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PubMed2004

Structure and segregation of the bacterial nucleoid.

In bacteria, chromosome segregation and DNA replication occur concurrently and there is no clear equivalent of a eukaryote mitotic spindle. Chromosome segregation can be viewed as a two-step process. As the first step, the origin of replication regions are segregated actively, probably by a mechanism involving an as yet unidentified motor protein or proteins, and held in position. The second step is the separation and migration of the rest of the chromosome probably driven by forces generated from various cellular processes such as DNA replication, transcription and transertion.

Bacillus subtilis↗
PubMed2005

Correlation between the genetic diversity of nosocomial pathogens and their survival time in intensive care units.

Bacteria differ in their ability to survive in the hospital environment outside the human host. Species remaining viable and infectious have a higher chance of being transmitted, giving them a fitness advantage in hospitals. This differential fitness could be expected to alter the genetic population structure of bacterial populations in hospitals, and should be reflected by the relative abundance of several successful clones. The objective of this study was to test for a potential correlation between tenacity, i.e. environmental survival, and clonal abundance determined by the genetic diversity in different bacterial species from prospectively collected isolates of intensive care patients. A literature review was performed to identify mean environmental survival times for the most important pathogens in intensive care units (ICUs): Staphylococcus aureus, enterococci, Acetinobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Escherichia coli, Klebsiella pneumoniae and Stenotrophomonas maltophilia. To determine the genetic diversity of the natural population of these species in ICUs, a prospective 18-month study was conducted in five units with median nosocomial infection rates. All clinical isolates were collected, and highly discriminatory DNA fingerprinting techniques were used to identify specific clones. A diversity index for each species was calculated as the number of distinguishable genotypes in the population divided by size. The correlation between survival times and the diversity indices for the individual pathogens was investigated using non-parametric methods. Although 21 studies were identified in the literature, only two were relevant. They showed median survival times between 1.5 days (P. aeruginosa) and 60.0 days (Enterococcus faecium). During the prospective ICU study, 1264 pathogens were investigated and simple diversity indices between 49.1 (Enterococcus faecalis) and 89.8 (E. coli) were found. A correlation between survival times and the diversity indices for the individual pathogens was found (correlation coefficient 0.821, P=0.024). Environmental survival may be an important factor contributing to the ecological fitness of some nosocomial pathogens in ICUs. Infection control measures should consider this finding.

Bacterial Infections↗
PubMed2004

Periodicities of 10-11bp as indicators of the supercoiled state of genomic DNA.

DNA sequences contain information about the bendability and native conformation of DNA. For example, a repetition of certain dinucleotides at distances of 10-11bp supports wrapping around nucleosomes and supercoiled structures of bacterial DNA. We analyzed 86 eubacterial genomes, 16 archaea, and six genomes of higher eukaryotes. First, we discuss whether or not the observed periodicities represent indeed bendability signals. This claim is confirmed since: (1) dinucleotide signals are of comparable size to mononucleotide signals, (2) the signals are present in non-coding DNA as well, and (3) repeat masking has only a minor effect on 10-11bp periodicities. Moreover, the periodicities persist up to 150bp, comparable to the nucleosome size. We show that doublet peaks in Caenorhabditis elegans and some prokaryotes can be traced back to long-ranging modulations. In mammalian genomes, we find consistently spectral peaks as observed earlier in human chromosomes 20, 21 and 22. It has been shown in previous studies that archaea have periods of 10bp, whereas eubacteria exhibit 11bp periodicities. These differences reflect different supercoiled states of microbial DNA. Is the period of 10bp an archaeal or a thermophilic feature? This question is addressed by relating periodicities to optimal growth temperatures. It turns out that the archaea Methanopyrus kandleri (t(opt)=80 degrees C) and a Halobacterium strain (t(opt)=42 degrees C) both have longer periods of about 11bp. Eubacterial genomes have consistently periods around 11bp indicative of negative supercoiling.

Animals↗
PubMed2006

Structural and physical aspects of bacterial chromosome segregation.

Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase separate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid-binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. Specific DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diffusion coefficient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be effectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. Differences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diffusion coefficient could explain the linear positioning of genes in the nucleoid and that differential transcriptional activity could induce different mobilities between either replichores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move specific chromosomal regions and the nucleoid as a whole without invoking a dedicated mechanism.

Chromosome Segregation↗
PubMed1991

Potentiometric and spectral studies with the two-subunit cytochrome aa3 from Paracoccus denitrificans. Comparison with the 13-subunit beef heart enzyme.

