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World Journal of Microbiology and Biotechnology (v.23, #11)

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.) by Rafik Errakhi; Francois Bouteau; Ahmed Lebrihi; Mustapha Barakate (pp. 1503-1509).
Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.

Keywords: Streptomyces ; Biological control; Sugar beet damping-off; Sclerotium rolfsii

Development of soil amoeba Dictyostelium discoideum as an expression system for recombinant human erythropoietin by Bhavesh Vats; Harish Padh (pp. 1511-1518).
Erythropoietin (EPO) is the most important biopharmaceutical product at present being produced by recombinant DNA technology from mammalian cell lines. Other available expression systems have not been useful in producing this protein due to the requirement of N-glycosylation for in vivo activity. In order to develop an alternative expression system, the human epo gene was expressed in the cellular slime mold Dictyostelium discoideum. The 2.43 kbp epo gene from the mammalian expression vector was cloned in the Dictyostelium expression cassette and used to transform cells. Positive clones were selected on the basis of antibiotic resistance. The clones were screened for the presence of the transgene. The copies of the gene inserted in the genome were identified and the transcript too was ascertained. The protein was identified by immuno-blotting and appears to be glycosylated though differently from that of humans or CHO cell lines. It has not yet been tested in bioassay.

Keywords: Dictyostelium discoideum ; EPO; Heterologous expression; Therapeutic proteins

Improvement of laccase production from Ganoderma sp. KU-Alk4 by medium engineering by Churapa Teerapatsakul; Roberto Parra; Christopher Bucke; Lerluck Chitradon (pp. 1519-1527).
To engineer the production of laccase by Ganoderma sp. KU-Alk4, a newly isolated white-rot fungus, a seven-level Box−Behnken factorial design was employed to optimize the culture medium composition. A mathematical model was developed to show the effect of each medium component and their interactions on the production of laccase activity in submerged fermentation. The model estimated the optimal concentrations of glycerol, yeast extract and veratryl alcohol as 40, 0.22 g/l and 0.85 mM, respectively, with the medium pH of 6.0. These predicted conditions were verified by validation experiments. The optimized medium gave laccase activity of 240 U/ml, which is 12 times higher than that produced in non-optimized medium. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for Ganoderma sp. KU-Alk4, resulted the high laccase production.

Keywords: Box–Behnken design; Ganoderma ; Glycerol; Laccase; Media optimization

Oxalotrophic Paracoccus alcaliphilus isolated from Amorphophallus sp. rhizoplane by Kolandaswamy Anbazhagan; Chelliah Edward Raja; Govindan Sadasivam Selvam (pp. 1529-1535).
A new type of oxalate-utilizing Paracoccus sp. was isolated from Amorphophallus rhizoplane. Basic physiological tests and 16S rDNA sequencing was performed to identify the strain. Optimum growth was observed at 37 °C and pH 8.0 in minimal oxalate (0.5% disodium oxalate) with doubling time of 7 h. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, were Paracoccus alcaliphilus KCT002, P. alcaliphilus JCM 7364 (TK1015) and P. seriniphilus MBT-A4 with 99.85, 98.35 and 97.31% sequence similarity respectively. Oxalate was metabolized via the serine pathway, as evidenced by the presence of oxalyl-coA reductase, l-serine glyoxylate aminotransferase and hydroxypyruvate reductase.

Keywords: Amorphophallus sp.; Paracoccus alcaliphilus ; Oxalotroph; Rhizoplane; Oxalate metabolism; Serine pathway

Keratinases vis-à-vis conventional proteases and feather degradation by Priya Ramnani; Rani Gupta (pp. 1537-1540).
Keratinases degrade feather in presence of a suitable reducing agent. Here we have demonstrated that conventional serine and cysteine proteases (subtilsin, chymotrypsin and papain) which selectively cleave proteins at the hydrophobic P1 residues also degrade feathers in presence of a suitable reducing agent in the form of live cells or chemical reductants. Further, trypsin and pepsin were also shown to degrade feather after cleaving hydrophobic residues of feathers following 2 h pre-treatment by any of the proteases.

Keywords: Bacillus subtilis WB600; Chymotrypsin; Disulfide reductases; Feather degradation; Keratinase RG1; Papain; Subtilisin; Trypsin

