Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-()-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.     

Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolates.     

Verrucosispora maris sp. nov., a novel deep-sea actinomycete isolated from a marine sediment which produces abyssomicins

Verrucosispora isolate AB-18-032^T, the abyssomicin- and proximicin-producing actinomycete, has chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora. The organism formed a distinct phyletic line in the Verrucosispora 16S rRNA gene tree sharing similarities of 99.7%, 98.7% and 98.9% with Verrucosispora gifhornensis DSM 44337^T, Verrucosispora lutea YIM 013^T and Verrucosispora sediminis MS 426^T, respectively. It was readily distinguished from the two latter species using a range of phenotypic features and from V. gifhornensis DSM 44337^T, its nearest phylogenetic neighbor, by a DNA G+C content of 65.5 mol% obtained by thermal denaturation and fluorometry and DNA:DNA relatedness values of 64.0% and 65.0% using renaturation and fluorometric methods, respectively. It is apparent from the combined genotypic and phenotypic data that strain AB-18-032^T should be classified in the genus Verrucosispora as a new species. The name Verrucosispora maris sp. nov. is proposed for this taxon with isolate AB-18-032^T (= DSM 45365^T = NRRL B-24793^T) as the type strain.     

The ground-state Na+ affinities of a series of substituted acetophenones: a DFT study

A detailed study of Na⁺ affinities of a series of para-substituted acetophenones and their O–Na⁺ counterparts was performed using density functional theory [Becke, Lee, Yang and Parr (B3LYP)] method using 6-311G(d,p) basis sets with complete geometry optimisation. The gas-phase O–Na⁺ complex formation turns out to be an exothermic case and the local stereochemical disposition of Na⁺ is found to be almost the same in each case. The presence of the para-substituent is seen to cause very little change in the Na⁺ affinity relative to the unsubstituted acetophenones. Electron-releasing p-substituents increase it by 0.0105 hartree and electron-withdrawing p-substituents decrease it by 0.011 hartree. Computed Na⁺ affinities are sought to be correlated with a number of computed system parameters such as the net charge on the Na⁺ and the carbonyl oxygen of the Na⁺ complexes and the net charge on the carbonyl oxygen of the free bases. The energetics, structural and electronic properties of the complexes indicate that the interaction between the Na⁺ ion and a carbonyl base is predominantly an ion–dipole attraction and the ion-induced dipole interaction as well rather than a covalent interaction.     

Cooperative nonenzymic base recognition. Kinetics of the binding of a base monomer to a complementary polynucleotide template

The kinetics of double-helix formation by poly U and the complementary monomer N-6,9-dimethyladenine (m⁶m⁹A) has been measured using a new fast temperature-jump apparatus. The cooperative binding kinetics are complicated by the extensive self-association of the monomers, but a satisfactory analysis using average relaxation times was possible in terms of three different models. Application of a model which considers only monomer binding yields the upper limit for the binding rate constant of an m⁶m⁹A monomer next to an already bound monomer on a poly U strand: (2 ± 0.4) × 10⁸ M^?1sec^?1. A lower limit is found by using a model which allows for binding of all m⁶m⁹A stacks to poly U with equal rate constants: (3 ± 0.3) × 10⁷ M^?1sec^?1. A third model with “weighted” rate constants consistent with the data: (7.5 ± 1.0) × 10⁷ M^?1sec^?1. The rate of cooperative binding of m⁶m⁹A to the trimer UpUpU has also been measured. The rate constants obtained with the trimer agree with those obtained with the polymer for each of the three models within experimental error.     

