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Archives of Microbiology (v.191, #5)

Heterologous expression of the msp2 gene from Marasmius scorodonius by Kateryna Zelena; Holger Zorn; Manfred Nimtz; Ralf Günter Berger (pp. 397-402).

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.

Keywords: Basidiomycete; E. coli ; Heterologous expression; In vitro refolding; Peroxidase

Levels and localization of mechanosensitive channel proteins in Bacillus subtilis by Paul G. Wahome; Ann E. Cowan; Barbara Setlow; Peter Setlow (pp. 403-414).

Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock ≥0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells’ osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.

Keywords: Bacillus ; Mechanosensitive channels; Osmoregulation; Osmotic downshock; Sporulation; Turgor pressure

Comparative effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions by Zahir Ahmad Zahir; Usman Ghani; Muhammad Naveed; Sajid Mahmood Nadeem; Hafiz Naeem Asghar (pp. 415-424).

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.

Keywords: Salinity; Pseudomonas ; Serratia ; ACC-deaminase; Ethylene; Triticum aestivum L.

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli by Sanath Kumar; Ammini Parvathi; Ricardo L. Hernandez; Kathleen M. Cadle; Manuel F. Varela (pp. 425-429).

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

Keywords: MurA; Fosfomycin; Antibiotic resistance; Vibrio fischeri ; MIC

Chromium uptake, retention and reduction in photosynthetic Euglena gracilis by J. D. García-García; J. S. Rodríguez-Zavala; R. Jasso-Chávez; D. Mendoza-Cozatl; Rafael Moreno-Sánchez (pp. 431-440).

Photosynthetic Euglena gracilis grown with different K₂CrO₄ concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl₃. Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K _m of 1.1 mM, V _m of 16 nmol (h × 10⁷ cells)⁻¹, and K _i sulfate of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K _m = 0.83 mM, V _m = 15.9 nmol (h × 10⁷cells)⁻¹ and K _i chromate = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 × 10⁷ cells at 1 mM K₂CrO₄) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 × 10⁷ cells at 1 mM CrCl₃). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.

Keywords: Chromium uptake; Sulfate uptake; Chromium accumulation; Chromium reduction; Cysteine; Glutathione

Localization and characterization of VVA0331, a 489-kDa RTX-like protein, in Vibrio vulnificus YJ016 by Li-Fang Chou; Hwei-Ling Peng; Yu-Chung Yang; Min-Chieh Kuo; Hwan-You Chang (pp. 441-450).

Vibrio vulnificus YJ016 contains three genes encoding proteins homologous to repeats-in-toxin proteins. One of these genes, vva0331, possesses a long open reading frame of 13,971 bp in length and resides on the small chromosome between two gene clusters encoding a type I secretion system and several regulatory proteins, respectively. Bioinformatic analysis revealed that VVA0331 consist of nineteen 87-amino acid repeats, two Arg-Gly-Asp motifs, four cysteine residues, an outer membrane protein domain, a polysaccharide-binding site and several motifs related to cell adhesions. These features are distinct from those of typical repeat-in-toxins and autotransporter adhesins. Real-time quantitative PCR analysis indicates that vva0331 gene expression is activated at 30°C and regulated by iron. In addition, VVA0331 is present primarily in a secreted form as determined by cell fractionation assay and Western blot analysis. No significant difference in Hep2 cell adherence, cytotoxicity, and virulence was observed between the wild type and vva0331 mutant strains. In contrast, these strains exhibited apparently different outer membrane protein profiles, and antiserum raised against C-terminal region of VVA0331 reacted with an 85-kDa outer membrane protein of V. vulnificus YJ016.

Keywords: Vibrio vulnificus ; RTX; Agglutination; Outer membrane proteins

CsrA interacting small RNAs in Haemophilus spp genomes: a theoretical analysis by Jayavel Sridhar; Kanagaraj Sekar; Ziauddin Ahamed Rafi (pp. 451-459).

