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At least 1,711 records · Page 95Linked to original sources
PubMed1985

Negative supercoiling induces spontaneous unwinding of a bacterial promoter.

We have examined the influence of negative supercoiling on the DNA structure of a bacterial promoter (tyrT from Escherichia coli), the transcriptional activity of which is strongly enhanced by torsional stress in vitro. Certain regions of this promoter become sensitive to digestion by single-strand-specific S1 nuclease as a consequence of negative superhelicity. These regions occur with high frequency (1 per 30-50 bp) and are normally centered on a TpA doublet. The major positions of cleavage are located in and around the -10 sequence TATGATG, the unwinding of which is a prerequisite for gene expression. An apparently trivial change in the -10 sequence from TATGATG to TATGAAG reduces both transcriptional activity and S1 nuclease sensitivity at least 10-fold. Thus the nuclease sensitivity of the promoter correlates strongly with its biological function; and both of these phenomena correlate with certain sequence-dependent structural properties of the DNA.

DNA Restriction Enzymes↗
PubMed2004

Mutations in the nucleotide-binding domain of MutS homologs uncouple cell death from cell survival.

After genotoxic insult, the decision to repair or undergo cell death is pivotal for undamaged cell survival, and requires a highly controlled coordination of both pathways. Disruption of this regulation results in tumorigenesis and failure of cancer therapy. Mismatch repair (MMR) proteins have a unique role by contributing to both pathways, though direct evidence for their function in the DNA damage response is ambiguous. We report separation of function mutants in the ATPase domains of yeast MutS homologous (MSH) proteins that uncouple MMR-dependent DNA repair from damage response to cisplatin. While mutations in the ATPase domain have devastating effects on the mutation rate of the cell, ATPase processing is mostly dispensable for the cell death phenotype; only limited processing by the MSH6 subunit is required in DNA damage response. Different DNA binding patterns and nucleotide sensitivity of Msh2/Msh6-DNA adduct and protein-mismatch complexes, respectively, suggest that the presence of different DNA lesions influences the requirement for ATP. Limited proteolysis of purified protein gives first indications for differences in nucleotide-induced conformational changes in the presence of platinated DNA. Structural modeling of bacterial MutS proteins reinforces nucleotide-dependent differences in structures that contribute to the distinction between DNA damage response and repair. Our results demonstrate the uncoupling of MMR-dependent damage response from repair and present first indications for the involvement of distinct conformational changes in MSH proteins in this process. These data present evidence for a mechanism of MMR-dependent damage response that differs from MMR; these results have strong implications for the chemotherapeutic treatment of MMR-defective tumors.

Adenosine Triphosphatases↗
PubMed1995

AB5 toxins.

Crystal structures of shiga and pertussis toxins have recently revealed a remarkable degree of structural homology among the members of the AB5 class of bacterial toxins. Other structures have provided a detailed view of the molecular basis of receptor binding specificity of cholera toxin, and of the heat-labile enterotoxin of Escherichia coli. These structures also provide tantalizing, but as yet incomplete, information on the site of ADP-ribosylation in the homologous A-subunits of the Escherichia coli heat-labile toxin, cholera toxin, and pertussis toxin.

Bacterial Toxins↗
PubMed2003

Antisense inhibition of Escherichia coli RNase P RNA: mechanistic aspects.

The ribonucleoprotein enzyme RNase P catalyzes endonucleolytic 5'-maturation of tRNA primary transcripts in all domains of life. The indispensability of RNase P for bacterial cell growth and the large differences in structure and function between bacterial and eukaryotic RNase P enzymes comply with the basic requirements for a bacterial enzyme to be suitable as a potential novel drug target. We have identified RNA oligonucleotides that start to show an inhibitory effect on bacterial RNase P RNAs of the structural type A (for example, the Escherichia coli or Klebsiella pneumoniae enzymes) at subnanomolar concentrations in our in vitro precursor tRNA (ptRNA) processing assay. These oligonucleotides are directed against the so-called P15 loop region of RNase P RNA known to interact with the 3'-CCA portion of ptRNA substrates. Lead probing experiments demonstrate that a complementary RNA or DNA 14-mer fully invades the P15 loop region and thereby disrupts local structure in the catalytic core of RNase P RNA. Binding of the RNA 14-mer is essentially irreversible because of a very low dissociation rate. The association rate of this oligonucleotide is on the order of 10(4) M(-1) s(-1) and is thus comparable to those of many other artificial antisense oligonucleotides. The remarkable inhibition efficacy is attributable to the dual effect of direct interference with substrate binding to the RNase P RNA active site and induction of misfolding of the catalytic core of RNase P RNA. Based on our findings, the P15 loop region of bacterial RNase P RNAs of the structural type A can be considered the "Achilles' heel" of the ribozyme and therefore represents a promising target for combatting multiresistant bacterial pathogens.

