1,721,021 research outputs found
Dynamics of Promoter Occupancy by the Bacterial DNA-Binding Protein H-NS; Transcriptional Repression Viewed in the Milliseconds Time Scale
No full textHistone-like Nucleoid Structuring protein (H-NS) is a well-characterized DNA-binding protein, known to condensate DNA and to act as a transcriptional repressor on bacterial Gram-negative genes. The proposed mechanism of repression is thought to occur via binding of the protein at multiple binding sites, followed by oligomerization of the protein and spreading to neighbor regions. These events induce a distortion of the double helix axis, leading to DNA curvature. As a consequence, the activity of RNA polymerase at the promoter sites is inhibited due to entrapping or to exclusion from the target sequence. We have investigated the interaction of H-NS with the promoter region of VirF, a gene responsible for the multistep pathogenicity cascade of Shigella flexneri. The results of footprinting experiments carried out at equilibrium in the presence of H-NS or its monomeric DNA binding domain (H-NSctd), indicate the presence of more than ten primary binding sites. These nucleation points have been indexed over a millisecond time scale by quantitative analyses of time-resolved hydroxyl radical footprinting experiments. Furthermore, fluorescent polarization studies carried out with six DNA fragments (22mer) corresponding to slow, medium and fast binding sites allowed us to determine the kinetic parameters of the interaction between H-NS and the isolated DNA targets. The combination of biomolecular and biophysical approaches will lead to a molecular model where the DNA-protein interactions, analysed on a temporal scale, provide a dynamic view of the mechanism of gene repression
Screening for Microbial Strains Degrading Glass Fiber Acrylic Composite Filters
No full textIn this study we have assessed the capacity of five fungal and two bacterial species to biodegrade glass fiber acrylic composite filters which are utilized in air conditioners. The strains used were Trichoderma harzianum (2 strains), Trichoderma koningii, Penicillium spp., Aspergillus niger, Pseudomonas aeruginosa and Pseudomonas fluorescens. Pre-sterilized filters were incubated in solid or liquid media at 30 C for 21 days. Biodegradability was monitored by evaluating microbial colonization, increase in biomass and weight loss of filters coupons. Among the species under investigation, the two strains of T harzianum (MYA198 and BCC5828) showed the best biodegradability performance and were used to analyse total carbon and esterase activity. Our results clearly indicate that cells grown in the presence of shredded filters display a hydrolytic activity and lead to a consistent removal of the organic portion of the tested filters. This study suggests that a solid state fermentation process in suitable bioreactors based on T. harzianum species could be a suitable approach to acrylic composite filter biodegradation
Probing the relation between protein–protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS
No full textWe have investigated the relationship between oligomerization in solution and DNA binding for the bacterial
nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a
H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly
more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization
in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter
region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments
show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than
for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional
repression.Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between
oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the
notion that cooperative binding of H-NS to DNA is related to protein–protein interactions. These results emphasize
the relative contributions of protein–protein interactions and substrate-dependent oligomerization in the
control of gene repression operated by H-NS
SaDA: From Sampling to Data Analysis—An Extensible Open Source Infrastructure for Rapid, Robust and Automated Management and Analysis of Modern Ecological High-Throughput Microarray Data
Full text linkOne of the most crucial characteristics of day-to-day laboratory information management is the collection, storage and retrieval of information about research subjects and environmental or biomedical samples. An efficient link between sample data and experimental results is absolutely important for the successful outcome of a collaborative project. Currently available software solutions are largely limited to large scale, expensive commercial Laboratory Information Management Systems (LIMS). Acquiring such LIMS indeed can bring laboratory information management to a higher level, but most of the times this requires a sufficient investment of money, time and technical efforts. There is a clear need for a light weighted open source system which can easily be managed on local servers and handled by individual researchers. Here we present a software named SaDA for storing, retrieving and analyzing data originated from microorganism monitoring experiments. SaDA is fully integrated in the management of environmental samples, oligonucleotide sequences, microarray data and the subsequent downstream analysis procedures. It is simple and generic software, and can be extended and customized for various environmental and biomedical studies
Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs
No full textThe use of antibiotics has revolutionized medicine, greatly improving our capacity to save millions of lives from otherwise deadly bacterial infections. Unfortunately, the health-associated benefits provided by antibiotics have been counteracted by bacteria developing or acquiring resistance mechanisms. The negative impact to public health is now considered of high risk due to the rapid spreading of multi-resistant strains. More than 60 % of clinically relevant antibiotics of natural origin target the ribosome, the supramolecular enzyme which translates the genetic information into proteins. Although many of these antibiotics bind the small ribosomal subunit, only a few are reported to inhibit the initiation of protein synthesis, with none reaching commercial availability. Counterintuitively, translation initiation is the most divergent phase of protein synthesis between prokaryotes and eukaryotes, a fact which is a solid premise for the successful identification of drugs with reduced probability of undesired effects to the host. Such a paradox is one of its kind and deserves special attention. In this review, we explore the inhibitors that bind the 30S ribosomal subunit focusing on both the compounds with proved effects on the translation initiation step and the underreported translation initiation inhibitors. In addition, we explore recent screening tests and approaches to discover new drugs targeting translation
Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions
Full text linkThe most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA)
The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms.
Full text linkDiatoms are major contributors to global
carbon fixation and constitute a significant portion of
biofilms found in lotic ecosystems. Despite their
widespread abundance and the fact that extensive
studies have been performed on morphological features
of frustules, molecular tools for the identification
of diatoms are not commonly available. This study
focuses on the development of oligonucleotide probes
for the detection of diatom species relevant to water
quality assessment. The selected panel of diatoms covers all the species found in water of varying quality
from the rivers of central-East Apennine (Italy). Small
subunit rRNA-targeted probes were applied to a
microarray platform as well as to a new technique
termed Primer–Probe, with the aim of obtaining a
molecular tool suitable for accurate identification of
both single and mixed species diatom populations. The
Primer–Probe technique together with dot-blot assays
proved to be ideal for the preliminary screening of a
large set of DNA oligonucleotides designed by ARB
software. It was shown that microarrays, as a promising
technology for rapid and simultaneous detection
of a wide range of species-specific genetic markers,
can be adapted to monitor changes within a diatom
community. It is suggested that microarrays will
provide a molecular basis for microbial identification
to support standard microscopy techniques used by
ecologists and environmental scientists for monitoring
water quality
Detection of Morganella morganii bound to a plastic substrate in surface water
Full text linkObjectives: Around the globe, escalation in rare opportunistic microbial infections is alarming as they are heading steadily towards ’superbug’ status. In aquatic ecosystems, plastic fosters multidrug-resistant pathogenic bacteria and plays a significant role in trafficking antibiotic-resistant genes. In this study, we focused on a multidrug-resistant bacterial strain isolated from microbial communities found on plastic substrates of a volcanic lake in central Italy. Methods: Extended-spectrum beta-lactamase-producing strains were isolated from both raw water and plastic substrates for a comparative investigation using microbiological and molecular methods, and an- tibiotic susceptibility profiling was performed against a panel of ten antibiotics. Results: Molecular identification and Basic Local Alignment Search Tool analysis confirmed an almost identical sequencing pattern of two isolated strains and their homology with Morganella morganii . An- tibiotic susceptibility tests revealed their resistance to almost all tested antibiotics. Class 1 integron- associated gene (intI1) and seven antibiotic resistance genes were detected in both strains, confirming their superbug status. Conclusion: To our knowledge, this is the first study on the characterization of extended-spectrum beta- lactamase-producing M. morganii isolated from the biofilm of plastic substrates, depicting the poten- tial toxicity of plastic in harbouring and dispersing virulent, multidrug-resistant, opportunistic human pathogens
The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending.
No full textEngineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics
No full textA number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling
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