LPS-based conjugate vaccines composed of O-polysaccharide from Pseudomonas aeruginosa IATS 6 and 11 bound to a carrier protein by Munir Al-Zeer; Hussein Masoud (pp. 1541-1549).
Lipopolysaccharide (LPS) is the major surface antigen of Pseudomonas aeruginosa and elicits protective antibodies in animals. No cross reaction was observed between LPSs of P. aeruginosa International Antigenic Typing Scheme (IATS) 6 and 11 strains using rabbit polyclonal antibodies raised against the whole cells. The O-polysaccharides (O-PSs) from IATS 6 and 11, the antigenic determinant of LPS, were directly coupled to bovine serum albumin (BSA) by carbodiimide mediated condensation reaction. The molar ratios of saccharide repeating units to BSA in the prepared conjugates were 15:1 and 26:1 for IATS 6 and 11 conjugates, respectively. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL), Freund, and Alum adjuvants. The conjugates emulsified with MPL adjuvant elicited the highest IgM antibody, followed by Freund. While both MPL and Freund adjuvants elicited high IgG antibody. Good correlation was observed between the IgG and IgM levels with the bactericidal activities of the sera against homologous strains. In addition, immunization of mice with the prepared conjugates emulsified with MPL and Freund adjuvants provided high protection against ten times the LD50 of P. aeruginsoa IATS 6 and 11, which showed a good correlations with the IgG titer.

Keywords: Adjuvant; Conjugate vaccine; ELISA; Lipopolysaccharide; Pseudomonas aeruginosa

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases by J. W. Zhang; R. Y. Zeng (pp. 1551-1557).
Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.

Keywords: α-Amylase; Deep sea sediment; Prydz Bay; Psychrotrophic

Novel laccases of Ganoderma sp. KU-Alk4, regulated by different glucose concentration in alkaline media by Churapa Teerapatsakul; Naoki Abe; Christopher Bucke; Ngampong Kongkathip; Saeree Jareonkitmongkol; Lerluck Chitradon (pp. 1559-1567).
Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs differed in their molecular mass, ranging from 53 to 112 kDa. KULac 2 was a new laccase that had a different N-terminal amino acid sequence from other laccases previously reported. All the isozymes had optimum pH at 3.5 and were stable over the wide range of pH, 3.0–10.0, especially in alkaline pH. It is noteworthy that the activities of the four KULacs with 2,6-dimethoxyphenol were extremely high up to 90°C. They retained 100% of their activities at 60°C for 1 h.

Keywords: Ganoderma ; Glucose effect; Isozyme; Laccase; Purification

Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent by V. P. Zambare; S. S. Nilegaonkar; P. P. Kanekar (pp. 1569-1574).
The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.

Keywords: Bacillus cereus ; Alkaline protease; Dehairing; Buffalo hide; Leather processing

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters by Saswati Nayak; Radha Prasanna; Boddupalli M. Prasanna; Dina B. Sahoo (pp. 1575-1584).
A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.

Keywords: Anabaena ; Diversity; Morphology; Nitrogen fixation; Pigments; Protein profiles

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions by Sarvesh Kumar Soni; Jayant Malhar Khire (pp. 1585-1593).
Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH2PO4 (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg2+, Mn2+, Ca2+ and Fe3+ ions and inhibited by Zn2+ and Cd2+ ions while Phy II activity was moderately stimulated by Fe3+ ions and was inhibited by Hg2+, Mn2+ and Zn2+ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.

Keywords: Phytase; Aspergillus niger ; Submerged fermentation; Phytate degradation; Poultry feed supplement

Nitrate removal with bacterial cells attached to quartz sand and zeolite from salty wastewaters by Lucija Foglar; Laszlo Sipos; Nenad Bolf (pp. 1595-1603).
A mixed bacterial culture was acclimated to the removal of high nitrate-N concentrations (100–750 mg NO3 -N L−1) from salty wastewaters. The experiments were carried out under anoxic conditions in the presence of 0.5, 1.5 and 3% (w/v) NaCl at different temperatures. The acclimated mixed bacterial culture was attached to quartz sand and zeolite. Denitrification was monitored in a continuous-flow bioreactor at different hydraulic retention times (HRT). Nitrate removal with cells attached to quartz sand and zeolite was completed at HRT of 167 h and 25 h respectively. Then brine denitrification with bacterial cells attached to zeolite was monitored for 85 days. Under the increased nitrate loading rate, nitrate removal was above 90%. Furthermore, during denitrification, not more than 0.5 mg NO2 -N L−1 could be produced. It can be concluded that nitrate removal with the cells attached to zeolite is economically and operationally a promising solution to denitrification of brine wastewaters.

Keywords: Denitrification; Spent brine; Mixed bacterial culture; Anoxic conditions; Quartz sand; Zeolite

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions by Kamila Myszka; Katarzyna Czaczyk; Marcin T. Schmidt; Anna M. Olejnik (pp. 1605-1612).
The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.

