Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria.

Iron (Fe) oxidation is one of Earth's major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. IMPORTANCE Iron is practically ubiquitous across Earth's environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or "rusting," can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins; thus, these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron oxidizers, including acidophilic and phototrophic iron oxidizers, suggesting that this fused cytochrome-porin structure is especially well adapted for iron oxidation.

Gut microbial beta-glucuronidase and glycerol/diol dehydratase activity contribute to dietary heterocyclic amine biotransformation.

BACKGROUND: Consuming red and processed meat has been associated with an increased risk of colorectal cancer (CRC), which is partly attributed to exposure to carcinogens such as heterocyclic amines (HCA) formed during cooking and preservation processes. The interaction of gut microbes and HCA can result in altered bioactivities and it has been shown previously that human gut microbiota can transform mutagenic HCA to a glycerol conjugate with reduced mutagenic potential. However, the major form of HCA in the colon are glucuronides (HCA-G) and it is not known whether these metabolites, via stepwise microbial hydrolysis and acrolein conjugation, are viable precursors for glycerol conjugated metabolites. We hypothesized that such a process could be concurrently catalyzed by bacterial beta-glucuronidase (B-GUS) and glycerol/diol dehydratase (GDH) activity. We therefore investigated how the HCA-G PhIP-N2-ß-D-glucuronide (PhIP-G), a representative liver metabolite of PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), which is the most abundant carcinogenic HCA in well-cooked meat, is transformed by enzymatic activity of human gut microbial representatives of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. RESULTS: We employed a combination of growth and enzymatic assays, and a bioanalysis approach combined with metagenomics. B-GUS of Faecalibacterium prausnitzii converted PhIP-G to PhIP and GDH of Flavonifractor plautii, Blautia obeum, Eubacterium hallii, and Lactobacillus reuteri converted PhIP to PhIP-M1 in the presence of glycerol. In addition, B-GUS- and GDH-positive bacteria cooperatively converted PhIP-G to PhIP-M1. A screen of genes encoding B-GUS and GDH was performed for fecal microbiome data from healthy individuals (n = 103) and from CRC patients (n = 53), which revealed a decrease in abundance of taxa with confirmed GDH and HCA transformation activity in CRC patients. CONCLUSIONS: This study for the first time demonstrates that gut microbes mediate the stepwise transformation of PhIP-G to PhIP-M1 via the intermediate production of PhIP. Findings from this study suggest that targeted manipulation with gut microbes bearing specific functions, or dietary glycerol supplementation might modify gut microbial activity to reduce HCA-induced CRC risk.

Epilithic Biofilms in Lake Baikal: Screening and Diversity of PKS and NRPS Genes in the Genomes of Heterotrophic Bacteria.

A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidetes . To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus ) was 34% and NRPS genes were found in 78%. In Proteobacteria , PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria , respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus) was 34% and NRPS genes were found in 78%. In Proteobacteria, PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria, respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.

Mutation of Phenylalanine-223 to Leucine Enhances Transformation of Benzo[a]pyrene by Ring-Hydroxylating Dioxygenase of Sphingobium sp. FB3 by increasing Accessibility of the Catalytic Site.

Burning of agricultural biomass generates polycyclic aromatic hydrocarbons (PAHs) including the carcinogen benzo[a]pyrene, of which the catabolism is primarily initiated by a ring-hydroxylating dioxygenase (RHD). This study explores catalytic site accessibility and its role in preferential catabolism of some PAHs over others. The genes flnA1f, flnA2f, flnA3, and flnA4, encoding the oxygenase α and ß subunits, ferredoxin, and ferredoxin reductase, respectively, of the RHD enzyme complex (FlnA) were cloned from Sphingobium sp. FB3 and coexpressed in E. coli BL21. The FlnA effectively transformed fluoranthene but not benzo[a]pyrene. Substitution of the bulky phenylalanine-223 by leucine reduces the steric constraint in the substrate entrance to make the catalytic site of FlnA more accessible to large substrates, as visualized by 3D modeling, and allows the FlnA mutant to efficiently transform benzo[a]pyrene. Accessibility of the catalytic site to PAHs is a mechanism of RHD substrate specificity. The results shed light on why some PAHs are more recalcitrant than others.

