Microbial Diversity and Community Structure on Corroding Concretes

The objective of this study was to analyze bacterial diversity in two different concrete samples to understand the dominant types of bacteria that may contribute to concrete corrosion. Two concrete samples, HN-1 from the sunny side and HN-2 from dark and damp side, were collected from Zijin Mountain in Nanjing and genomic DNA was extracted. The partial bacterial 16S rRNA gene fragment was PCR amplified and two clone libraries were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). Phylogenetic trees of bacterial 16S rDNA nucleotide sequences were constructed. Sample HN-1 and HN-2 contained 21 OTUs and 26 OTUs, respectively. Proteobacteria and Planctomycetes were the predominant bacteria in both samples, and they are distributed among Herbaspirillum, Archangium, Phyllobacteriaceae and Planctomycetaceae. Cyanobacteria and Rubrobacter sp. are dominant in HN-1; while Acidobacteriaceae, Adhaeribacter sp. and Nitrospira sp. are predominant in HN-2. This distribution pattern was consistent with local environmental conditions of these two samples. The inferred physiological characteristics of these bacteria, based on relatedness of the DNA clone sequences to cultivated species, revealed different mechanisms of concrete corrosion depending on the local environmental conditions.     

Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres

Two strains HN-1^T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the two strains were 99.9 and 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1^T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472^T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1^T with P. stellifer DSM 14472^T was 90.69, which was below the recommended threshold value (<?95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1^T and P. stellifer DSM 14472^T, which was lower than the recommended threshold value (dDDH?<?70%). The strain HN-1^T contain anteiso-C_15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472^T, strain HN-1^T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1^T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1^T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.

     

Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N₇ guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N₇ position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N₇ guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 μM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3′-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-56, having an activity of d-glucose isomerization was isolated from soil, and was identified to be similar to Aerobacter aerogenes (Kruse) Beijerink. d-Glucose-isomerizing activity was induced when HN-56 was precultured in the media containing d-xylose, d-mannose, lactate, especially d-mannitol. Paper chromatography showed that the ketose formed in reaction system containing d-glucose was d-fructose alone. The optimum pH for the reaction was 6.5~7.0. Sulfhydryl reagents inhibit the reaction, but metal inhibitors affect little if any. With the washed living cells as enzyme source, only arsenate could accumulate d-fructose. In addition, the cells grown with d-mannitol and d-mannose showed no activity of d-xylose isomerase.     

Studies on d-Glucose-isomerizing Enzyme of Bacillus coagulans,Strain HN-68

Cells of Bacillus coagulans, strain HN-68 grown on the medium containing d-glucose, did not show any measurable d-glucose-isomerizing activity. However, when d-glucose-grown cells were shaked for a few hours in an induction medium containing d-xylose, induced formation of d-glucose-isomerizing enzyme occurred in the cells. Cell weight and number of survival cells showed only an increase of 30 and 10%, respectively during 6 hr induction.

The induced formation of d-glucose-isomerizing enzyme required organic nitrogen such as polypeptone in addition to d-xylose. Development of the maximum activity was observed when the concentration of d-xylose and polypeptone were 2 and 3%, respectively. Initial velocity of induced formation of d-glucose-isomerizing enzyme increased in proportion to the decrease of initial pH values of the induction medium, i.e., at 2 hr induction, activity at pH 4.5 was 5-fold increase than that at pH 8.0.

Induced formation of d-glucose-isomerizing enzyme was inhibited strongly by addition of chloramphenicol, tetracycline, streptomycin, cyanide or azide, and was promoted by threonine plus glycine.     

A novel biotransformation of 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. for the purification of crude 2,6-naphthalene dicarboxylic acid

Crude 2,6-naphthalene dicarboxylic acid was purified by Pseudomonas sp. HN-72 which biotransformed the major impurity of 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid. The biotransformation yield reached 100% when the reaction was performed at 40°C for 1 h, in 200 ml KH₂PO₄/KOH buffer (50 mM, pH 8.0), with 0.2% (w/v) crude 2,6-naphthalene dicarboxylic acid and 2.5 mg dry cell wt/ml.     

X-Ray diffraction of oriented amylose fibers. II. Structure of V amyloses

Oriented amylose fibers in the V form were prepared and subjected to x-ray analysis. Unit cells and the probable space group of P2₁2₁2₁ were determined for the V anhydrous and V hydrate forms of amylose; the analysis confirms previous predictions of these structures based on x-ray powder patterns. Chain folding in V amyloses is discussed in view of crystallographic evidence and folding experiments conducted with space-filling models. Reported also is evidence for amylose helices having diameters intermediate between 13.0 and 13.7 A.     

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2

Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.     

