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At least 1,099 records · Page 61Linked to original sources
PubMed2005

Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.

Cloning the whole 3.5-megabase (Mb) genome of the photosynthetic bacterium Synechocystis PCC6803 into the 4.2-Mb genome of the mesophilic bacterium Bacillus subtilis 168 resulted in a 7.7-Mb composite genome. We succeeded in such unprecedented large-size cloning by progressively assembling and editing contiguous DNA regions that cover the entire Synechocystis genome. The strain containing the two sets of genome grew only in the B. subtilis culture medium where all of the cloning procedures were carried out. The high structural stability of the cloned Synechocystis genome was closely associated with the symmetry of the bacterial genome structure of the DNA replication origin (oriC) and its termination (terC) and the exclusivity of Synechocystis ribosomal RNA operon genes (rrnA and rrnB). Given the significant diversity in genome structure observed upon horizontal DNA transfer in nature, our stable laboratory-generated composite genome raised fundamental questions concerning two complete genomes in one cell. Our megasize DNA cloning method, designated megacloning, may be generally applicable to other genomes or genome loci of free-living organisms.

Bacillus subtilis↗
PubMed2005

Flocculation of colloidal clay by bacterial polysaccharides: effect of macromolecule charge and structure.

The molecular mechanism of montmorillonite flocculation by bacterial polysaccharides was investigated, with special emphasis on the effect of carboxylic charges in the macromolecules on the mechanisms of interaction with the clay surface. An indirect way to quantify the energy of interaction was used, by comparing the flocculation ability of variously acidic polysaccharides. Data on tensile strength of aggregates in diluted suspension were collected by timed size measurements in the domain 0.1-600 microm, using laser diffraction. The flow behavior of settled aggregates was studied by rheology measurements. Flocculation of colloidal clay suspension by polysaccharides requires cancelling of the electrostatic repulsions by salts, which allows approach of clay surfaces close enough to be bridged by adsorbing macromolecules. The amount of acidic charges of the polysaccharides, and especially their location in the molecular structure, governs the bridging mechanism and the resulting tensile strength of the aggregates. The exposure of carboxylate groups located on side chains strongly promotes flocculation. In turn, charges located on the backbone of the polysaccharide are less accessible to interaction, and the flocculation ability of such polysaccharides is lowered. Measurements at different pH indicate that adsorption of acidic polysaccharides occurs via electrostatic interactions on the amphoteric edge surface of clay platelets, whereas neutral polysaccharides rather adsorb via weak interactions. Increased tensile strength in diluted aggregates due to strong surface interactions results in proportionally increased viscosity of the concentrated aggregates.

Aluminum Silicates↗
PubMedDate not supplied

The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.

Animals↗
PubMed2003

Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies.

Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.

Bacterial Proteins↗
PubMed2001

Aerobic biological treatment of a pharmaceutical wastewater: effect of temperature on cod removal and bacterial community development.

The effect of temperature was studied on the efficiency of soluble COD removal and bacterial community development during the aerobic biological treatment of a pharmaceutical wastewater. Using wastewater and bacterial inoculum obtained from the full-scale facility treating this wastewater, batch laboratory cultures were operated at 5 degrees C intervals from 30 degrees C to 70 C. Following four culture transfers to allow for bacterial acclimation, residual soluble COD levels were measured and bacterial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. Soluble COD removal efficiency declined as temperature increased from 30 degrees C (62%) to 60 degrees C (38%). Biological treatment of this wastewater failed to occur at temperatures higher than 60 C. Gradual shifts in bacterial community structure were detected as temperature increased, including a concomitant reduction in the number of different bacterial populations. The impact of temperature on a two-stage biological treatment process was also compared. Better soluble COD removal was achieved when both reactors were operated at 30 degrees C compared to a system where the two stages were consecutively operated at 55 degrees C and 30 degrees C. These results indicate that operation of aerobic biological wastewater treatment reactors at elevated temperatures can have adverse effects on process performance.

DNA, Bacterial↗
PubMed2001

Transcription elongation complex: structure and function.

Our understanding of the mechanisms of transcription has been greatly advanced by recent determination of the X-ray structure of bacterial RNA polymerase. Using crosslinking approaches, extensive mapping of DNA and RNA contacts onto this structure allowed tracking of the path of nucleic acids through the transcription elongation complex. The resulting structural model of the transcription elongation complex is linked to the functional one, which is based on numerous data accumulated during previous studies of RNA synthesis. An integrated structure-function model allows the rational explanation of termination and pausing and provides new insights into the mechanisms of transcription.

DNA↗
PubMed2002

Variation in human intestinal microbiota with age.

The large intestinal microbiota plays an important role in normal bowel function and the maintenance of host health, through the formation of short chain fatty acids, modulation of immune system reactivity and development of colonisation resistance. However, the effects of ageing on bacterial community structure in the colon are not well documented. Aim of this study is to assess bacterial species diversity in the human faecal microbiota with respect to age and Clostridium difficile infection. Bacterial populations were quantified from stool samples obtained from children (16 months to seven years), young adults (21-34 years), healthy elderly people (67-88 years) and patients diagnosed with Clostridium difficile-associated diarrhoea (68-73 years). Microbial diversity was assessed to species level for samples from the latter three subject groups. Marked interindividual variations occurred in microbial composition at genus and species levels. The faecal microbiota of children was found to be bacteriologically less complex whilst advancing age was associated with decreased bifidobacteria and increased bacteroides species diversity. Changes in microbial composition with age or disease will alter the metabolic capacity of the gut microbiota and has important implications for therapies aimed at modulating the large intestinal microbiota.