Bacillus subtilis↗
PubMed2003

The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites.

Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.

Binding Sites↗
PubMed2006

Interactions of earthworms with Atrazine-degrading bacteria in an agricultural soil.

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.

Amidohydrolases↗
PubMed1992

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.

Crystallization↗
PubMed1986

Chemical structure and biological activity relationship of bacterial cell walls and muramyl peptides.

The biological activities of the cell walls of bacteria having different types of peptidoglycans, and those of stereoisomers and analogs of muramyl dipeptide (MDP), of N-acetylglucosaminyl-beta(1-4)-N-acetylmuramyl tetrapeptides having different L- and D-amino acids at the COOH-terminus, and of 6-O-acyl-MDPs were examined to elucidate the relationship between structure and activity. Replacement of the L-alanine residue of MDP with glycine and replacement of the D-isoglutamine residue with L-isoglutamine, L-glutamic acid, and D-isoasparagine, but not with D-glutamic acid, caused a marked decrease in the biological activities of the MDP molecule. Test disaccharide tetrapeptides, irrespective of the configuration of COOH-terminal amino acid, showed strong immunoadjuvant activity and stimulation of macrophages, whereas those having COOH-terminal L-amino acids exhibited greater pyrogenicity, induction of acute joint inflammation, and hemorrhagic necrosis at a primed site than those having COOH-terminal D-amino acids. Introduction of an alpha-branched higher fatty acid to the muramic acid residue resulted in the disappearance of pyrogenicity after i.v. injection, an increase of adjuvanticity, and a loss of dependence on administration vehicles. The lack of the immunopotentiating activity (adjuvanticity) in cell walls from group B-type bacterial species was explained by the combined inhibitory effects of the replacement of the L-alanine residue by glycine and involvement of the alpha-carboxyl group of the D-glutamic acid residue in linking with neighboring peptide subunits.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2001

Structure of TrwB, a gatekeeper in bacterial conjugation.

Bacterial conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of alpha/beta topology, similar to RecA and DNA ring helicases, and an all-alpha domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F(1)-ATPase. A central 20 A wide channel traverses the hexamer, thus connecting cytoplasm with periplasm.

Bacterial Proteins↗
PubMed1981

[Changes in the structural state of boundary lipids in bacterial membrane under effect of the membranotropic antibiotic gramicidin S].

Using data from measurements of excimerization at different wave-lengths of fluorescence excitation of the fluorescent probe pyrene in isolated M. lysodeikticus membranes and using the phenomenon of energy transfer in the protein tryptophanyl-pyrene system, a method for monitoring the probe population localized in the immediate proximity to protein structures, has been developed. This method allows to detect the existence within the physiological temperature range of a specific lipid population or a boundary lipid vicinal to the membrane proteins. The changes in this region under membrane treatment with the cyclodecapeptide antibiotic gramicidin S were studied. Incubation of membranes with the antibiotic increases the efficiency of quenching of membrane protein fluorescence by the quenchers localized in the membrane phase, i.e. 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxasolidinyloxyl, cetylpyridinium chloride and pyrene, and changes the protein chromophore accessibility for J-. The lateral mobility of pyrene during gramicidin S adsorption on the lipid and protein-lipid membranes is decreased. The results obtained are interpreted in favour of partial disturbance in the interaction of the boundary lipid and membrane proteins under effects of gramicidin S.

