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We prompted to characterize a wastewater bacterium, Pseudoxanthomonas mexicana GTZY, that efficiently transforms toxic mercury and arsenic, explores its bioremediation capability, and reveals their relevant gene resistance operons. The isolated strain was characterized by its phylogenetic, biochemical, and phenotypic properties. The strain GTZY potentially removed 84.3% of mercury and their mercury volatilization (Hg(II) to Hg(0)) was confirmed using the X-ray film method, and its respective merA gene was PCR amplified. In addition, strain GTZY efficiently removed arsenate (68.5%) and arsenite (63.2%), and showed resistance up to > 175 and > 55 mM, respectively. Their genomic annotations disclosed the linkage of Tn2-transposon and int1 in both ends of mer operon (merAPTR). The co-existence of arsP and arsH proteins in its intrinsic ars operon (arsCPRH) was extremely diverse from its ancestral species. We believe that the mercury resistance-conferring mer operon of P. mexicana GTZY presumably derived horizontally from other species in the reactor, while the arsenic resistance-conferring intrinsic ars operon was highly diversified and evolved from its ancestral species. By considering the potential of the strain GTZY to transform heavy metals, this can be used to recover contaminated sites.

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OBJECTIVES: Elucidating the recent evolutionary history of clinically important antibiotic resistance genes may inform measures to delay the future emergence of additional resistance genes in clinics. This study investigated the recent origin of blaAIM-1, a metallo-ß-lactamase gene found in Pseudomonas aeruginosa, and the possible role of ISCR15 in its mobilisation and transfer into clinical species. METHODS: Comparative genomics were used to identify the recent origin of blaAIM. Mobilisation attempts were performed under different conditions by cloning ISCR15 and the blaAIM-1-like gene in Escherichia coli. RESULTS: Several blaAIM-1 homologues were identified in the Pseudoxanthomonas genus, with conserved synteny of the locus between species and absence of elements associated with mobility. The closest AIM-1 homologue (97.7% amino acid identity) was found in a Pseudoxanthomonas mexicana (P. mexicana) strain. Cloning the blaAIM-like gene in Escherichia coli resulted in high resistance towards carbapenems. While blaAIM-1 is surrounded by ISCR15 elements in clinical strains, in vitro experiments failed to demonstrate their role as mobilising elements. CONCLUSIONS: This study presents evidence that P. mexicana, an environmental species occasionally associated with infections, is the origin of the B3 metallo-ß-lactamase AIM-1. The presence of terIS, a plausible recognition site for ISCR15, in other parts of the P. mexicana genome suggests a more complex and yet not understood mobilisation mechanism.

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The emergence of antibiotic resistance in retort to environmental pollutants during wastewater treatment still remains elusive. Here, we first to investigate the emergence of antibiotic resistance in an environmental non-pathogenic bacterium, Pseudoxanthomonas mexicana isolated from a lab-scale bioreactor treating wastewater containing streptomycin. The molecular mechanism of antibiotic resistance development was evaluated in its genomic, transcriptional, and proteomic levels. The streptomycin resistant (SR) strain showed strong resistance to streptomycin (MIC > 600 µg/mL) as well to sulfamethoxazole, ampicillin, and kanamycin (≥250 µg/mL). A 13.4 kb class-1-integron array consisting of a new arrangement of gene cassette (IS6100-sul1-aadA2-catB3-aacA1-2-aadB-int1-IS256-int) linked with Tn5393c transposon was identified in the SR strain, which has only been reported in clinical pathogens so far. iTRAQ-LC-MS/MS proteomics revealed 22 up-regulated proteins in the SR strain growing under 100 mg L-1 streptomycin, involving antibiotic resistance, toxin production, stress response, and ribosomal protein synthesis. At the mRNA level, elevated expressions of ARGs (strA, strB, and aadB) and 30S-ribosomal protein genes (rpsA and rpsU) were observed in the SR strain. The results highlighted the genomic plasticity and multifaceted regulatory mechanism employed by P. mexicana in adaptation to high-level streptomycin during biological wastewater treatment.

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Pseudoxanthomonas indica H32, constitutes the active component of a promising bioproduct candidate with biopesticide and biofertilizing activities. However, suitable fermentation conditions should be established to manufacture this product. Accordingly, the aim of this study was to optimize the fermentation conditions of bacterium Pseudoxanthomonas indica H32 in a 3 L bioreactor. The relevant parameters for strain growth, temperature (25-40 °C) and pH (6-9), were optimized using the response surface methodology (RSM). Additionally, the influence of air flow (0.5-1.5 vvm) and impeller stirrer speed (4.17-14.17 s - 1) in the volumetric oxygen transfer coefficient (KLa) were evaluated and then, in the P. indica H32 growth. The response variables were dry cell weight (DCW) and specific growth rate (µ). The results showed that P. indica H32 growth was significantly affected by the culture medium pH (p < 0.005). The optimum pH and temperature values were found at 7.4 and 34 °C, respectively. The conditions that increased KLa, were 12.17 s - 1 and 1.5 vvm, with a KLa value of 0.104 s - 1. This conditions ensured optimum strain growth of 5.496 g/L of DCW (5.E1010 CFU/mL), whereas µ was 0.403 h - 1. The findings of this study are a step forward in the development of P. indica H32 as biopesticide and biofertilizer.