Adult↗
PubMed2002

Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java, Indonesia.

In Java, Indonesia, many nutrient-poor soils are intensively reforested with Pinus merkusii (pine). Information on nutrient cycles and microorganisms involved in these cycles will benefit the management of these important forests. Here, seasonal effects on the stratification of bacterial community structure in the soil profile of a tropical pine forest are described, and differences in bacterial communities are related to chemical and physical soil parameters. Culture-independent community profiles of litter, fragmented litter and mineral soil layers were made by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA-specific polymerase chain reaction (PCR) fragments. The community profiles of the different soil layers clustered separately, correlating with significant differences in organic matter content between the three layers. The bacterial communities appeared to be stable during the wet season of 1998. The drought in 1997, caused by the El Niño climatic effect, did not influence the bacterial communities in fragmentation and mineral soil, although moisture content and other soil parameters were markedly lower than in the wet season. However, communities in litter were influenced by drought. In the litter layer, the moisture content was significantly lower than in the fragmentation and mineral layers during the dry season. A clone library was made from a litter sample taken during the wet season. Partial sequencing of 74 clones and linking the DGGE banding positions of these clones to bands in the DGGE profile of the sample from which the clone library was derived showed considerable bacterial diversity. Alpha-proteobacteria (40.5% of the clones, of which 57% belonged to the Rhizobium-Agrobacterium group) and high-G+C content, Gram-positive bacteria (36.5%) dominated the clone library.

Bacteria↗
PubMed2003

Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil.

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.

Bacteria↗
PubMed2006

Na+ channel pharmacology and molecular mechanisms of gating.

Electrogenesis of efficiently propagated action potentials requires synchronized opening of transmembrane Na+ channels possessing a sodium selectivity-filter, a high-throughput ion-conductance pathway, and voltage-dependent gating functions. These properties of the Na+ channel have long been the target of molecular analysis. Several toxins and drugs, known to selectively bind to Na+ channels, have been used as pharmacological tools to investigate Na+ channel properties either electrophysiologically or chemically. Recent analyses of the protein crystal structure of bacterial voltage-dependent K+ channels have provided important clues to the identity of mobile structures involved in channel gating. The new information may be applicable to Na+ channels, and may well require a total revision of our understanding of gating mechanisms of sodium channels. Several experiments challenge the emerging view that channel gating by S6 transmembrane segments is triggered by signals from voltage sensors floating in membrane lipid. Herein, we review the various toxin and drug molecules that affect the gating behavior of Na+ channels in this new structural framework, by characterizing the binding sites of these toxins, and assessing the pharmacological effects resulting from changes in the structure of the toxin or sodium channel.

Animals↗
PubMed2004

Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey.

Predation at the lowest trophic level, i.e. between bacteria, is poorly understood, hindering efforts to assess its impact on the structure of bacterial communities. The interaction of Bdellovibrio and Bacteriovorax (Bdellovibrio and like organisms, BLOs), a group of obligate, ubiquitous predatory bacteria, with their Gram-negative bacterial prey results in the multiplication of the predator and in the lysis, but not in the eradication, of the prey. We show that the residual, surviving populations of prey cells exposed to predation stress differ from the populations before exposure, as they exhibit increased resistance to predation. This resistance was demonstrated in a number of Gram-negative prey. Moreover, predation resistance is not specific for the BLO strain experienced by the prey. The phenomenon does not stem from a mutation but is a plastic response associated with a phenotypic change, and it disappears upon removal of the predator. As resistance to predation is not total, this mechanism can ensure survival of both predator and prey.

Adaptation, Physiological↗
PubMed2006

Design of a minimal polypeptide unit for bacteriochlorophyll binding and self-assembly based on photosynthetic bacterial light-harvesting proteins.

We introduce LH1beta24, a minimal 24 amino acid polypeptide that binds and assembles bacteriochlorophylls (BChls) in micelles of octyl beta-glucoside (OG) into complexes with spectral properties that resemble those of B820, a universal intermediate in the assembly of native purple bacterial light-harvesting complexes (LHs). LH1beta24 was designed by a survey of sequences and crystal structures of bacterial LH proteins from different organisms combined with currently available information from in vitro reconstitution studies and genetically modified LHs in vivo. We took as a template for the design sphbeta31, a truncated 31 amino acid analogue of the native beta-apoprotein from the core LH complex of Rhodobacter sphaeroides. This peptide self-assembles with BChls to form B820 and, upon cooling and lowering OG concentration, forms red-shifted B850 spectral species that are considered analogous to native LH complexes. We find that LH1beta24 self-assembles with BChl in OG to form homodimeric B820-type subunits comprising two LH1beta24 and two BChl molecules per subunit. We demonstrate, by modeling the structure using the highly homologous structure of LH2 from Rhodospirillum molischianum, that it has the minimal size for BChl binding. Additionally, we have compared the self-assembly of sphbeta31 and LH1beta24 with BChls and discovered that the association enthalpies and entropies of both species are similar to those measured for native LH1 from Rhodospirillum rubrum. However, sphbeta31 readily aggregates into intermediate higher oligomeric species and further to form B850 species; moreover, the assembly process of these oligomers is not reversible, and they are apparently large nonspecific BChl-peptide coaggregates rather than well-defined nativelike LH complexes. Similar aggregates were observed during LH1beta24 assembly, but these were formed less readily and required lower temperatures than sphbeta31. In view of these results, we reevaluate previous in vitro reconstitution studies and propose alternative templates for new designs.