Estimating the Growth Rate of a Bacterial Species in a Complex Mixture by Hybridization of Genomic DNA

Abstract Advances in molecular techniques have enabled new approaches to identifying bacteria. However, once identified, there is no quantitative information on the in situ growth rate of the species, mainly because the technology has not been available. The quantitative incorporation of [methyl-³H]thymidine into dividing bacteria is coupled with a molecular (hybridization) method, to determine the growth rate of bacterial species in situ. The basis of this molecular method is a reverse gene probe—natural populations are labeled in situ with [methyl-³H]thymidine. The probe (³H-Tdr-DNA) is captured, using a hybridization procedure, on a positively charged nylon membrane on which is attached non-labeled target DNA. Two bacterial species, Bacillus cereus and Zoogloea ramigera, were used to demonstrate the principle in laboratory cultures and in a municipal activate sludge treatment process. The DNA of the dividing bacteria in activated sludge was radioactively labeled with [methyl-³H]thymidine, and the DNA of Z. ramigera was recovered using a DNA hybridization method. The recovered radioactively-labeled DNA was used to estimate the growth rate (0.03 × 10⁹ cells · ml⁻¹· h⁻¹) of Z. ramigera in situ. Simultaneously applying these two powerful molecular-based methods could potentially be used to study bacterial population dynamics in situ. Received: 10 September 1997; Accepted: 5 January 1998     

Development of a nonequilibrium microwave discharge at the end of a cylindrical electrode in nitrogen at reduced pressures

Excitation of a microwave discharge at the end of a cylindrical electrode in nitrogen at a pressure of 1 Torr and incident powers of 60–140 W was investigated experimentally by using K-008 and K-011 video cameras and analyzing oscillograms of discharge emission. The times during which the discharge is established in the radial and axial directions are found to be on the order of 10⁻⁴ and 10⁻² s, respectively. The results obtained are analyzed using one-dimensional simulations of a discharge in nitrogen in a quasistatic approximation. The kinetic scheme includes 50 processes involving electrons, ions, and excited molecules and atoms. The time evolution of the concentrations of molecular nitrogen in the N₂(C ³II _u ) and N₂(B ³II _g ) states, responsible for the recorded discharge emission, is compared with the experimental data.     

Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter

Abstract

The aim of this study was to quantify algal colonisation on anthropogenic surfaces (viz. building facades and roof tiles) using chlorophyll a (chl a) as a specific biomarker. Chl a was estimated as the initial fluorescence F₀ of ‘dark adapted’ algae using a pulse-modulated fluorometer (PAM-2000). Four isolates of aeroterrestrial green algae and one aquatic isolate were included in this study. The chl a concentration and F₀ showed an exponential relationship in the tested range between 0 and 400 mg chl a m^?2. The relationship was linear at chl a concentrations <20 mg m^?2. Exponential and linear models are presented for the single isolates with large coefficients of determination (exponential: r² > 0.94, linear: r² > 0.92). The specific power of this fluorometric method is the detection of initial algal colonisation on surfaces in thin or young biofilms down to 3.5 mg chl a m^?2, which corresponds to an abundances of the investigated isolates between 0.2 and 1.5 million cells cm^?2.     

To what extent is a constant volume design worse than a minimum volume design for a series of CSTR's?

The balance equations for substrate in a cascade of CSTR's undergoing an enzyme-catalyzed reaction following Michaelis-Menten kinetics are developed in dimensionless form. Analytical expressions relating the intermediate concentrations are independently obtained for the cases of minimum overall volume and constant volume. The fractional deviations between the overall volumes following these two design criteria are calculated and presented for several values of the relevant parameters. For situations of practical interest, the fractional deviation is below 10%. Increasing values of the Michaelis-Menten parameter, K _m(or decreasing values of the number of reactors in the cascade, N) lead to lower values of the maximum deviation; this maximum deviation is attained at lower conversions of substrate when K _mis increased or N decreased.List of Symbols C _{S, i}mol.m^–3 concentration of substrate at the outlet of the i-th reactor - C _* ^{S, i} normalized concentration of substrate at the outlet of the i-th reactor - C _* ^{S, i, eq} normalized concentration of substrate at the outlet of the i-th reactor using the design criterion of constant volume - C _* ^{S, i, opt} normalized concentration of substrate at the outlet of the i-th reactor using the design criterion of minimum overall volume - C _{S, 0} mol.m^–3 concentration of substrate at the inlet to the first reactor - Da _i Damköhler number for the i-th reactor - Da _eq constant Damköhler number for each reactor of the cascade - Da _{tot, eq} overall Damköhler number for the cascade assuming equal-sized reactors - Da _{tot, min} minimum overall Damköhler number for the cascade - Er fractional deviation between the overall volumes using the two different design criteria - K _mmol. m^–3 Michaelis-Menten constant - K _* ^M dimensionless Michaelis-Menten constant - N number of reactors of the cascade - Q m³. s^–1 volumetric flow rate - V _im³ volume of the i-th reactor - v _max mol. m^–3. s^–1 reaction rate under saturation conditions of the enzyme with substrate - V _{tot, opt} m³ minimum overall volume of the cascade - V _{tot, eq} m³ overall volume of the cascade assuming equal-sized reactors     