Purification and Characterisation of κ-Carrageenan Oligosaccharides Prepared by κ-Carrageenase from Thalassospira sp. Fjfst-332.

κ-Carrageenan oligosaccharides (KCOs) are promising agents for treating inflammatory diseases. However, the lack of purification and structural elucidation of KCOs has limited structure-function evaluation. In this study, using a system coupling medium pressure liquid chromatography (MPLC) with an evaporative light scattering detector (ELSD), four types of KCOs were separated. The total yield of the four KCO powders was ∼5.02% after purification (KCOs/κ-carrageenan, w/w). Their structural identities were characterised by ESI-MS, CID-MS/MS and NMR, as κ-neocarrabiose (α-DA-1,3-G4Srα/ß), κ-neocarratetraose (α-DA-1,3-ß-G4S-1,4-α-DA-1,3-G4Srα/ß), κ-neocarrahexaose (α-DA-1,3-ß-G4S-1,4-α-DA-1,3-ß-G4S-1,4-α-DA-1,3-G4Srα/ß) and heterozygous κ/ι-neocarrahexaose (α-DA/DA2S-1,3-ß-G4S-1,4-α-DA-1,3-ß-G4S-1,4-α-DA-1,3-G4Srα/ß). KCOs showed no cytotoxicity in RAW264.7 macrophages, and the anti-inflammatory activity was closely correlated with the degree of polymerisation and the number of sulfated groups. κ/ι-Neocarrahexaose exhibited the highest inhibition of ROS (Reactive Oxygen Species) production in LPS-induced RAW264.7 macrophages. The MPLC-ELSD system provides a platform for large-scale fabrication of purified KCOs and affords a route to these compounds that may regulate immune defense.

Iron Hydroperoxide Intermediate in Superoxide Reductase: Protonation or Dissociation First? MM Dynamics and QM/MM Metadynamics Study.

Superoxide reductase is a mononuclear iron enzyme involved in superoxide radical detoxification in some bacteria. Its catalytic mechanism is associated with the remarkable formation of a ferric hydroperoxide Fe3+-OOH intermediate, which is specifically protonated on its proximal oxygen to generate the reaction product H2O2. Here, we present a computational study of the protonation mechanism of the Fe3+-OOH intermediate, at different levels of theory. This was performed on the whole system (solvated protein) using well-tempered metadynamics at the QM/MM (B3LYP/AmberFF99SB) level. Enabled by the development of a new set of force field parameters for the active site, a conformational MM study of the Fe3+-OOH species gave insights into its solvation pattern, in addition to generating the two starting conformations for the ab initio metadynamics setup. Two different protonation mechanisms for the Fe3+-OOH intermediate have been found depending on the starting structure. Whereas a possible mechanism involves at first the protonation of the hydroperoxide ligand and then dissociation of H2O2, the most probable one starts with an unexpected dissociation of the HOO- ligand from the iron, followed by its protonation. This favored reactivity was specifically linked to the influence of both the nearby conserved lysine 48 residue and the microsolvatation on the charge distribution of the oxygens of the HOO- ligand. These data highlight the crucial role of the whole environment, solvent, and protein, to describe accurately this second protonation step in superoxide reductase. This is clearly not possible with smaller models unable to reproduce correctly the mechanistically determinant charge distribution.

Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico.

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of ß-Aryl Ether Bonds in Lignin.

