The Expression and Diversity of Catalases in Isolates of Genus Comamonas in Response to the Oxidative Stress of a Polluted Environment

We have evaluated the role of monofunctional heme-containing catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Our results indicate that this constitutively expressed catalase represents the major source for the defence of Comamonas terrigena cells against toxic peroxides but the cells can express also a second form of catalase that is bigger and its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Comamonas terrigena but in those strains that were not exposed to oxidative stress, no such sequence motif could be detected. The results obtained underline the importance of catalase expression in the defence mechanism against oxidative stress in bacterial cells.     

Oxidative Stress Responses Accompanying Photoinactivation of Catalase in NaCI-treated Rye Leaves

When segments of rye leaves (Secale cereale L.) grown at 90 μmol m^?2 s^?1 PAR were incubated at a higher photon flux of 400–500 μ mol m^?2 s^?1 PAR in the presence of 0.2-0.6 M NaCl, a preferential loss of catalase activity was induced. The extent of this decline increased with the concentration of NaCl. In addition, the accumulation of alternative antioxidative components, such as ascorbate, glutathione, glutathione reductase, or peroxidase, was inhibited. The total content of H₂O₂ was, however, lower in catalase-depleted than in untreated control leaves. The occurrence of strong oxidative stress in NaCl-treated leaves was indicated by marked declines in the ratios of reduced to oxidized ascorbate and glutathione and by the degradation of chlorophyll in light. The specific elimination of catalase activity by the inhibitor aminotriazole was also accompanied by a rapid decline in the ratio of reduced to oxidized glutathione but other symptoms of oxidative stress were much less severe than in the presence of NaCl. However, all symptoms of photooxidative damage seen in NaCl-treated leaves were closely mimicked by treatment with the translation inhibitor, cycloheximlde, in light. The results suggest that NaCl-induced oxidative damage in light was predominantly mediated by the inhibition of protein synthesis. By this inhibition the resynthesis of catalase, which has a high turnover in light, was blocked and the leaves were thus depleted of catalase activity and, in addition, the intensification of alternative antioxidative systems was also prevented.     

Responses of Methanotrophic Activity in Soils and Cultures to Water Stress

Diffusive gas transport at high water contents and physiological water stress at low water contents limited atmospheric methane consumption rates during experimental manipulations of soil water content and water potential. Maximum rates of atmospheric methane consumption occurred at a soil water content of 25% (grams per gram [dry weight]) and a water potential of about -0.2 MPa. In contrast, uptake rates were highest at a water content of 38% and a water potential of -0.03 MPa when methane was initially present at 200 ppm. Uptake rates of atmospheric and elevated methane decreased when water potentials were reduced by adding either ionic or nonionic solutes to soils with a fixed water content. Uptake rates during these manipulations were lower when sodium chloride or potassium chloride was used to adjust water potential rather than sucrose. The response of methane consumption by soils to water potential was somewhat less pronounced than the response of methanotrophic cultures (e.g., Methylosinus trichosporium OB3b, Methylomonas rubra [= M. methanica], an isolate from a freshwater peat, and an isolate from an intertidal marine mudflat). However, unlike soils, methanotrophic cultures exhibited a stronger adverse response to nonionic solutes than to sodium chloride.     

Diversity of Bacterial Isolates from Commercial and Homemade Composts

The diversity of heterotrophic bacterial isolates of three commercial and two homemade composts was studied. The commercial composts were produced from poultry litter (PC), sewage sludge (SC), municipal solid waste (MC), and homemade composts (thermal compost [DC] and vermicompost [VC]) from food wastes. The taxonomic and physiological diversity of the heterotrophic culturable bacteria was assessed using phenotypic and genotypic characterization and the analysis of the partial 16S rRNA gene sequence. Composts DC and SC presented the higher genotypic diversity, as could be inferred from the number of distinct genotypic patterns observed, 28 and 21, respectively. Gram-positive bacteria, mainly Firmicutes, were predominant in all the composts. Some organisms related with taxa rarely reported in composts, as Rhodanobacter spathiphylli, Moraxella osloensis, Lysobacter, Corynebacterium, Pigmentiphaga kullae, and new taxa were also isolated. The highest relative proportion of isolates able to degrade starch was found in compost SC (>70%), to degrade gelatine in compost DC (>70%), to degrade Tween 80 in compost PC (>90%), and to degrade poly-epsilon-caprolactones in compost DC (>80%). Compost MC presented the lowest relative proportions of isolates able to degrade starch (<25%), gelatine (<20%), and poly-epsilon-caprolactone (<40%). When compared with the others, the homemade composts presented higher relative proportions of Gram-positive isolates able to inhibit the target organisms Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, or Pseudomonas aeruginosa. In compost MC, none of the Gram-positive isolates was able to inhibit those targets.     

Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm^-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k_cat/K_m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK_a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.     

Identification of Crucial Amino Acids of Bacterial Peptide Deformylases Affecting Enzymatic Activity in Response to Oxidative Stress

Peptide deformylase (PDF) is an essential bacterial metalloprotease involved in deformylation of N-formyl group from nascent polypeptide chains during protein synthesis. Iron-containing variants of this enzyme from Salmonella enterica serovar Typhimurium (sPDF) and Mycobacterium tuberculosis (mPDF), although inhibited by oxidizing agents like H₂O₂, exhibited strikingly different 50% inhibitory concentrations (IC₅₀s) that ranged from nanomolar (sPDF) to millimolar (mPDF) levels. Furthermore, the metal dissociation rate was higher in sPDF than mPDF. We hypothesized that a restriction in entry of environmental oxygen or oxidizing agents into the active site of mPDF might be the cause for such discrepancies between two enzymes. Since the active-site residues of the two proteins are similar, we evaluated the role of the oxidation-prone noncatalytic residue(s) in the process. Amino acid sequence analysis revealed that Cys-130 of sPDF corresponds to Met-145 of mPDF and that they are away from the active sites. Swapping methionine with cysteine in mPDF, the M145C protein displayed a drastic decrease in the IC₅₀ for H₂O₂ and an increased metal dissociation rate compared to the wild type. Matrix-assisted laser desorption ionization (MALDI) analysis of a trypsin-digested fragment containing Cys-145 of the M145C protein also indicated its increased susceptibility to oxidation. To incorporate residues identical to those of mPDF, we created a double mutant of sPDF (C130M-V63C) that showed increased IC₅₀ for H₂O₂ compared to the wild type. Interestingly, the oxidation state of cysteines in C130M-V63C was unaffected during H₂O₂ treatment. Taken together, our results unambiguously established the critical role of noncatalytic cysteine/methionine for enzymatic sensitivity to H₂O₂ and, thus, for conferring behavioral distinction of bacterial PDFs under oxidative stress conditions.     

A Chaperone Function of NO CATALASE ACTIVITY1 Is Required to Maintain Catalase Activity and for Multiple Stress Responses in Arabidopsis

Catalases are key regulators of reactive oxygen species homeostasis in plant cells. However, the regulation of catalase activity is not well understood. In this study, we isolated an Arabidopsis thaliana mutant, no catalase activity1-3 (nca1-3) that is hypersensitive to many abiotic stress treatments. The mutated gene was identified by map-based cloning as NCA1, which encodes a protein containing an N-terminal RING-finger domain and a C-terminal tetratricopeptide repeat-like helical domain. NCA1 interacts with and increases catalase activity maximally in a 240-kD complex in planta. In vitro, NCA1 interacts with CATALASE2 (CAT2) in a 1:1 molar ratio, and the NCA1 C terminus is essential for this interaction. CAT2 activity increased 10-fold in the presence of NCA1, and zinc ion binding of the NCA1 N terminus is required for this increase. NCA1 has chaperone protein activity that may maintain the folding of catalase in a functional state. NCA1 is a cytosol-located protein. Expression of NCA1 in the mitochondrion of the nca1-3 mutant does not rescue the abiotic stress phenotypes of the mutant, while expression in the cytosol or peroxisome does. Our results suggest that NCA1 is essential for catalase activity.     

一株具有IMPDH抑制活性的稀有放线菌的筛选和鉴定

从重庆南川药用植物乌头根际土中分离到一株具有次黄嘌呤核苷磷酸脱氢酶(IMPDH)抑制活性的根际稀有放线菌株.通过菌株形态特征、培养特征观察、生理生化特征测定、细胞化学组分分析及16S rRNA基因序列同源性分析等多相分类特征研究,确定I06-03147属于动孢囊菌属(Kineosporia)的菌株.     

Broad Diversity and Newly Cultured Bacterial Isolates from Enrichment of Pig Feces on Complex Polysaccharides

One of the fascinating functions of mammalian intestinal microbiota is fermentation of plant cell wall components. Eight-week continuous culture enrichments of pig feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 575 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented in the bacterial isolates: Firmicutes (242), Bacteroidetes (185), Proteobacteria (65), Fusobacteria (55), Actinobacteria (23), and Synergistetes (5). The majority of the bacterial isolates had ≥97 % similarity to cultured bacteria with sequences in the RDP, but 179 isolates represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families of Lachnospiraceae, Enterococcaceae, Staphylococcaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. Many of the Firmicutes and Bacteroidetes isolates were identified as species that possess enzymes that ferment plant cell wall components, and the rest likely support these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 30 % of the isolates not represented in culture, there are new opportunities to study genomic and metabolic capacities of these members of the complex intestinal microbiota.     