Amino Acid Sequence↗
PubMed2002

DNA-lipid interactions in vitro and in vivo.

The data on lipid-nucleic interactions and their role in vitro and in vivo are presented. The results of study of DNA-lipid complexes in absence and in presence of divalent metal cations (triple complexes) are discussed. The triple complexes represent the generation of cellular structures such as pore complexes of eucaryotes and "Bayer's junctions" of procaryotes. The participation of triple complexes in the formation of structure of bacterial and eucaryotic nucleoid and nuclear matrix is analysed. A model of formation of triple complexes and cellular structures and their role in DNA-lipid interactions are discussed.

Animals↗
PubMed2002

Redefining bacterial populations: a post-genomic reformation.

Sexual reproduction and recombination are essential for the survival of most eukaryotic populations. Until recently, the impact of these processes on the structure of bacterial populations has been largely overlooked. The advent of large-scale whole-genome sequencing and the concomitant development of molecular tools, such as microarray technology, facilitate the sensitive detection of recombination events in bacteria. These techniques are revealing that bacterial populations are comprised of isolates that show a surprisingly wide spectrum of genetic diversity at the DNA level. Our new awareness of this genetic diversity is increasing our understanding of population structures and of how these affect host pathogen relationships.

Bacteria↗
PubMed2005

Activation-coupled inactivation in the bacterial potassium channel KcsA.

X-ray structures of the bacterial K+ channel KcsA have led to unparalleled progress in our understanding of ion channel structures. The KcsA channel has therefore been a prototypic model used to study the structural basis of ion channel function, including the gating mechanism. This channel was previously found to close at near-neutral intracellular pH (pH(i)) and to open at acidic pH(i). Here, we report the presence of a previously unknown channel inactivation process that occurs after the KcsA channel is activated. In our experiments, mammalian cells transfected with a codon-optimized synthetic gene encoding the KcsA protein expressed K+-selective channels that activated in response to a decrease in pH(i). Using patch-clamp and rapid solution exchange techniques, we observed that the KcsA channels inactivated within hundreds of milliseconds after channel activation. At all tested pHs, inactivation always accompanied activation, and it was profoundly accelerated in the same pH range at which activation increased steeply. Recovery from inactivation was observed, and its extent depended on the pH(i) and the amount of time that the channel was inactive. KcsA channel inactivation can be described by a kinetic model in which pH(i) controls inactivation through pH-dependent activation. This heretofore-undocumented inactivation process increases the complexity of KcsA channel function, but it also offers a potential model for studying the structural correspondence of ion channel inactivation.

Animals↗
PubMed2005

Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7.

We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Phi-values and compare the results with the experimentally determined Phi-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.

Biophysical Phenomena↗
PubMed2005

Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils.

Molecular approaches have revealed considerable diversity and uncultured novelty in natural prokaryotic populations, but not direct links between the new genotypes detected and ecosystem processes. Here we describe the influence of the structure of communities of ammonia-oxidizing bacteria on nitrogen cycling in microcosms containing natural and managed grasslands and amended with artificial sheep urine, a major factor determining local ammonia concentrations in these environments. Nitrification kinetics were assessed by analysis of changes in urea, ammonia, nitrite and nitrate concentrations and ammonia oxidizer communities were characterized by analysis of 16S rRNA genes amplified from extracted DNA using ammonia oxidizer-specific primers. In natural soils, ammonia oxidizer community structure determined the delay preceding nitrification, which depended on the relative abundance of two Nitrosospira clusters, termed 3a and 3b. In batch cultures, pure culture and enrichment culture representatives of Nitrosospira 3a were sensitive to high ammonia concentration, while Nitrosospira cluster 3b representatives and Nitrosomonas europaea were tolerant. Delays in nitrification occurred in natural soils dominated by Nitrosospira cluster 3a and resulted from the time required for growth of low concentrations of Nitrosospira cluster 3b. In microcosms dominated by Nitrosospira cluster 3b and Nitrosomonas, no substantial delays were observed. In managed soils, no delays in nitrification were detected, regardless of initial ammonia oxidizer community structure, most probably resulting from higher ammonia oxidizer cell concentrations. The data therefore demonstrate a direct link between bacterial community structure, physiological diversity and ecosystem function.

Ammonia↗
PubMed2006

Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III.

Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.

Amino Acid Sequence↗