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via ß-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent ß-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because ß-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic ß-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

Differentiation of 1-aminocyclopropane-1-carboxylate (ACC) deaminase from its homologs is the key for identifying bacteria containing ACC deaminase.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase-mediated reduction of ethylene generation in plants under abiotic stresses is a key mechanism by which bacteria can promote plant growth. Misidentification of ACC deaminase and the ACC deaminase structure gene (acdS) can lead to overestimation of the number of bacteria containing ACC deaminase and their function in ecosystems. Previous non-specific amplification of acdS homologs has led to an overestimation of the horizontal transfer of acdS genes. Here, we designed consensus-degenerate hybrid oligonucleotide primers (acdSf3, acdSr3 and acdSr4) based on differentiating the key residues in ACC deaminases from those of homologs for specific amplification of partial acdS genes. PCR amplification, sequencing and phylogenetic analysis identified acdS genes from a wide range of proteobacteria and actinobacteria. PCR amplification and a genomic search did not find the acdS gene in bacteria belonging to Pseudomonas stutzeri or in the genera Enterobacter, Klebsiella or Bacillus. We showed that differentiating the acdS gene and ACC deaminase from their homologs was crucial for the molecular identification of bacteria containing ACC deaminase and for understanding the evolution of the acdS gene. We provide an effective method for screening and identifying bacteria containing ACC deaminase.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase.

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.

Use of limited proteolysis and mutagenesis to identify folding domains and sequence motifs critical for wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase activity.

Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.

Crystallization and preliminary X-ray diffraction studies of the reduced form of the terminal oxygenase component of the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase.

The initial reaction of bacterial carbazole degradation is catalysed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase, ferredoxin and ferredoxin reductase components. The reduced form of the terminal oxygenase component was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals diffracted to a resolution of 1.74 Šand belonged to space group P6(5), with unit-cell parameters a = b = 92.0, c = 243.6 Å. The asymmetric unit contained a trimer of terminal oxygenase molecules.

Hydrogen bonding to the cysteine ligand of superoxide reductase: acid-base control of the reaction intermediates.

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe(2+) center in a [His4Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe(3+)-S(Cys) and S-Cß(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2 (•-) were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pK a of the first reaction intermediate, recently proposed to be an Fe(2+)-O2 (•-) species. These data were supported by density functional theory calculations conducted on three models of the Fe(2+)-O2 (•-) intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe(2+)-O2 (•-) reaction intermediate to form an Fe(3+)-OOH species.

The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization.

CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO2] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K(M) value of 0.69±0.14 mM ATP and an apparent V(max) value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO2] cluster assembly are discussed.

Molecular characterization of 'Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

This study was undertaken to characterize the alpha subgroup of the proteobacteria causing the huanglongbing (HLB) disease of citrus from three different ecological zones of Kenya namely the Lower highlands (LH2, LH3, 1800-1900 m above sea level); Upper midlands (UM3, UM4, 1390-1475m), Lower midlands (LM5, LM4, LM3 of 1290-1340-1390m), by isolation and sequencing DNA encoding the L10 and L12 ribosomal proteins and the intergenic region. A 7I6-basepair DNA fragment was amplified and sequenced and consisted of 536 basepairs of DNA encoding the L10 protein, 44 basepairs of DNA intergenic region and 136 basepairs of DNA that partially encodes the L12 protein. Sequences of rpL10/L12 protein genes from Kenyan strains were 98 percent and 81 percent similar to the South African 'Candidatus Liberibacter africanus strain Nelspruit' and the Asian 'Candidatus Liberibacter asiaticus' strains, respectively. The intergenic rDNA sequence of Kenyan strain from UM and LM showed 84 percent similarity with 'Candidatus L. africanus strain Nelspruit' and 50 percent similarity with 'Candidatus L. asiaticus' strain. However, the LH strain had an 11- basepairs deletion, while the LM4 had a 5-basepair deletion in the intergenic region compared to 'Candidatus L. africanus strain Nelspruit'. The L10 amino acid sequence was 100 percent homologous among HLB bacteria obtained from the agro-ecological zones in Kenya and the L10 protein sequence was also homologus to 'Candidatus L. africanus strain Nelspruit'. Nevertheless, the L10 amino acid sequence of 'Candidatus L. asiaticus' and the 'Candidatus L. africanus subsp. capensis' differed from the Kenyan strains by 18.36 percent and 11.82 percent, respectively. Phylogenetic analysis of both the L10/L12 rDNA sequences and the L10 amino acid sequences clustered the Kenyan strains of the 'Candidatus Liberibacter' species with members of alpha subdivision of proteobacteria.