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Culture medium composition is one of the most important parameters to analyze in biotechnological processes with industrial purposes. The aim of this study was to design of a culture medium for optimal growth of the bacterium Pseudoxanthomonas indica H32 allowing its production as biopesticide and biofertilizer. The influence of several carbon and nitrogen sources and their molar ratios on P. indica H32 growth was investigated. The effect of different micronutrients such as mineral salts and vitamin on P. indica H32 growth was determined as well. A mixture design based on Design-Expert 10.0 Software was performed to optimize the culture medium concentration. Finally, in the designed medium, an attribute of the biological mechanism of action of the P. indica H32 against nematodes, was evaluated: the hydrogen sulfide production. It was found that tested carbon/nitrogen ratios were not a significant influence on P. indica H32 growth. Growth of P. indica H32 was favored with use of sucrose, yeast extract and phosphate buffer without the addition of any tested micronutrients. An optimal concentration of 10 g/L sucrose and 5 g/L yeast extract were obtained at a cost of 0.10 $/L. In this concentration, the specific growth rate (µ) and maximal optical density (Xmax) were equal to 0.439 h- 1 and 8.00 respectively. It was evidenced that under the culture conditions used, P. indica H32 produced hydrogen sulfide. The designed medium led to a 1.08 $/L reduction of costs in comparison to LB medium. These results were critical to carry on with biotechnological development of P. indica H32 as a bioproduct.

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Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2'…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Šresolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

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Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

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Microbial degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is the most promising way to clean up DDT residues found in the environment. In this paper, a bacterium designated as wax, which was capable of co-metabolizing DDT with other carbon sources, was isolated from a long-term DDT-contaminated soil sample by an enrichment culture technique. The new isolate was identified as a member of the Pseudoxanthomonas sp., based on its morphological, physiological and biochemical properties, as well as by 16S rRNA gene analysis. In the presence of 100 mg l-1 glucose, the wax strain could degrade over 95 percent of the total DDT, at a concentration of 20 mg l-1, in 72 hours, and could degrade over 60 percent of the total DDT, at a concentration of 100 mg l-1, in 144 hours. The wax strain had the highest degradation efficiency among all of the documented DDT-degrading bacteria. The wax strain could efficiently degrade DDT at temperatures ranging from 20 to 37ºC, and with initial pH values ranging from 7 to 9. The bacterium could also simultaneously co-metabolize 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE), and other organochlorine compounds. The wax strain could also completely remove 20 mg kg-1 of DDT from both sterile and non-sterile soils in 20 days. This study demonstrates the significant potential use of Pseudoxanthomonas sp. wax for the bioremediation of DDT in the environment.

Assuntos

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Microbial degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is the most promising way to clean up DDT residues found in the environment. In this paper, a bacterium designated as wax, which was capable of co-metabolizing DDT with other carbon sources, was isolated from a long-term DDT-contaminated soil sample by an enrichment culture technique. The new isolate was identified as a member of the Pseudoxanthomonas sp., based on its morphological, physiological and biochemical properties, as well as by 16S rRNA gene analysis. In the presence of 100 mg l(-1) glucose, the wax strain could degrade over 95% of the total DDT, at a concentration of 20 mg l(-1), in 72 hours, and could degrade over 60% of the total DDT, at a concentration of 100 mg l(-1), in 144 hours. The wax strain had the highest degradation efficiency among all of the documented DDT-degrading bacteria. The wax strain could efficiently degrade DDT at temperatures ranging from 20 to 37°C, and with initial pH values ranging from 7 to 9. The bacterium could also simultaneously co-metabolize 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE), and other organochlorine compounds. The wax strain could also completely remove 20 mg kg(-1) of DDT from both sterile and non-sterile soils in 20 days. This study demonstrates the significant potential use of Pseudoxanthomonas sp. wax for the bioremediation of DDT in the environment.

RESUMO

Microbial degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is the most promising way to clean up DDT residues found in the environment. In this paper, a bacterium designated as wax, which was capable of co-metabolizing DDT with other carbon sources, was isolated from a long-term DDT-contaminated soil sample by an enrichment culture technique. The new isolate was identified as a member of the Pseudoxanthomonas sp., based on its morphological, physiological and biochemical properties, as well as by 16S rRNA gene analysis. In the presence of 100 mg l-1 glucose, the wax strain could degrade over 95% of the total DDT, at a concentration of 20 mg l-1, in 72 hours, and could degrade over 60% of the total DDT, at a concentration of 100 mg l-1, in 144 hours. The wax strain had the highest degradation efficiency among all of the documented DDT-degrading bacteria. The wax strain could efficiently degrade DDT at temperatures ranging from 20 to 37ºC, and with initial pH values ranging from 7 to 9. The bacterium could also simultaneously co-metabolize 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE), and other organochlorine compounds. The wax strain could also completely remove 20 mg kg-1 of DDT from both sterile and non-sterile soils in 20 days. This study demonstrates the significant potential use of Pseudoxanthomonas sp. wax for the bioremediation of DDT in the environment.