The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Draft genome sequences of halotolerant Halomonas spp. SpR1 and SpR8, potential plant growth-promoting bacteria associated with Salicornia rhizosphere in a hydrothermal lagoon ecosystem of the Altiplano, Northern Chile.

Halotolerant Halomonas spp. SpR1 and SpR8 are potential plant growth-promoting bacteria (PGPB) isolated from Salicornia rhizosphere in a Chilean Altiplano hydrothermal lagoon. We report draft genomes of Halomonas sp. SpR1 (5.17Mb) and Halomonas sp. SpR8 (4.47 Mb). Both represent potentially novel independent species closely related to Halomonas boliviensis DSM 15516T.

The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans

Background Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxy-phenylacetate (3-HPA). Methods The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. Results The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. Conclusions The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

GenoVi, an open-source automated circular genome visualizer for bacteria and archaea.

The increase in microbial sequenced genomes from pure cultures and metagenomic samples reflects the current attainability of whole-genome and shotgun sequencing methods. However, software for genome visualization still lacks automation, integration of different analyses, and customizable options for non-experienced users. In this study, we introduce GenoVi, a Python command-line tool able to create custom circular genome representations for the analysis and visualization of microbial genomes and sequence elements. It is designed to work with complete or draft genomes, featuring customizable options including 25 different built-in color palettes (including 5 color-blind safe palettes), text formatting options, and automatic scaling for complete genomes or sequence elements with more than one replicon/sequence. Using a Genbank format file as the input file or multiple files within a directory, GenoVi (i) visualizes genomic features from the GenBank annotation file, (ii) integrates a Cluster of Orthologs Group (COG) categories analysis using DeepNOG, (iii) automatically scales the visualization of each replicon of complete genomes or multiple sequence elements, (iv) and generates COG histograms, COG frequency heatmaps and output tables including general stats of each replicon or contig processed. GenoVi's potential was assessed by analyzing single and multiple genomes of Bacteria and Archaea. Paraburkholderia genomes were analyzed to obtain a fast classification of replicons in large multipartite genomes. GenoVi works as an easy-to-use command-line tool and provides customizable options to automatically generate genomic maps for scientific publications, educational resources, and outreach activities. GenoVi is freely available and can be downloaded from https://github.com/robotoD/GenoVi.

PromoterLCNN: A Light CNN-Based Promoter Prediction and Classification Model.

Promoter identification is a fundamental step in understanding bacterial gene regulation mechanisms. However, accurate and fast classification of bacterial promoters continues to be challenging. New methods based on deep convolutional networks have been applied to identify and classify bacterial promoters recognized by sigma (σ) factors and RNA polymerase subunits which increase affinity to specific DNA sequences to modulate transcription and respond to nutritional or environmental changes. This work presents a new multiclass promoter prediction model by using convolutional neural networks (CNNs), denoted as PromoterLCNN, which classifies Escherichia coli promoters into subclasses σ70, σ24, σ32, σ38, σ28, and σ54. We present a light, fast, and simple two-stage multiclass CNN architecture for promoter identification and classification. Training and testing were performed on a benchmark dataset, part of RegulonDB. Comparative performance of PromoterLCNN against other CNN-based classifiers using four parameters (Acc, Sn, Sp, MCC) resulted in similar or better performance than those that commonly use cascade architecture, reducing time by approximately 30-90% for training, prediction, and hyperparameter optimization without compromising classification quality.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400.

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic-degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H2O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZoxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H2O2. The effects of these oxidants on gene expression (qRT-PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR, ahpC, ahpF, kat, trxB, dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli. The LB400 genome also harbors the soxR, fumC, acnA, sodB, fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli. The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa ΔsoxR. Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR, ahpC1, katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR, fumC, ahpC1, sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR-regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium.

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H 2 O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZ oxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H 2 O2. The effects of these oxidants on gene expression (qRT PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR , ahpC , ahpF , kat , trxB , dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli . The LB400 genome also harbors the soxR, fumC , acnA , sodB , fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli . The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa Δ soxR . Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR , ahpC1 , katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR , fumC , ahpC1 , sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species.

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner-Doudoroff (ED), pentose-phosphate (PP), and lower Embden-Meyerhoff-Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via ß-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

Draft Genome Sequences of Two Pseudomonas Strains That Are Able To Use Furan Derivatives as Their Sole Carbon Source.

