Andrimid production at low temperature by a psychrotolerant Serratia proteamaculans strain

Andrimid, a known non-ribosomal pseudo-peptide antibiotic, was isolated from a psychrotolerant Serratia proteamaculans strain. The antibiotic peptide was produced at low temperature (8 °C) in a 7.5 l BIOFLO 101 bioreactor under batch culture mode. Andrimid activity from S. proteamaculans culture was only detected at 25 °C and below and potent antibacterial activity was revealed against both, pathogenic and non-pathogenic bacteria. Minimal inhibitory concentration values determined by microdilution experiments varied in the range between 0.01 and 0.78 μg/ml. Antimicrobial purification and structure elucidation were carried out by LC-MS/MS and ¹H/¹³C NMR approaches. The effects on the ultrastructure of sensitive Escherichia coli 35,218 cells were observed by transmission electron microscopy at different inhibition stages. This work demonstrated the significance of bioprospection from cold environments through the screening of microorganisms with ability to produce cold-active biomolecules of biotechnological interest. S. proteamaculans 136 was revealed as a novel microbial source for andrimid production at low temperatures, showing biotechnological potential to be applied in cryopreservation, food or cosmetic industries against pathogenic bacteria.     

Denitrification by thermophilic soil bacteria with ethanol as substrate in a USB reactor

Summary Thermophilic biological denitrification was studied in a laboratory-upflow sludge blanket reactor fed with ethanol as carbon and energy source. High denitrification efficiency (>98%) was obtained at an ethanol: nitrate ratio >2 and at a hydraulic retention time (HRT) of 5 hours. The performance of the system with respect to nitrate removal was very satisfactory (>95%), even at high nitrate (235 mg NO₃-N/L) and hydraulic (3 hours HRT) loading rates applied.A stable sludge was formed by spherical granules 1 to 3 mm in diameter with a content of 25,8 g VS/L and were almost exclusively composed of bacteria belonging to the genus Bacillus.     

Simul taneous production of alpha and beta amylases byBacillus subtilis MIR-5 in batch and continuous culture

Summary Simultaneous production of - and -amylase was studied in batch and continuous culture using starch as substrate inB. subtilis MIR-5. By mainipulating the cultural condition, both enzymes could then be produced by the same strain.Footnote: dedicated to 75^th aniverssary of Dr. Raul E. Trucco.     

Regulation by Membrane Fluidity of the Allosteric Behavior of the (Ca2+)-Adenosine Triphosphatase from Escherichia coli

The allosteric properties of the membrane-bound (Ca(2+))-adenosine triphosphatase of an unsaturated fatty acid auxotroph of Escherichia coli were studied in membranes with different fatty acid compositions. The Hill coefficient of the inhibition by Na(+) ranged from 1.4, in the case where the auxotroph was grown with cis-vaccenic acid as supplement, to 2.8 when grown on linolenic acid. The results indicate that no fatty acid is particularly involved in the allosteric phenomena. A correlation between the values of the Hill coefficient and the double bond index or the ratio of the double bond index saturated to the fatty acids of the membrane was found. These facts are interpreted as a modulation by the membrane fluidity of the allosteric behavior of the membrane-bound enzyme. The general biological character of this phenomenon is discussed in this paper.     

Isolation and evaluation of tannin-degrading fungal strains from the Mexican desert

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source.     

Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment

ETR-3 (also know as BRUNOL3, NAPOR, and CUGBP2) is one of six members of the CELF (CUG-BP1- and ETR-3-like factor) family of splicing regulators. ETR-3 regulates splicing by direct binding to the pre-mRNA. We performed systematic evolution of ligands by exponential enrichment (SELEX) to identify the preferred binding sequence of ETR-3. After five rounds of SELEX, ETR-3 selected UG-rich sequences, in particular UG repeats and UGUU motifs. Either of these selected motifs was able to restore ETR-3 binding and responsiveness to a nonresponsive splicing reporter in vivo. Moreover, this effect was not specific to ETR-3 since minigenes containing either of the two motifs were responsive to two other CELF proteins (CUG-BP1 and CELF4), indicating that different members of the CELF family can mediate their effects via a common binding site. Using the SELEX-identified motifs to search the human genome, we identified several possible new ETR-3 targets. We created minigenes for two of these genes, the CFTR and MTMR1 genes, and confirmed that ETR-3 regulates their splicing patterns. For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins.     

Can wild Triatoma infestans foci in Bolivia jeopardize Chagas disease control efforts?

The expected success of Chagas disease control programs in the Southern Cone countries relied on the assumption that Triatoma infestans, the main domestic vector, did not maintain silvatic foci except in the Cochabamba valley in Bolivia. Recent fieldwork revealed that wild populations of this vector are much more widespread throughout Bolivia than previously thought. Therefore, it is important to find out whether these silvatic populations could jeopardize control efforts in Bolivia, and to investigate their possible occurrence in neighboring regions of Paraguay and Argentina.     

A simple solid phase assay for the detection of 2,4-D in soil

Contaminated soils are usually characterized using chemical analyses. However, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. Thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. Although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies have been carried out in slurries or soil extracts rather than soil itself. The following study presents the development of a simple solid-phase bioassay for the direct detection of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) in soil using Ralstonia eutropha JMP 134-32, a luxCDABE-based 2,4-D whole cell bioreporter. The bioreporter was spotted onto glass microfibre filter discs that allowed its retrieval and analysis after exposure to 2,4-D amended soils. These disc-fixed cells responded in a concentration dependent manner to 2,4-D in solution (0-25 mg/L) and in spiked soil (0-50 mg/kg). The influence of environmental factors on bioavailability was demonstrated in soil with a low moisture content which prevented 2,4-D-induced bioluminescence but which did not affect bioluminescence from already induced cells. This rapid and low cost bioassay provides a proof of concept demonstrating that retrievable disk-fixed cells can be induced in soil, thus providing a measure of solid-phase bioavailability. This method overcomes some of the limitations associated with the inoculation and monitoring of bioreporters directly in soil. Additionally, this simple system should be amenable to use with other bioreporters.     

Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.     

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain

A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.