Xenobiotics enhance laccase activity in alkali-tolerant gama-proteobacterium JB / Xenobióticos aumentam a atividade de lacase em gama-Proteobacterium JB alcali-tolerante

Various genotoxic textile dyes, xenobiotics, substrates (10 µM) and agrochemicals (100 µg/ml) were tested for enhancement of alkalophilic laccase activity inã-proteobacterium JB. Neutral Red, Indigo Carmine, Naphthol Base Bordears and Sulphast Ruby dyes increased the activity by 3.7, 2.7, 2.6 and 2.3 fold respectively. Xenobiotics/substrates like p-toluidine, 8-hydroxyquinoline and anthracine increased it by 3.4, 2.8 and 2.3 fold respectively. Atrazine and trycyclozole pesticides enhanced the activityby 1.95 and 1.5 fold respectively.

Vários corantes têxteis genotóxicos, xenobióticos, substratos (10 mM) e agroquímicos (100 mM/mL) foram testados quanto ao aumento da atividade de lacase em ã-Proteobacterium JB. Os corantes Neutral Red, Indigo Carmine, Naphtol Base Bordears e Sulphast Ruby aumentaram a atividade em 3,7, 2,7, 2,6 e 2,3 vezes, respectivamente. Xenobióticos/substratos como p-toluidina, 8-hidroxiquinolina e antracina aumentaram a atividade em 3,4, 2,8 e 2,3 vezes, respectivamente. Atrazina e pesticidas triciclozol aumentaram a atividade em 1,95 e 1,5 vezes, respectivamente.

Novel s-triazine-degrading bacteria isolated from agricultural soils of central Chile for herbicide bioremediation

s-Triazine-degrading bacterial strains were isolated from long-term simazine-treated agricultural soils of central Chile. The number of culturable heterotrophic bacteria of these agricultural soils (7 x 10(6) CFU/g of dry soil) was not affected by simazine application on field. The simazine-degrading bacterial strains P51, P52 and C53 were isolated by enrichment in minimal medium using simazine as the sole nitrogen source. Resting cells of strains P51 and P52 degraded >80 percent of simazine within 48 hrs, whereas strain C53 was able to remove >60 percent of the herbicide. The atzA and atzD genes of the s-triazine upper and lower catabolic pathways were detected in strains P51 and C53, while only atzD gene was observed in strain P52. To compare the bacterial 16S rRNA gene sequence structure, ARDRA were performed using the restriction enzymes Msp1 and Hha1. ARDRA indicated that strain P52 was a different ribotype than C53 and P51 strains. For further characterization the novel isolates were identified by 16S rRNA gene sequencing. Strains C53 and P51 belong to the genus Stenotrophomonas and the strain P52 belongs to the genus Arthrobacter . s -Triazine-degrading bacterial strains isolated from contaminated soils could be used as biocatalysts for bioremediation of these herbicides.

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification.

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.

"Candidatus Accumulibacter" population structure in enhanced biological phosphorus removal sludges as revealed by polyphosphate kinase genes.

We investigated the fine-scale population structure of the "Candidatus Accumulibacter" lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of "Candidatus Accumulibacter" 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the "Candidatus Accumulibacter" lineage. Sequences from at least five clades of "Candidatus Accumulibacter" were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using "Candidatus Accumulibacter"-specific 16S rRNA and "Candidatus Accumulibacter" clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total "Candidatus Accumulibacter" lineage and the relative distributions and abundances of the five "Candidatus Accumulibacter" clades. The qPCR-based estimation of the total "Candidatus Accumulibacter" fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined "Candidatus Accumulibacter" clades. The relative distributions of "Candidatus Accumulibacter" clades varied among different EBPR systems and also temporally within a system. Our results suggest that the "Candidatus Accumulibacter" lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study.

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.