Cell Membrane↗
PubMed1978

The presence in faecal extracts of bacterial cell wall components resembling viral structures.

Electron microscopy of faecal extracts frequently shows organized structures which have a superficial resemblance to viral material in that it is more heterogenous and consists mainly of vesicles and tubules covered by subunits arranged in different patterns. Evidence is presented that this kind of material is derived from detached outer layers of bacterial cell walls.

Bacteria↗
PubMed2001

Projection structure and oligomeric properties of a bacterial core protein translocase.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

Bacterial Proteins↗
PubMed1998

A structural feature in the central channel of the bacterial flagellar FliF ring complex is implicated in type III protein export.

The FliF ring complex, which consists of the M-S ring and a proximal portion of the rod of the flagellar basal body, is the base structure for the bacterial flagellar assembly. The FliF ring is also thought to be part of the export apparatus for flagellar proteins from its amino acid sequence homology to proteins involved in type III protein export systems. We established a new purification procedure for the FliF ring particles and carried out electron microscopic image analyses in their two distinct forms: well-dispersed single particles in the presence of salt and ordered monolayer arrays of hexagonal packing formed in the absence of salt. In both cases, the axial projection maps showed a common feature, a pair of concentric rings: the inner ring corresponds to the proximal rod; the outer ring represents the thick, edge portion of the M-S ring. However, the central channel of the FliF ring, the putative pathway for the flagellar protein export, appeared to show distinct structural features in the two forms. This suggests that a domain of FliF partially occupies the central channel to be involved in the export and gate mechanism, and the domain changes its conformation depending on the ionic strength.

Amino Acid Sequence↗
PubMedDate not supplied

[Preparation of protoplast-like structures of Pseudomonas putida and their reversion to bacterial forms].

A technique is proposed for the preparation of Pseudomonas putida protoplast-like structures by treating the cells with lysozyme in a hypertonic medium containing mono-and divalent cations. Pretreatment of the cells at the logarithmic growth phase with sucrose (0.5 M) for 90 min at the pH 7.9 to 8.0 is a prerequisite for the formation of 'protoplasts'. Under these conditions, the yield of 'protoplasts' reached 99.9% of the total cell number. The protoplast-like structures are capable of reversing into the original bacterial forms in a minimal hypertonic medium containing amino acids which are components of the cell wall. The level of reversion reaches 10% for certain strains.

Bacteriological Techniques↗
PubMed1988

Probing of DNA polymorphic structure in the cell with osmium tetroxide.

It is shown that osmium tetroxide, 2,2'-bipyridine can be applied as a probe of DNA structure in a bacterial cell. Using this probe we demonstrate (a) presence of structural distortions at the junctions between the right-handed B and left-handed Z DNA in a recombinant plasmid pRW751 and (b) unusual structure of the d(A-T)16 insert in pAT32 plasmid in E. coli cells and in in vitro.

DNA Restriction Enzymes↗
PubMed2004

Structure and function of TolC: the bacterial exit duct for proteins and drugs.

The bacterial TolC protein plays a common role in the expulsion of diverse molecules, which include protein toxins and antibacterial drugs, from the cell. TolC is a trimeric 12-stranded alpha/beta barrel, comprising an alpha-helical trans-periplasmic tunnel embedded in the outer membrane by a contiguous beta-barrel channel. This structure establishes a 140 A long single pore fundamentally different to other membrane proteins and presents an exit duct to substrates, large and small, engaged at specific inner membrane translocases. TolC is open to the outside medium but is closed at its periplasmic entrance. When TolC is recruited by a substrate-laden translocase, the entrance is opened to allow substrate passage through a contiguous machinery spanning the entire cell envelope, from the cytosol to the external environment. Transition to the transient open state is achieved by an iris-like mechanism in which entrance alpha-helices undergo an untwisting realignment, thought to be stabilized by interaction with periplasmic helices of the translocase. TolC family proteins are ubiquitous among gram-negative bacteria, and the conserved entrance aperture presents a possible cheomotherapeutic target in multidrug-resistant pathogens.

Anti-Bacterial Agents↗