Isolation and characterization of phosphate solubilizing bacteria from corn rhizosfer

The aim of this study was to isolate and characterize the Phosphate solubilizing bacteria from the rhizosphere of Zea mays L., Jeneponto Regency. This research was conducted in several stages; i.e, sampling, medium preparation, sample dilution, isolation, characterization in the form of gram staining, biochemical tests, and quantitative tests of phosphate solubility. Soil samples were diluted in 0.9% NaCl and soil containing microbes was isolated on the Picovskaya medium. Three isolates were obtained which could dissolve phosphate, namely J2KN1, J3KR2, and J3TG3 isolates. The isolates were generally round in shape with raised elevations, white, slimy, smooth, shiny surface, milky white, shape like coccus and bacillus, and gram-negative. Some of the isolates had positive motility, indole, voges, methyl red, glucose, and sucrose fermentation in the biochemical test. The quantitative tests of the ability to dissolve phosphate showed that J2KN1 isolate had the highest concentration of 51.1 μM, and the J3KR1 and J3TG3 isolates had a concentration of 45.2 μM and 37.6 μM, respectively.

Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone–butanol–ethanol production

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n -butanol production. The combined expression of StSUT1 , encoding a sucrose transporter from potato ( Solanum tuberosum ), and SUC2 , encoding a sucrose invertase from Saccharomyces cerevisiae , remarkably enhanced n -butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium . The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

Degenerate primed randomly amplified polymorphic DNA (DP-RAPD) fingerprinting of bacteriophages of Vibrio harveyi from shrimp hatcheries in Southern India

Vibrio harveyi is a significant pathogen of shrimp. Seventy-six bacteriophages infecting luminescent V. harveyi were isolated from a total of 194 water samples drawn from various sources of shrimp hatcheries located in South East coast and Andaman island of India. Degenerate primed randomly amplified polymorphic DNA (DP- RAPD) fingerprinting of these bacteriophages was carried out to determine their genetic relatedness. Similarity matrix based on Dice coefficient followed by construction of dendrogram by unweighted pair group method with arithmetic averages (UPGMA) revealed 12 major clusters. One phage was randomly selected from each of these major clusters for transmission electron microscopic observations. Eleven of them had an icosahedral head (46-115 nm) with a long non- contractile tail (132-329 nm), belonging to the Siphoviridae family and two phages had a short tail (15-27 nm), belonging to the family Podoviridae. The phylogenetic analysis of the phages using DP-RAPD fingerprinting correlated to some extent to the phenotypic nature of the host specifically with regard to sucrose fermentation and source of isolation. However, phages specifically infecting V. harveyi and those belonging to different families did not cluster together in the DP-PCR cluster analysis. Hence, the genetic diversity of phages infecting same host with respect to phenotypic difference was revealed by the DP-RAPD applied in this study.

A Convenient Fluorescence-Based Assay for the Detection of Sucrose Transport and the Introduction of a Sucrose Transporter from Potato into Clostridium Strains

A convenient and effective sucrose transport assay for Clostridium strains is needed. Traditional methods, such as 14C-sucrose isotope labelling, use radioactive materials and are not convenient for many laboratories. Here, a sucrose transporter from potato was introduced into Clostridium, and a fluorescence assay based on esculin was used for the analysis of sucrose transport in Clostridium strains. This showed that the heterologously expressed potato sucrose transporter is functional in Clostridium. Recombinant engineering of high-level sucrose transport would aid sucrose fermentation in Clostridium strains. The assay described herein provides an important technological platform for studying sucrose transporter function following heterologous expression in Clostridium.