Toward a plant-based proxy for the isotope ratio of atmospheric water vapor

Atmospheric water vapor is a major component of the global hydrological cycle, but the isotopic balance of vapor is largely unknown. Here, using models and observations, we show that the leaf water δ¹⁸O in the tropical Crassulacean acid metabolism (CAM) epiphyte Tillandsia usneoides is controlled by the δ¹⁸O of atmospheric water vapor in a predictable manner, irrespective of precipitation inputs. By taking the leaf‐water‐atmospheric signature as recorded in plant organic material, we have reconstructed the atmospheric water vapor δ¹⁸O signature for Miami, FL, USA between 1878 and 2005 using contemporary and herbarium specimens. T. usneoides ranges from Virginia, USA southwards through the tropics to Argentina, and the CAM epiphytic lifeform is widespread in other species. Therefore, there is significant potential for using epiphytes to reconstruct the isotope ratio of atmospheric water (both δ¹⁸O and δD) for spatial scales that span over 60° of latitude and temporal scales that cover the last century of global temperature increase.     

Vibrio plantisponsor sp. nov., a diazotrophic bacterium isolated from a mangrove associated wild rice (Porteresia coarctata Tateoka)

Two Gram negative, facultatively anaerobic, halophilic, motile, slightly curved rod-shaped bacterial strains MSSRF60^T and MSSRF64 were isolated from the roots of a mangrove-associated wild rice collected in the Pichavaram mangroves, India. These strains possess the key functional nitrogenase gene nifH. Phylogenetic analysis based on the 16S rRNA, recA, gapA, mreB, gyrB and pyrH, gene sequences revealed that strains MSSRF60^T and MSSRF64 belong to the genus Vibrio, and had the highest sequence similarity with the type strains of Vibrio diazotrophicus LMG 7893^T (99.7, 94.8, 98.5, 97.9, 94.0 and 90.7%, respectively), Vibrio areninigrae J74^T (98.2, 87.5, 91.5, 88.9, 86.5 and 84.6% respectively) and Vibrio hispanicus LMG 13240^T (97.8, 87.1, 91.7, 89.8, 84.1 and 81.9%, respectively). The fatty acid composition too confirmed the affiliation of strains MSSRF60^T and MSSRF64 to the genus Vibrio. These strains can be differentiated from the most closely related Vibrio species by several phenotypic traits. The DNA G + C content of strain MSSRF60^T was 41.8 mol%. Based on phenotypic, chemotaxonomic, genotypic (multilocus sequence analysis using five genes and genomic fingerprinting using BOX-PCR) and DNA–DNA hybridization analyses, strains MSSRF60^T and MSSRF64 represent a novel species of the genus Vibrio, for which the name Vibrio plantipsonsor sp. nov. is proposed. The type strain is MSSRF60^T (=DSM 21026^T = LMG 24470^T = CAIM 1392^T).     