Previous work from this laboratory has revealed a complex and interactive redox behavior for the active metal centers in beef heart cytochrome aa3. All of these centers are contained in two of the 13 subunits which make up the enzyme. The isolated cytochrome aa3 of Paracoccus denitrificans contains only two subunits. The purpose of the current investigation was to see if the complex redox behavior is dependent on the presence of the additional 11 peptides that are present in the mammalian enzyme. In this paper we report that the structurally simpler bacterial enzyme displays a redox behavior which is very similar to that seen with the mammalian enzyme. Therefore, the observed redox behavior does not depend on interactions involving the additional peptides.

Animals↗
PubMed2001

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.

17-Hydroxysteroid Dehydrogenases↗
PubMed1998

Distinct mechanisms of antibody-mediated enzymatic reactivation in beta-galactosidase molecular sensors.

The antibody-mediated reactivation of engineered Escherichia coli beta-galactosidases [Benito et al. (1996) J. Biol. Chem. 271, 21251-21256] has been thoughtfully investigated in three recombinant molecular sensors. Proteins M278VP1, JX772A and JX795A display the highly antigenic G-H loop peptide segment of foot-and-mouth disease virus VP1 protein, accommodated in different solvent-exposed loops of the assembled tetramer. These chimaeric enzymes exhibit a significant increase in enzymatic activity upon binding of either monoclonal antibodies or sera directed against the inserted viral peptide. In JX772A but not in M278VP1, the Fab 3E5 antibody fragment promotes reactivation to the same extent as the complete antibody. On the other hand, M278VP1 Km is reduced by more than 50% in the presence of activating serum, this parameter remains invariable in JX772A and it is only slightly modified in JX795A. In these last two proteins, significant k(cat) variations can account for the increased enzymatic activity. Alternative reactivation mechanisms in the different beta-galactosidase probes are discussed in the context of the bacterial enzyme structure and its tolerance to antibody-induced conformational modifications.

Amino Acid Sequence↗
PubMed1988

Management of associated soft-tissue injury.

Initially, soft-tissue injuries associated with vascular trauma require management according to the general surgical principles of wound care. Antimicrobial prophylaxis should be instituted. The mechanism and extent of injury must be determined, and debridement and irrigation should be completed in the operating room with the intent to preserve vital structures, decrease bacterial contamination, and render the wound free of devitalized tissue. Prompt coverage of exposed vascular repairs is vital in order to minimize the chance for infection and to protect the repair from desiccation and subsequent trauma. Early primary closure of heavily contaminated wounds can have disastrous results. Therefore, questionable wounds should be managed by delayed primary closure after repeated irrigation and debridements, with the definitive decision as to the timing of wound closure being based on quantitative cultures. The plastic and reconstructive surgeon can aid the vascular surgeon in the primary or secondary closure of such wounds by bringing healthy, vascularized muscle to cover the vascular repair. Vascularized muscle has proved superior in the coverage and healing of contaminated wounds. If conditions permit, this can be most readily accomplished by transfer of local muscle. Musculocutaneous flaps are of special value in cases that require increased durability or where extended coverage is necessary. With either of these flap techniques, the surgeon must be thoroughly familiar with the anatomy, blood supply, and function of the local muscles. Free-tissue transfer is to be utilized when local tissue is severely damaged in a way that precludes the use of adjacent muscle or myocutaneous flaps. Strict adherence to the principles and techniques of microsurgery, thorough evaluation of the zone of injury and recipient blood vessels, and understanding of the principles governing local muscle transfer are essential for a successful outcome.

Bacterial Infections↗
PubMed1999

New molecular complexes of heterocyclic bis-adducts with bacterial lectins: synthesis and structure-activity relationship studies.

A strongly antitumour effect has been discovered for lectins of Bacillus bacteria [Bacillus subtilis 668(1 + 2)IMV, Bacillus polymyxa 102(1 + 2) KGU] and for their molecular complexes with some heterocyclic bis-adducts of unsubstituted benzimidazole and 6-methyluracile for the first time. These were tested on the tumours: Lymphosarcoma Plissa, Sarcoma 45, Carcinosarcoma Yokera 256. A new convenient method for the preparation of the heterocyclic bisadducts of imidazole, benzimidazole, uraciles with 1,1,1-trifluoro-2-bromo-2-chloroethane is described. The reactions are catalysed by the 18-crown-6-complex.

Amino Acids↗
PubMed1978

[An acquired B antigen in a subject of A2 group].

Almost all cases of acquired B antigen have been found in individuals. This report concerns a group A2 patient with acquired B antigen. The family was not available for genetic studies. Nevertheless two facts were in favour of A2 genotype: 10 after acetylation of the patient's red cells the A1 reactivity which usually reappears or is increased on acquired B cells, did not appear. 20 The study of the serum alpha-N acetyl galactosaminyl transferase showed an optimal activity at pH7, as it is usually found in A2. The respective roles of anti acquired B antibodies, group A specific structures and bacterial enzyme properties in the occurrence of acquired B antigen are discussed.

