Plasmid-mediated extended-spectrum beta-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1

Six non-clonally related enterobacterial isolates producing a same extended-spectrum beta-lactamase CTX-M-15 were isolated in 1999 from patients hospitalized in a New Delhi hospital. CTX-M-15 differed from CTX-M-3 by an asparagine to glycine substitution in position ABL238. Its gene was located on large plasmids varying in size. In each case, a same insertion sequence ISEcp1 was identified upstream of the 5' end of bla(CTX-M-15). Typical -35 and -10 promoter sequences of Enterobacteriaceae were identified in the 3' end of ISEcp1. The location of ISEcp1 upstream of plasmid-mediated CTX-M-type beta-lactamase genes may contribute to their spread or/and their expression.     

In vivo qualitative changes of 31P NMR in stressed maize roots vis-à-vis carbon substrate determining the degree of stress

High resolution 31P nuclear magnetic resonance spectroscopy was used to investigate the changes in phosphate metabolism and intracellular pH in intact maize (Zea mays L) root segments to hyper osmotic shock. The results were compared with the happenings under field conditions, when the stress was given gradually. Effect of sugar substrate on adaptation of tissue to both kinds of situations was also studied. The hyper osmotic shock resulted in large vacuolar alkalinization and a decrease in pH across tonoplast membrane. There was gradual build up of phosphocholine and decrease in glucose 6P and UPDG levels. In gradual stress, the root segments were able to adapt to the stress and maintained pH gradient across tonoplast, with marginal alkalinization of vacuoles. The presence of sugar substrate reduced the impact of stress significantly, commensurate with the increased activity of plasmalemma H(+)-ATPase. The latter providing the driving force for uptake of organic molecules and ions required for osmoregulation.     

Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. Strain SE19 by in vitro transposition

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.     

Industrial wastewater bioreactors: sources of novel microorganisms for biotechnology

Microorganisms exist in nature as members of complex, mixed communities. The microbial communities in industrial wastewater bioreactors can be used as model systems to study the evolution of new metabolic pathways in natural ecosystems. The evolution of microbial metabolic capability in these bioreactors is presumably analogous to phenomena that occur in natural ecosystems. The microorganisms in these bioreactors compete for different carbon sources and constantly have to evolve new metabolic capabilities for survival. Thus, industrial bioreactors should be a rich source of novel biocatalysts.     

Crystal structure of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

Biphenyl dioxygenase is the enzyme that catalyzes the stereospecific dioxygenation of the aromatic ring. This enzyme has attracted the attention of researchers due to its ability to oxidize polychlorinated biphenyls, which is one of the serious environmental contaminants. We determined the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1 in substrate-free and complex forms. These crystal structures revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. Our analysis of the crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which, respectively, seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket. The cooperative actions of residues in the three groups seem to determine the substrate specificity of the enzyme.     

The stability of Seeman JX DNA topoisomers of paranemic crossover (PX) molecules as a function of crossover number

We use molecular dynamics simulations in explicit water and salt (Na⁺) to determine the effect of varying the number of crossover points on the structure and stability of the PX65 paranemic crossover DNA molecule and its JXM topoisomers (M denotes the number of missing crossover points), recently synthesized by the Seeman group at New York University. We find that PX65, with six crossover points, is the most stable, and that the stability decreases monotonically with the number of crossover points PX65 > JX1 > JX2 > JX3 > JX4, with 6, 5, 4, 3 and 2 crossover points, respectively. Thus, for PX65/JX1, the strain energy is ~3 kcal/mol/bp, while it is ~13 kcal/mol/bp for JX2, JX3 and JX4. Another measure of the stability is the change in the structure from the minimum energy structure to the equilibrium structure at 300 K, denoted as root-mean-square deviation in coordinates (CRMSD). We find that CRMSD is ~3.5 Å for PX65, increases to 6 Å for JX1 and increases to 10 Å for JX2/JX3/JX4. As the number of crossover points decreases, the distance between the two double helical domains of the PX/JX molecules increases from ~20 Å for PX65 to 23 Å for JX4. This indicates that JX2, JX3 and JX4 are less likely to form, at least in with Na⁺. However, in all the cases, the two double helical domains have average helicoidal parameters similar to a typical B-DNA of similar length and base sequence.     

