculated plants of both genotypes. Integrative use of a combination of PGPR strains and SA appears to be a promising and eco-friendly strategy for reducing moisture stress in plants., Competing Interests: The authors have declared that no competing interests exist.

The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ Similar observations were made for the Methylophilaceae However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model. IMPORTANCE The metabolism of methane is an important part of the global carbon cycle. While deciphering the community function and the potential role of the different functional guilds is very difficult when considering native complex communities, synthetic communities, built of species originating from a study site in question, present a simplified model and allow specific questions to be addressed as to carbon, nitrogen, and other nutrient transfer among species in a controlled system. This study applies an ecophysiological approach, as a proof of principle, to an already well-studied model system, contributing to a better understanding of microbial community function and microbial ecosystem processes., (Copyright © 2019 Yu et al.)

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B 12 -dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases., (Copyright © 2019. Published by Elsevier Ltd.)

Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To explore the secondary metabolite biosynthetic potential of Rubrivivax benzoatilyticus JA2 using a new metabolite mining strategy., Results: Combination of precursor-feeding and altered growth conditions were used to mine new biomolecules. Strain JA2 utilised L-phenylalanine as sole source of nitrogen and showed pigments production only under phenylalanine-amended aerobic cultures. Stable isotope based precursor feeding studies indicated the blue pigment consists of 4-phenyl rings derived from L-phenylalanine. The purified blue pigment displayed characteristic visible-absorption and pH-dependent color variations. Precursor-feeding under altered growth conditions activated the plausible novel aromatic pigment production in strain JA2., Conclusion: Our approach unraveled the previously unknown pigment synthesis in strain JA2 and demonstrated the potential of mining strategy in discovering the hidden secondary metabolite repertoire in microorganisms.

The genus of Parasutterella has been defined as a core component of the human and mouse gut microbiota, and has been correlated with various health outcomes. However, like most core microbes in the gastrointestinal tract (GIT), very little is known about the biology of Parasutterella and its role in intestinal ecology. In this study, Parasutterella was isolated from the mouse GIT and characterized in vitro and in vivo. Mouse, rat, and human Parasutterella isolates were all asaccharolytic and producers of succinate. The murine isolate stably colonized the mouse GIT without shifting bacterial composition. Notable changes in microbial-derived metabolites were aromatic amino acid, bilirubin, purine, and bile acid derivatives. The impacted bile acid profile was consistent with altered expression of ileal bile acid transporter genes and hepatic bile acid synthesis genes, supporting the potential role of Parasutterella in bile acid maintenance and cholesterol metabolism. The successful colonization of Parasutterella with a single environmental exposure to conventional adult mice demonstrates that it fills the ecological niche in the GIT and contributes to metabolic functionalities. This experiment provides the first indication of the role of Parasutterella in the GIT, beyond correlation, and provides insight into how it may contribute to host health.