ABO Blood-Group System↗
PubMed1993

Hemagglutination by Pasteurellaceae isolated from rodents.

Pasteurellaceae notably P. pneumotropica, have been associated with severe outbreaks of respiratory disease in several species of rodents. Host-specific parasitism of Pasteurellaceae in rodents has hardly been studied. Since host tropism in many bacteria involves adhesive mechanisms, we examined the hemagglutinating (HA) properties of 44 isolates from different rodent species (mouse (15) rat (8), hamster (9), gerbil (10) and Mastomys (2)). Only 13 mouse isolates and the 2 Mastomys isolates hemagglutinated human (type O Rh+) and canine red blood cells (RBCs). No HA was found using RBCs from 10 other animal species. HA was not inhibited by simple sugars and glycoconjugates, but was completely inhibited by heating of bacterial cells for 10 min at 80 or 100 degrees C, partially inhibited by glutaraldehyde and inhibited in a dose-dependent mode by NaIO4, suggesting the involvement of bacterial polysaccharide structures in the HA process. Enrichment procedures did not reveal the presence of HA- subpopulations in HA+ isolates or the presence of HA+ subpopulations in HA- isolates. Electron microscopy revealed the presence of fimbriae both in HA+ and HA- isolates. A regularly structured (RS) layer was detected on cells of part of the HA+ isolates only. Our results suggest that Pasteurellaceae of mice and Mastomys may be related and differ from isolates isolated from other rodent species.

Animals↗
PubMed1998

Cytochrome P450 monooxygenases.

The field of P450 research is progressing rapidly. The active sites of structurally characterized bacterial P450 enzymes have been rationally re-engineered to alter both substrate specificity and selectivity of substrate oxidation. Many human P450 enzymes have been functionally expressed and new methods of substrate turnover described. These developments, arising from our increased understanding of the chemistry and molecular biology of P450 enzymes, open up new and exciting research directions.

Bacterial Proteins↗
PubMed1999

Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more.

Many Gram-negative bacteria that cause disease in either mammals or plants share a strategy of delivering toxic proteins into the cytoplasm of host cells known as type III secretion. Recent advances have provided a glimpse at the molecular nature of these lethal injection machines. Several groups have reported fibrous structures on bacterial surfaces that appear to be extensions of type III machines and necessary for toxin injection into host cells. Other research revealed complex mechanisms of secretion substrate recognition that presumably function to direct toxins to different locations during infection.

Animals↗
PubMed2001

Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase.

The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1). The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+). This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.

Animals↗
PubMed1998

The family 1 beta-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism.

Comparisons of catalytic mechanisms have not previously been performed for homologous enzymes from hyperthermophilic and mesophilic sources. Here, the beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was recombinantly produced in Escherichia coli and shown to have biophyscial and biochemical properties identical to those of the wild-type enzyme. Moreover, the recombinant enzyme was subjected to a detailed kinetic investigation at 95 degreesC to compare its catalytic mechanism to that determined at 37 degreesC for the beta-glucosidase (abg) from the mesophilic bacterium, Agrobacterium faecalis [Kempton, J., and Withers, S. G. (1992) Biochemistry 31, 9961]. These enzymes have amino acid sequences that are 33% identical and have been classified as family 1 glycosyl hydrolases on the basis of amino acid sequence similarities. Both enzymes have similar broad specificities for both sugar and aglycone moieties and exhibit nearly identical pH dependences for their kinetic parameters with several different substrates. Bronsted plots were constructed for bgl at several temperatures using a series of aryl glucoside substrates. These plots were concave downward at all temperatures, indicating that bgl utilized a two-step mechanism similar to that of abg and that the rate-limiting step in this mechanism did not change with temperature for any given aryl glucoside. The Bronsted coefficient for bgl at 95 degreesC (beta1g = -0.7) was identical to that for abg at 37 degreesC and implies that these enzymes utilize nearly identical transition states, at least in regard to charge accumulation on the departing glycosidic oxygen. In addition, a high correlation coefficient (rho = 0.97) for the linear free energy relationship between these two enzymes and similar inhibition constants for these two enzymes with several ground state and transition state analogue inhibitors further indicate that these enzymes stabilize similar transition states. The mechanistic similarities between these two enzymes are noteworthy in light of the large difference in their temperature optima. This suggests that, in the presumed evolution that occurred between the hyperthermophilic archaeal enzyme and the mesophilic bacterial enzyme, structural modifications must have been selected which maintained the integrity of the active site structure and, therefore, the specificity of transition state interactions, while adapting the overall protein structure to permit function at the appropriate temperature.

1-Deoxynojirimycin↗