Clues about the ancestral roles of plant MADS-box genes from a functional analysis of moss homologues

Classic MIKC-type MADS-box genes (MIKC ^c genes) are indispensable elements in the genetic programming of pattern formation, including the segmental organisation of angiosperm flowers, in seed plants. Since little is known about the functions of MIKC ^c genes in non-seed plants, a functional analysis of moss MIKC ^c homologues was performed using the genetically amenable, simple model plant, Physcomitrella patens. Expression of moss homologues was knocked down using an antisense RNA approach or abolished by generating transformants with gene knockouts. The knocked down (“antisense”) transformants displayed a multifaceted mutant phenotype comprising delayed gametangia formation, diminished sporophyte yield and, in the most extremely affected cases, abnormal sporophyte development and altered leaf morphogenesis. Knocked out transformants were phenotypically normal. Analysis of in situ MIKC ^c gene expression using transgenic strains containing MIKC ^c promoter–GUS fusions showed that these genes are generally expressed ubiquitously in vegetative and reproductive tissues. We conclude that MIKC ^c genes play significant roles in morphogenetic programming of the moss. Functional redundancy characterises some members of the gene group. Our findings provide clues concerning the ancestral roles of some MIKC ^c genes that may be represented in the genomes of diverse extant plant taxa. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acetone biodegradation in a trickle-bed biofilter

Laboratory scale-studies on acetone biodegradation in a trickle-bed biofilter were carried out using using two identically sized columns, one of which was packed with coconut fibre and the other with plexiglass chips. The columns were inoculated with two strains of bacteria: Burkholderia cepacia (syn. Pseudomonas cepacia) and Acinetobacter baumannii. The continuous process of air purification was carried out at incremental acetone concentrations from 0.3 to 2.5 g m⁻³ air and air flow-rates from 0.1 to 0.3 m³ h⁻¹. A maximum acetone elimination capacity of 95.8 g m⁻³ filter bed h⁻¹ was achieved at air flow-rate of 36 m h⁻¹.     

Seed-shedding in a Scottish heath community

Abstract. For a 28-week period in late 1987 and early 1988, a study of seed-shedding by several heath species was carried out at the Muir of Dinnet in northeastern Scotland. The dominant species in the heath is Calluna vulgaris. Seed-shedding in Calluna began in early September 1987 and was completed in April 1988, with the period of maximum shedding falling between early November and late December 1987. The total numbers of seeds/m² deposited in stands of Calluna in its four growth-phases were: pioneer, 18 910; building, 169010; mature, 198580; degenerate, 33900. Substantial loss of potential seeds results from the shedding of immature flowers. A control area close to, but outside the area of Calluna dominance had a deposition rate of 770 Calluna seeds/m², indicating sufficient seed to colonise nearby available habitats. Seed rain was also recorded for several other heathland species: Erica cinerea, E. tetralix, Carex spp. and Betula pendula. Seeds of Erica cinerea were deposited in all the Calluna stands, 32670/m² in the pioneer stand, 17600/m² in building, 3720/m² in mature, 210/m² in degenerate (120/m² in the control area). Numbers were greatest at the start of the sampling period, declining thereafter. This applied also to Betula seeds. Erica tetralix occurred in the degenerate Calluna stand and yielded 640 seeds/m² (400/m² in the control area). Seeds of Carex spp. were obtained in the control: 4110/m². The method of sampling has a significant effect on the figures obtained. A method using tube collectors, emptied frequently, is recommended.     

Chromium ions phytoaccumulation by three floating aquatic macrophytes from a nutrient medium

In the present work, the trivalent and hexavalent chromium phytoaccumulation by three living free floating aquatic macrophytes Salvinia auriculata, Pistia stratiotes, and Eicchornia crassipes was investigated in greenhouse. These plants were grown in hydroponic solutions supplied with non-toxic Cr³⁺ and Cr⁶⁺ chromium concentrations, performing six collections of nutrient media and plants in time from a batch system. The total chromium concentrations into Cr-doped hydroponic media and dry roots and aerial parts were assayed, by using the Synchrotron radiation X-ray fluorescence technique. The aquatic plant-based chromium removal data were described by using a nonstructural kinetic model, obtaining different bioaccumulation rate, ranging from 0.015 to 0.837 l mg⁻¹ d⁻¹. The Cr³⁺ removal efficiency was about 90%, 50%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata, respectively; while it was rather different for Cr⁶⁺ one, with values about 50%, 70%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata.     

