Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Campylobacter spp., Giardia spp., Cryptosporidium spp., Noroviruses, and Indicator Organisms in Surface Water in Southwestern Finland, 2000-2001

A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli, Clostridium perfringens, and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni, 25.0% contained Campylobacter lari, 4.2% contained Campylobacter coli, and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly (P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli, and C. perfringens had significant bivariate nonparametric Spearman's rank order correlation coefficients (P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 × 10⁸, 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.     

Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.     

Confirmation of the chromosomal localization of human lamp genes and their exclusion as candidate genes for Salla disease

Summary Salla disease is an inherited lysosomal storage disorder caused by accumulation of free sialic acid in the lysosomes. Lamp genes, lamp A and lamp B (lysosome associated membrane proteins), are the first known genes encoding for human lysosomal membrane proteins. Absence of linkage in a large group of families shows that lamp genes are not involved in Salla disease. The lamp genes were localized, using Southern hybridization in hamster — human hybrid cell panels, to chromosomes 13 (lamp A) and X (lamp B).     

Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting

Fibrobacter succinogenes S85 grew rapidly on cellobiose (0.31 h⁻¹) and the absolute rate of increase in fermentation acids was 0.68 h⁻¹. Cultures that were provided with ball-milled cellulose initially produced fermentation acids and microbial protein as fast as those provided with cellobiose, but the absolute cellulose digestion rate eventually declined. If the inoculum size was increased, the kinetics decayed from first to zero order (with respect to cells) even sooner, but in each case the absolute rate declined after only 20 to 30% of the cellulose had been fermented. Congo red binding indicated that the cellulose surface area of individual cellulose particles was not decreasing, and the transition of ball-milled cellulose digestion corresponded with the appearance of unbound cells in the culture supernatant. When bound cells from partially digested cellulose were removed and the cellulose was re-incubated with a fresh inoculum, the initial absolute fermentation rate was as high as the one observed for undigested cellulose and cellobiose. Based on these results, cellulose digestion by F. succinogenes S85 appears to be constrained by cellulose surface area rather than cellulase activity per se. Received: 19 January 2000 / Received revision: 18 April 2000 / Accepted: 1 May 2000     

First report of leaf spot disease caused by Corynespora torulosa MCC-1368 on cotton plant from India

In present study, the leaf spot disease of cotton plant emerged in the North Maharashtra region of India was reported. The fungal phytopathogen associated with inducing the leaf spot disease symptoms was isolated and characterised. The isolated fungus was identified as Corynespora torulosa (Deposition accession number, MCC-1368; Genbank accession no. MF462072) based on morphological and cultural characteristics and molecular analysis of ITS region. The pathogenicity of fungal phytopathogen was verified by Koch’s postulates. To our knowledge, this is the first report of incidence of leaf spot disease caused by Corynespora torulosa on cotton plant.     

Create and preserve: Proteostasis in development and aging is governed by Cdc48/p97/VCP

The AAA-ATPase Cdc48 (also called p97 or VCP) acts as a key regulator in proteolytic pathways, coordinating recruitment and targeting of substrate proteins to the 26S proteasome or lysosomal degradation. However, in contrast to the well-known function in ubiquitin-dependent cellular processes, the physiological relevance of Cdc48 in organismic development and maintenance of protein homeostasis is less understood. Therefore, studies on multicellular model organisms help to decipher how Cdc48-dependent proteolysis is regulated in time and space to meet developmental requirements. Given the importance of developmental regulation and tissue maintenance, defects in Cdc48 activity have been linked to several human pathologies including protein aggregation diseases. Thus, addressing the underlying disease mechanisms not only contributes to our understanding on the organism-wide function of Cdc48 but also facilitates the design of specific medical therapies. In this review, we will portray the role of Cdc48 in the context of multicellular organisms, pointing out its importance for developmental processes, tissue surveillance, and disease prevention. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.