Predicted 3-D structures for mouse I7 and rat I7 olfactory receptors and comparison of predicted odor recognition profiles with experiment

The first step in the perception of an odor is the activation of one or more olfactory receptors (ORs) following binding of the odorant molecule to the OR. In order to initiate the process of determining how the molecular level receptor-odorant interactions are related to odor perception, we used the MembStruk computational method to predict the three-dimensional (3-D) structure of the I7 OR for both mouse and rat. We then used the HierDock ligand docking computational method to predict the binding site and binding energy for the library of 56 odorants to these receptors for which experiment response data are now available. We find that the predicted 3-D structures of the mouse and rat I7 OR lead to predictions of odorant binding that are in good agreement with the experimental results, thus validating the accuracy of both the 3-D structure and the predicted binding site. In particular we predict that heptanal and octanal both bind strongly to both mouse and rat I7 ORs, which conflicts with the older literature but agrees with recent experiments. To provide the basis of additional validations of our 3-D structures, we also report the odorant binding site for a new odorant (8-hydroxy-octanal) with a novel functionality designed to bind strongly to mouse I7. Such validated computational methods should be very useful in predicting the structure and function of many other ORs.     

Making sense of olfaction through predictions of the 3-D structure and function of olfactory receptors

We used the MembStruk first principles computational technique to predict the three-dimensional (3-D) structure of six mouse olfactory receptors (S6, S18, S19, S25, S46 and S50) for which experimental odorant recognition profiles are available for a set of 24 odorants (4-9 carbons aliphatic alcohols, acids, bromo-acids and diacids). We used the HierDock method to scan each predicted OR structure for potential odorant binding site(s) and to calculate binding energies of each odorant in these binding sites. The calculated binding affinity profiles are in good agreement with experimental activation profiles, validating the predicted 3-D structures and the predicted binding sites. For each of the six ORs, the binding site is located between trans-membrane domains (TMs) 3-6, with contributions from extracellular loops 2 and 3. In particular, we find six residue positions in TM3 and TM6 to be consistently involved in the binding modes of the odorants. Indeed, the differences in the experimental recognition profiles can be explained on the basis of these critical residues alone. These predictions are also consistent with mutation data on ligand binding for catecholamine receptors and sequence hypervariability studies for ORs. Based on this analysis, we defined amino acid patterns associated with the recognition of short aliphatic alcohols and mono-acids. Using these two sequence fingerprints to probe the alignment of 869 OR sequences from the mouse genome, we identified 34 OR sequences matching the fingerprint for aliphatic mono-acids and 36 corresponding to the recognition pattern for aliphatic alcohols. We suggest that these two sets of ORs might function as basic arrays for uniquely recognizing aliphatic alcohols and acids. We screened a library of 89 additional molecules against the six ORs and found that this set of ORs is likely to respond to aldehydes and esters with longer carbon chains than their currently known agonists. We also find that compounds associated with the flavor in foods are often among the best calculated binding affinities. This suggests that physiologic ligands for these ORs may be found among aldehydes and esters associated with flavor.     

Alterations in the TGFbeta signaling pathway in myogenic progenitors with age

Myogenic progenitors in adult muscle are necessary for the repair, maintenance and hypertrophy of post-mitotic muscle fibers. With age, fat deposition and fibrosis contribute to the decline in the integrity and functional capacity of muscles. In a previous study we reported increased accumulation of lipid in myogenic progenitors obtained from aged mice, accompanied by an up-regulation of genes involved in adipogenic differentiation. The present study was designed to extend our understanding of how aging affects the fate and gene expression profile of myogenic progenitors. Affymetrix murine U74 Genechip analysis was performed using RNA extracted from myogenic progenitors isolated from adult (8-month-old) and aged (24-month-old) DBA/2JNIA mice. The cells from the aged animals exhibited major alterations in the expression level of many genes directly or indirectly involved with the TGFbeta signaling pathway. Our data indicate that with age, myogenic progenitors acquire the paradoxical phenotype of being both TGFbeta activated based on overexpression of TGFbeta-inducible genes, but resistant to the differentiation-inhibiting effects of exogenous TGFbeta. The overexpression of TGFbeta-regulated genes, such as connective tissue growth factor, may play a role in increasing fibrosis in aging muscle.