The csrA is a carbon storage regulator gene that encodes a protein with multiple RNA interaction sites. Bacterial non-coding small RNAs like csrB, csrC and their counterparts in diverse bacterial genus are identified to control the regulatory activities of CsrA and its orthologs. An attempt has been made in this study to identify ‘novel’ non-coding small RNAs that are involved in the regulatory activities of csrA gene. All CsrA-interacting small RNAs are computationally fingerprinted to have multiple occurrence of 7-nucleotide CsrA interacting repeats [CAGGA(U/A/C)G] along with a 18-nucleotide upstream binding site. However, in several of the genomes like Haemophilus spp, the upstream binding site is not identified. The current methodology overcomes this difficulty by identifying small RNA-specific orphan transcriptional units within the intergenic regions of the genome. The results could identify all known CsrA-interacting small RNAs in E. coli, Vibrio cholerae and Pseudomonas aeruginosa genomes and additionally has picked six new possible CsrA-interacting small RNA regions in E. coli. Our computational analysis indicates that known rygD and rprA sRNAs in E. coli could possibly interact with CsrA proteins. The study is extended to three of the Haemophilus genomes that could identify seven new possible CsrA interacting small RNAs.

Keywords: Carbon storage regulator; Small RNA; Upstream binding site; Haemophilus

Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase by Nicola Steffan; Shu-Ming Li (pp. 461-466).

To create structural diversity of prenylated diketopiperazine derivatives, acceptance of cyclic dipeptides was tested using FgaPT2, a prenyltransferase from Aspergillus fumigatus, which catalyses the conversion of l-tryptophan to 4-dimethylallyl-l-tryptophan. It could be shown that seven tryptophan-containing cyclic dipeptides were accepted by FgaPT2 at high protein concentrations and regiospecifically converted to their C4 prenylated derivatives. The structures of the enzymatic products were elucidated by NMR and LC-MS analyses. This substrate promiscuity of a dimethylallyltryptophan synthase towards cyclic dipeptides increases the potential of the fungal indole prenyltransferases as tools for the production of biologically active compounds.

Keywords: Aspergillus ; Cyclic dipeptides; Diketopiperazine; Dimethylallyltryptophan synthase; Prenyltransferases; Substrate promiscuity

Phosphate accumulation of Acetobacter xylinum by Lubov P. Ryazanova; Natalia E. Suzina; Tatiana V. Kulakovskaya; Igor S. Kulaev (pp. 467-471).

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg²⁺ and glucose, or Mg²⁺ and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by α-ketoglutaric acid in combination with (NH₄)₂SO₄, or arginine, or glutamine, the catabolism of which results in formation of NH₄ ⁺ and α-ketoglutarate.

Keywords: Acetobacter xylinum ; Bacteria; Polyphosphate; Phosphate; Uptake; Localization; Magnesium

Expression of the yggE gene protects Escherichia coli from potassium tellurite-generated oxidative stress by Lillian G. Acuña; Iván L. Calderón; Alex O. Elías; Miguel E. Castro; Claudio C. Vásquez (pp. 473-476).

Potassium tellurite is highly toxic to most forms of life and specific bacterial tellurite defense mechanisms are not fully understood to date. Recent evidence suggests that tellurite would exert its toxic effects, at least in part, through the generation of superoxide anion that occurs concomitantly with intracellular tellurite (Te⁴⁺) reduction to elemental tellurium (Te^o). In this work the putative antioxidant role of YggE from Escherichia coli, a highly conserved protein in several bacterial species and whose function is still a matter of speculation, was studied. When exposed to tellurite, E. coli lacking yggE exhibited increased activity of superoxide dismutase and fumarase C, augmented levels of reactive oxygen species and high concentration of carbonyl groups in proteins. Upon genetic complementation with the homologous yggE gene these values were restored to those observed in the parental, isogenic, wild type strain, suggesting a direct participation of YggE in E. coli tolerance to tellurite.

Keywords: YggE; Tellurite; Oxidative stress; Superoxide

Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc by Aline C. Intorne; Marcos Vinicius V. de Oliveira; Mariana L. Lima; Juliana F. da Silva; Fábio L. Olivares; Gonçalo Apolinário de Souza Filho (pp. 477-483).

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca₅(PO₄)₃OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.

Keywords: Gluconacetobacter diazotrophicus ; Genomic functional analysis; Transposon mutant library; Phosphorus solubilization; Zinc solubilization

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Archives of Microbiology (v.191, #5)

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Archives of Microbiology (v.191, #5)