Cloning of soybean genes induced during hypersensitive cell death caused by syringolide elicitor

Syringolide elicitors produced by bacteria expressing Pseudomonas syringae pv. glycinea avirulence gene D (avrD) induce hypersensitive cell death (HCD) only in soybean (Glycine max [L.] Merr.) plants carrying the Rpg4 disease resistance gene. Employing a differential display method, we isolated 13 gene fragments induced in cultured cells of a soybean cultivar Harosoy (Rpg4) treated with syringolides. Several genes for isolated fragments were induced by syringolides in an rpg4 cultivar Acme as well as in Harosoy; however, the genes for seven fragments designated as SIH (for syringolide-induced/HCD associated) were induced exclusively or strongly in Harosoy. cDNA clones for SIH genes were obtained from a cDNA library of Harosoy treated with syringolide. Several sequences are homologous to proteins associated with plant defense responses. The SIH genes did not respond to a non-specific -glucan elicitor, which induces phytoalexin accumulation but not HCD, suggesting that the induction of the SIH genes is specific for the syringolide–Harosoy interaction. HCD and the induction of SIH genes by syringolides were independent of H₂O₂. On the other hand, Ca²⁺ was required for HCD and the induction of some SIH genes. These results suggest that the induction of SIH genes by syringolides could be activated through the syringolide-specific signaling pathway and the SIH gene products may play an important role(s) in the processes of HCD induced by syringolides.Abbreviations AOS active oxygen species - CHS chalcone synthase - DPI diphenylene iodonium - HCD hypersensitive cell death - HR hypersensitive response - PAL phenylalanine ammonia lyase - SID syringolide-induced/defense associated - SIG syringolide-induced/general - SIH for syringolide-induced/HCD associated - XET xyloglucan endotransglycosylase     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

X-ray diffraction of oriented amylose fibers. III. The structure of amylose–n-butanol complexes

Complexes of amylose with n-butanol were prepared both as crystalline precipitates and as oriented fibers. These complexes were subjected to x-ray analysis, their unit cells were calculated, and the space group of P2₁2₁2₁ was confirmed. n-Butanol complexes exist in both hydrated and anhydrous forms. There is no evidence for methanol, ethanol, or n-propanol structures similar to those shown by the n-butanol complex. The Complexes are unstable in the open air and revert to V-amylose hydrate on standing.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.     

Biotransformation of Heavy Metals from Soil in Synthetic Medium Enriched with Glucose and Shewanella sp. HN-41 at Various pH

The biotransformation of heavy metals from contaminated soil was examined using a facultative anaerobic bacterium Shewanella sp. HN-41. The experiments were carried out to assess the influence of glucose at various pH on the transformation of heavy metals from soil thorough solubilization. A preliminary study on the transformation of heavy metals from soil was first performed using a defined medium supplemented with glucose at 10, 20, and 30 mM to select the effective concentration. Among the three concentrations examined, glucose at 30 mM leached a highest level of metal ions. Therefore, 30 mM glucose was used as the representative carbon source for the subsequent experiments in a defined medium at various pH (5, 6, 7, 8, and 9). The organism HN-41 was not influenced by pH ranging from acidic to neutral and was able to metabolize all the metal elements from contaminated soil. The level of Fe, Cr, As, Mn, Pb, and Al solubilization ranged from 3 to 7664 mg kg^?1 at various initial pH. The rate of metal solubilization was found to be low at neutral pH compared with acidic and alkaline. These results are expected to assist in the development of heavy metal transformation processes for the decontamination of heavy metal-contaminated soil.     

Diversity of the Degradation of Panthenol by Microorganisms

Several microorganisms isolated from soil were found to grow in the medium containing panthenol. The results of the investigation of the degradative metabolism of this compound demonstrated that there are two different inducible pathways.

Strain 1041 produced 3-aminopropanol and β-alanine when grown with panthenol. 3-Aminopropanol plus pantoate, as well as panthenol, supported the growth of induced culture. Both washed cells and cell extract of the organism also produced 3-aminopropanol, which was then oxidized to β-alanine. No oxidation of panthenol to pantothenic acid was observed. Isolation and identification of the products were performed. These results led to the conclusion that panthenol is hydrolyzed to pantoic acid and 3-aminopropanol as the first step, which is then followed by oxidation to β-alanine.

Strain 1091 produced pantothenic acid, but not 3-aminopropanol, from panthenol. 3-Aminopropanol plus pantoate did not support the growth of the induced culture. No degradation of 3-aminopropanol was observed. Isolation and identification of pantothenic acid and a 3-methyl-2-benzothiazolone hydrazone derivative of the aldehyde form panthenol were performed. From the results, it was confirmed that panthenol is first oxidized to pantothenic acid, which is then hydrolyzed to β-alanine and pantoic acid.

Panthenol was also oxidized to pantothenic acid by Bacillus roseus AKU 0208. The enzyme was not induced in the presence of panthenol.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂ and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.     

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.