Pseudomonas sp. strains ALS1279 and ALS1131 were isolated from wastewater treatment facilities on the basis of their ability to use furfural, a key lignocellulose-derived inhibitor, as their only carbon source. Here, we present the draft genome sequences of both strains, which can shed light on catabolic pathways for furan compounds in pseudomonads.

Complete Genome Sequence of Hydrocarbon-Degrading Halotolerant Acinetobacter radioresistens DD78, Isolated from the Aconcagua River Mouth in Central Chile.

Acinetobacter radioresistens strain DD78 (= CCUG 69565) is a soil hydrocarbon-degrading and biosurfactant-producing bacterium isolated from chronically crude oil-polluted soil of the Aconcagua River mouth in Chile. The 3.25-Mb A. radioresistens DD78 genome (41.8% GC content) was completely sequenced, with 4 replicons, 2,970 coding sequences, and 77 tRNAs.

Long-chain flavodoxin FldX1 improves Paraburkholderia xenovorans LB400 tolerance to oxidative stress caused by paraquat and H2O2.

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.

Genomic and Physiological Traits of the Marine Bacterium Alcaligenes aquatilis QD168 Isolated From Quintero Bay, Central Chile, Reveal a Robust Adaptive Response to Environmental Stressors.

Alcaligenes aquatilis QD168 is a marine, aromatic hydrocarbon-degrading bacterium, isolated from an oil-polluted sediment of Quintero Bay, an industrial-coastal zone that has been chronically impacted by diverse pollutants. The aims of this study were to characterize the phylogenomic positions of Alcaligenes spp. and to characterize the genetic determinants and the physiological response of A. aquatilis QD168 to model environmental stressors (benzene, oxidizing agents, and salt). Phylogenomic analyses, using 35 housekeeping genes, clustered A. aquatilis QD168 with four other strains of Alcaligenes spp. (A. aquatilis BU33N, A. faecalis JQ135, A. faecalis UBA3227, and A. faecalis UBA7629). Genomic sequence analyses of A. aquatilis QD168 with 25 Alcaligenes spp., using ANIb, indicated that A. aquatilis BU33N is the closest related strain, with 96.8% ANIb similarity. Strain QD168 harbors 95 genes encoding proteins of seven central catabolic pathways, as well as sixteen peripheral catabolic pathways/reactions for aromatic compounds. A. aquatilis QD168 was able to grow on 3-hydroxybenzoate, 4-hydroxybenzoate, benzoate, benzene, 3-hydroxycinnamate, cinnamate, anthranilate, benzamide, 4-aminobenzoate, nicotinate, toluene, biphenyl and tryptophan, as sole carbon or nitrogen source. Benzene degradation was further analyzed by growth, metabolite identification and gene expression analyses. Benzene strongly induced the expression of the genes encoding phenol hydroxylase (dmpP) and catechol 1,2-dioxygenase (catA). Additionally, 30 genes encoding transcriptional regulators, scavenging enzymes, oxidative damage repair systems and isozymes involved in oxidative stress response were identified. Oxidative stress response of strain QD168 to hydrogen peroxide and paraquat was characterized, demonstrating that A. aquatilis QD168 is notably more resistant to paraquat than to H2O2. Genetic determinants (47 genes) for osmoprotective responses were identified, correlating with observed high halotolerance by strain QD168. The physiological adaptation of A. aquatilis QD168 to environmental stressors such as pollutants, oxidative stress and salinity may be exploited for bioremediation of oil-polluted saline sites.

Complete Genome Sequence of the Marine Hydrocarbon Degrader Alcaligenes aquatilis QD168, Isolated from Crude Oil-Polluted Sediment of Quintero Bay, Central Chile.

Alcaligenes aquatilis strain QD168 (= CCUG 69566) is a marine hydrocarbon-degrading bacterium isolated from crude oil-polluted sediment from Quintero Bay, Central Chile. Here, we present the 4.32-Mb complete genome sequence of strain QD168, with 3,892 coding sequences, 58 tRNAs, and a 56.3% G+C content.

Complete Genome Sequence of the Hydrocarbon-Degrading Strain Achromobacter sp. B7, Isolated during Petroleum Hydrocarbon Bioremediation in the Valparaiso Region, Chile.

Achromobacter sp. strain B7 (= CCUG 72081) was isolated from a diesel-polluted soil from the Valparaiso Region, Chile, subjected to bioremediation with a hydrocarbon-degrading enrichment. The complete genome sequence of Achromobacter sp. B7 has been determined to have a size of 6.24 Mb, 5,578 coding sequences, 57 tRNAs, and a G+C content of 64.8%.