Solution conformational preferences of a peptidic analogue of a natural macrolide

The conformational behavior in solution of a cyclic peptide with sequence cyclo-(Pro¹-Pro²-Dab³ (cHexA)ψ[N⁷HCO]-Leu⁴ψ[NHCO]-Suc⁵-Gly⁶-) has been throughly investigated with the combined use of nmr and molecular dynamic techniques. The compound, which has been modeled to mimic the FK506 macrolide bound to the FK506 binding protein structure, can be considered as a peptidic analogue of the FK506 system. The synthesis was carried out on a phenylacetoamidomethyl resin using an appropriate protocol for the peptide chain elongation. The conformational properties of the cyclic hexapeptide were studied in DMSO and water. The nmr data in DMSO and restrained molecular dynamics simulations show the presence of two contiguous cis peptide bonds involving the -Gly-Pro-Pro- segment. This segment in water exhibits conformational heterogeneity presenting at least two distinct conformational families, characterized the first by cis-cis and the second by a trans-cis Gly-Pro-Pro configuration; the trans-cis isomer was fully characterized. © 1997 John Wiley & Sons, Inc. Biopoly 42: 349–361, 1997     

Nitratireductor lucknowense sp. nov., a novel bacterium isolated from a pesticide contaminated soil

A straw-yellow pigmented bacterium, strain IITR-21^T was isolated from a pesticide contaminated site and characterized by using a polyphasic taxonomic approach. The organism had morphological and chemotaxonomic properties consistent with its classification in the genus Nitratireductor. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain IITR-21^T belongs to the genus Nitratireductor and was moderately related to Nitratireductor indicus C115^T (97.7%) and Nitratireductor pacificus pht-3B^T (97.4%), whereas sequence similarity value with the other species including the type species of the genus Nitratireductor, Nitratireductor aquibiodomus showed less than 97.0% similarity. However, the DNA–DNA relatedness values between strain IITR-21^T and the moderately related taxa N. indicus (59.1%) and N. pacificus (40.4%) were well below the 70% threshold value recommended for the delineation of bacterial species. The G+C content of the DNA was 62.4 mol%. Based on physiological, biochemical tests and genotypic differences between the strain IITR-21^T and the other two validly published species of the genus Nitratireductor, it is proposed that the strain be classified as a new species of Nitratireductor as Nitratireductor lucknowense sp. nov. The type strain is IITR-21^T (=MTCC 8354^T = DSM 24322^T).     

Synthesis and evaluation of a bifunctional chelate for development of Bi(III)-labeled radioimmunoconjugates

A new bifunctional ligand C-DEPA was designed and synthesized as a component for antibody-targeted radiation therapy (radioimmunotherapy, RIT) of cancer. C-DEPA was conjugated to a tumor targeting antibody, trastuzumab, and the corresponding C-DEPA-trastuzumab conjugate was evaluated for radiolabeling kinetics with ^205/6Bi. C-DEPA-trastuzumab conjugate rapidly bound ^205/6Bi, and ^205/6Bi-C-DEPA-trastuzumab conjugate was stable in human serum for 72 h. The in vitro radiolabeling kinetics and serum stability data suggest that C-DEPA is a potential chelate for preclinical RIT applications using ²¹²Bi and ²¹³Bi.     

Leaf area estimation in a sugar beet cultivar by linear models

An indirect method of leaf area measurement for Rizor sugar beet cultivar was tested. Leaves were sampled during two growing seasons in a Randomised Complete Block Design experiment. For 2002 samplings, leaf area [cm²] was linearly correlated with maximum leaf width [cm] using all leaf samples (r ² = 0.83, p < 0.001) or using the means of the 8 sampling occasions (r ² = 0.97, p < 0.001). Correlations between leaf area and leaf mid vein length [cm] were weaker (r ² = 0.75, p < 0.001 and r ² = 0.93, p < 0. 001, respectively). For 2003 samplings, the area estimated by the equations was highly correlated to the measured leaf area.     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.