Novel Proteomic changes in Yeast Mitochondria provide insights into mitochondrial functioning upon over-expression of human p53

Cancer cells display enhanced glycolytic activity and impaired oxidative phosphorylation even in the presence of adequate oxygen (Warburg effect). Mitochondrial physiology is a promising hit target for anti-cancer therapy because of its key role in Warburg effect and activating apoptosis in mammalian as well as yeast cells. Over-expression of human p53 in S.cerevisiae leads to cell cycle arrest and apotosis. In the present work we show that how S.cerevisiae escapes from p53 induced apoptosis in fermentable carbon source, whereas in case of non-fermentable carbon source this phenomenon is not observed. To shed the light on this aspect we performed a quantitative proteomic analysis of yeast mitochondria isolated from the cells grown on sucrose (fermentation) and glycerol (respiration) with and without p53 over-expression. Through this approach, we identified a total dataset of 1120 proteins with 1% FDR, of which 239(133+106) proteins are differentially experssed in both conditions. Interestingly, we observed that after over-expression of p53 in sucrose grown yeast cells, a complete set of pentose phosphate pathway (PPP) enzymes is up-regulated in the mitochondria that leads to enhanced mitochondrial NADPH production and ROS quenching. Increased association of a hexose transporter (HXT6) and a hexokinase (HXK2) with the mitochondria of fermenting yeast cells upon over-expression of p53, may direct glucose towards PPP inside the mitochondria. In conclusion, our results provide the evidence that up-regulated PPP inside the mitochondria is a key to evade apoptosis by S.cerevisiae upon p53 over-expression.

The Chaaracteristics Yeast Isolated from Commercial Kefir Grain, Indonesia

The objective of this study was to investigate yeast characteristics obtained from commercial grain kefir from Indonesian. Isolation based on macroscopic morphology and microscopic morphology. The first method of research is activation of kefir grain using 10% reconstitution milk, yeast growth on Potato Dextrose Agar (PDA-Agar) medium, yeast coloration by using Lactophenol cotton blue. Then, yeast identification was done by macroscopic and microscopic morphology. Macroscopic morphological observations are observations of colony morphology at the time of isolation and purification, including size, shape, texture, color, surface, elevation, and edges. Microscopic morphological observations include cell shape, budding (budding) which first make preparations with yeast coloring then observed with Zeiss Asio Imager A2 Microscope using Zeiss Axiocam HRC camera. Macroscopic observation of yeast size description colony very small, small, medium, large, colony form is round, the margin is raised, the elevation is entire, the texture is smooth and surface glistening, cream colony color, and yeast smell characteristic. Microscopic observation seen there is cell nucleus, oval, there is pseudohypa, budding, gram-positive, urea test negative, glucose, lactose, maltose fermentation test positive, sucrose fermentation test negative, growth test on liquid media growth in surface medium (pellicle), and bottom medium (sediment). Based on the morphological observations in macroscopic and microscopic yeast identified genus Saccharomyces.

Multidrug Resistance in Large-Plasmid-Associated Presumptive Enterohaemorrhagic Escherichia coli Isolated from Contaminated Lake Water

To determine the presence of enterohaemorrhagic Escherichia coli (EHEC), 50 water samples of three different lakes in Dhaka, Bangladesh were studied. A total of 90 strains were examined thoroughly to isolate sorbitol non-fermenting (NSF) E. coli. Twelve of 90 isolates (13.3%) from 5 samples were found to be sorbitol nonfermenter, which were further screened for rhamnose, cellobiose and sucrose fermentation. Eleven (12.2%) of 90 isolates also tested negative for cellobiose and sucrose fermentation, while only 3 (3.3%) of them were found to be rhamnose non-fermenting. Biochemical tests confirmed that 91.7% (11 of 12) of NSF strains were indole positive. Eleven NSF E. coli isolates survived at 45.5°C and mostly produced gas by fermentation within 24h. After antimicrobial susceptibility testing, 91.7% (n=11) of these isolates were found resistant to cephalexin, ampicillin and amoxicillin. Ceftazidime and nalidixic acid resistance was also observed in 75% (9 of 12) and 58.3% (7 of 12) isolates, respectively. Plasmid profile analysis revealed that nine (75%) strains harboured large plasmids of >35.8-140 megadaltons (MDa), four (33.3%) of which contained 60-MDa plasmid. Also, the large-plasmid-associated strains shared common behaviour in antimicrobial resistance and eight (88.9%) of these were resistant to four or more antibiotics. Our data suggest a possible association of source water contamination in Bangladesh with multidrug-resistant (MDR) EHEC, which is alarming to public health. DOI: http://dx.doi.org/10.3329/bjm.v28i1.11807 Bangladesh J Microbiol, Volume 28, Number 1, June 2011, pp 33-40