Phylogenetic Diversity and Characterization of Novel and Efficient Cellulase Producing Bacterial Isolates from Various Extreme Environments

A set of 300 bacterial strains isolated from various extreme environments were screened for the presence of cellulase activity on CMC agar plates. Phylogenetic analysis of the positive strain, based on 16S rRNA gene sequences indicated that the isolates were clustered within Firmicutes and Actinobacteria. A majority (17) of the isolates were identified as Bacillus, Paenibacillus, and Lysinibacillus sp., and the remaining three were identified as Arthobacter, Rhodococcus, and Bhargavaea cecembensis. Among the 20 positive isolates, 6 were evaluated for the production of cellulases on five different cellulosic substrates. Two isolates, B. cecembensis and Bacillus sp., based on maximum enzyme production on all cellulosic substrates, especially CMC and rice straw, were evaluated in terms of enzyme properties and kinetics. The enzymes of these two isolates are found to be active over broad range of pH and temperature. Such thermostable enzymes facilitate the development of efficient and cost-effective forms of the simultaneous saccharification and fermentation process converting lignocellulosic biomass into biofuels and value-added products.     

Bacterial Community and Diversity from the Watermelon Cultivated Soils through Next Generation Sequencing Approach

Knowledge and better understanding of functions of the microbial community are pivotal for crop management. This study was conducted to study bacterial structures including Acidovorax species community structures and diversity from the watermelon cultivated soils in different regions of South Korea. In this study, soil samples were collected from watermelon cultivation areas from various places of South Korea and microbiome analysis was performed to analyze bacterial communities including Acidovorax species community. Next generation sequencing (NGS) was performed by extracting genomic DNA from 92 soil samples from 8 different provinces using a fast genomic DNA extraction kit. NGS data analysis results revealed that, total, 39,367 operational taxonomic unit (OTU), were obtained. NGS data results revealed that, most dominant phylum in all the soil samples was Proteobacteria (37.3%). In addition, most abundant genus was Acidobacterium (1.8%) in all the samples. In order to analyze species diversity among the collected soil samples, OTUs, community diversity, and Shannon index were measured. Shannon (9.297) and inverse Simpson (0.996) were found to have the highest diversity scores in the greenhouse soil sample of Gyeonggi-do province (GG4). Results from NGS sequencing suggest that, most of the soil samples consists of similar trend of bacterial community and diversity. Environmental factors play a key role in shaping the bacterial community and diversity. In order to address this statement, further correlation analysis between soil physical and chemical parameters with dominant bacterial community will be carried out to observe their interactions.     

A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance

Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P . aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H₂O₂ toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H₂O₂-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.     

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with ¹³CH₄ at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of ¹³C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.     

Structural Change and Catalytic Activity of Horseradish Peroxidase in Oxidative Polymerization of Phenol

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.     

Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co^r, Zn^r, and Cd^r), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.     

Diversity and Structure of Bacterial Chemolithotrophic Communities in Pine Forest and Agroecosystem Soils

Obligate lithotrophs (e.g., ammonia oxidizers) and facultative lithotrophs (e.g., CO and hydrogen oxidizers) collectively comprise a phylogenetically diverse functional group that contributes significantly to carbon and nitrogen cycles in soils and plays important roles in trace gas dynamics (e.g., carbon monoxide and nitrous and nitric oxides) that affect tropospheric chemistry and radiative forcing. In spite of their diverse physiologies, facultative and obligate lithotrophs typically possess the Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisCO). In an effort designed to understand the structure of lithotrophic communities in soil, genomic DNA extracts from surface (0 to 2 cm) and subsurface (5 to 7 cm) soils have been obtained from two sites in a Georgia agroecosystem (peanut and cotton plots) and an unmanaged pine stand (>50 years old). The extracts have been used in PCR amplifications of the cbbL gene for the rubisCO large subunit protein. cbbL PCR products were cloned, sequenced, and subjected to phylogenetic and statistical analyses. Numerous novel lineages affiliated with the form IC clade (one of four form I rubisCO clades), which is typified by facultative lithotrophs, comprised lithotrophic communities from all soils. One of the form IC clone sequences clustered with a form IC clade of ammonia-oxidizing Nitrosospira. Distinct assemblages were obtained from each of the sites and from surface and subsurface soils. The results suggest that lithotrophic populations respond differentially to plant type and land use, perhaps forming characteristic associations. The paucity of clone sequences attributed to ammonia-oxidizing bacteria indicates that even though ammonia oxidation occurs in the various soils, the relevant populations are small compared to those of facultative lithotrophs.