Keywords: Adhesion; Biofilm; Extracellular molecules; Hydrophobicity; Proteus vulgaris ; Starvation

Identification of yeasts within Saccharomyces sensu stricto complex by PCR-fingerprinting by Scheila Karina Brito dos Santos; Anna Carla Moreira Basílio; Bereneuza Tavares Ramos Valente Brasileiro; Diogo Ardaillon Simões; Eurípedes Alves da Silva-Filho; Marcos de Morais Jr (pp. 1613-1620).
Biological relatedness makes species characterization of the industrially important Saccharomyces sensu stricto complex difficult. In this paper we present a set of PCR-fingerprinting markers based in Single Primer Amplification Reactions (SPAR) that, together with PCR-ribotyping and single gene RFLP analysis, can effectively identify individual species and fully characterize the hybrid nature of industrial isolates. With those markers, all six yeast species of the sensu stricto complex could be discriminated and we also identified errors in the previous taxonomic characterization of certain wine yeasts. The unique patterns generated by the SPAR markers could be useful in monitoring yeast populations during industrial fermentation processes and can be used to detect the appearance of yeast hybrids in these environments.

Keywords: Industrial yeast; Molecular taxonomy; PCR-fingerprinting; Saccharomyces sensu Stricto; SPAR; Yeast hybrids

Expression and characterization of inhA gene from Bacillus thuringiensis 8010 by Xiaomin Yu; Tianpei Huang; Zhipeng Huang; Charles A. Powell; Xiong Guan (pp. 1621-1625).
InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.

Keywords: Bacillus thuringiensis ; Bioassay; Expression; inhA gene; Sequence analysis

Anaerobic biodegradation of polychlorinated biphenyls by a microbial consortium originated from uncontaminated paddy soil by Daisuke Baba; Tsuyoshi Yasuta; Naoko Yoshida; Yuko Kimura; Katsuhide Miyake; Yasushi Inoue; Koki Toyota; Arata Katayama (pp. 1627-1636).
Polychlorinated biphenyls (PCBs) in Kanechlor-300 and -400 mixtures dissipated significantly compared with a sterilized control under anaerobic conditions in three Japanese paddy soils with no history of PCB contamination, demonstrating the anaerobic microbial degradation of PCBs. The PCB-degrading activity was maintained successfully in a static flooded soil medium for more than 3 years by serial transfer at intervals of 56 days (13 transfers). Ortho-, meta-, and para-substituted PCBs, 15.2 ± 9.9 mol% in total, were significantly degraded after 56 days of incubation. Analysis of menaquinones-6 and -7 and cloning of 16S rRNA gene fragments from a polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) profile indicated the predominance of Firmicutes in the consortium. A PCR-based identification of the gene fragments showed the frequent presence of Desulfitobacterium sp., but not Dehalobacter sp. or Dehalococcoides sp., in the consortium. It is proposed that Japanese paddy soils with no history of PCB contamination contain an anaerobic microbial consortium consisting predominantly of Firmicutes that have the potential for anaerobic degradation of PCB.

Keywords: Anaerobic microbial degradation; Dechlorination; Polychlorinated biphenyls; Paddy soils; Activity maintenance; Microbial consortium; Firmicutes

Photomechanical wave-assisted molecular delivery in oral biofilms by Makoto Ogura; Abraham D. Abernethy; Ryan D. Blissett; Karriann Ruggiero; Sovanda Som; J. Max Goodson; Ralph Kent; Apostolos G. Doukas; Nikolaos S. Soukos (pp. 1637-1646).
Photomechanical waves (PW), the product of an intense light beam interaction with a target material, enhance molecular delivery across biological membranes and skin. The ability to deliver methylene blue (MB), a fluorescent probe and photosensitizer, into bacterial biofilms was demonstrated by applying PW on saliva-derived multi-species biofilms that were developed on agar surfaces in 24-well plates. PW were generated with a Q-switched Nd:YAG laser and were directed into the biofilms in the presence of 25 μg/ml MB. The biofilms were then irradiated with red light at 665 nm. After illumination, adherent bacteria were scraped and spread over the surface of blood agar plates. Survival fractions were calculated by counting bacterial colonies. Microbial analysis was performed via a colony lift method and a DNA checkerboard assay using whole genomic probes to 40 oral microorganisms. Visual analysis by confocal scanning laser microscopy demonstrated that the application of PW enhanced the penetration depth of MB in biofilms. Exposure to MB, PW and light led to a significant reduction of the mean levels of log10 CFU counts compared with the group that received MB and light (P = 0.006). The DNA checkerboard assay showed some benefit from PW-assisted phototargeting in 25 biofilm microorganisms relative to phototreatment alone. Our data provide a basis for further exploration and optimization of PW parameters for complete eradication of microorganisms in oral microcosm biofilms.

Keywords: Confocal scanning laser microscopy; Drug delivery; Oral biofilms; Photodynamic therapy; Photomechanical waves; Stress waves; Ultrasound

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley by Dharmendra Kumar; Ramesh Chand; Lal C. Prasad; Arun K. Joshi (pp. 1647-1651).
We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.

Keywords: Bipolaris sorokiniana ; Cochliobolus sativus ; Hemibiotrophic; Spot blotch; Isolate

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