Studies on R-phycoerythrins from two Antarctic marine red algae and a mesophilic red alga

R-phycoerythrin was purified from two benthic red algae, Iridaea cordata and Phyllophora antarctica, obtained growing at ?2°C under thick sea ice off the coast of Antarctica. For the I. cordata protein, the molecular mass was 245,000 Da, and its secondary structure was 60% α helix, 17% β sheet, 16% turn, and 7% other. The light-harvesting faculties of the I. cordata protein resembled those of R-phycoerythrins from mesophilic red algae and were distinctive from the novel R-phycoerythrin from P. antarctica. Deconvolution of the visible absorption spectrum of R-phycoerythrin from I. cordata indicated a minimum of five component bands having maxima at 568, 558, 534, 496, and 481?nm. R-phycoerythrins from the mesophilic Porphyra tenera and psychrophilic Phyllophora antarctica had the same five bands. The protein from Phyllophora antarctica obtained its unique spectrum from a more intense component at 482?nm, and a less intense band at 533?nm. This change was probably produced by a replacement of phycoerythrobilin by phycourobilin. A temperature study of the circular dichroism CD was obtained for R-phycoerythrin from I. cordata from 4 to 80°C. Laser time-resolved fluorescence studies on R-phycoerythrin showed bilin to bilin energy transfer with a 60.2-ps lifetime, which should occur by the Förster resonance. The similarities in spectra between the proteins from I. cordata and Porphyra tenera and the different spectrum for the protein from Phyllophora antarctica show that only particular antarctic habitats require unique R-phycoerythrins.     

Construction of xylose-assimilating Saccharomyces cerevisiae

The xylose reductase gene originating from Pichia stipitis was subcloned on an expression vector with the enolase promoter and terminator from Saccharomyces cerevisiae. The transformants of S. cerevisiae harboring the resultant plasmids produced xylose reductase constitutively at a rate about 3 times higher than P. stipitis, but could not assimilate xylose due to the deficient conversion of xylitol to xylulose. The xylitol dehydrogenase gene was also isolated from the gene library of P. stipitis by plaque hybridization using a probe specific for its N-terminal amino acid sequence. The gene transferred into S. cerevisiae was well expressed. Furthermore, high expressions of the xylose reductase and xylitol dehydrogenase genes in S. cerevisiae were achieved by introducing both genes on the same or coexisting plasmids. The transformants could grow on a medium containing xylose as the sole carbon source, but ethanol production from xylose was less than that by P. stipitis and a significant amount of xylitol was excreted into the culture broth.     

Ethanol production by In situ xylose isomerization using recombinant Escherichia coli and fermentation using conventional Saccharomyces cerevisiae

Xylose isomerase from Geobacillus kaustophilus HTA426 was functionally expressed in Escherichia coli BL21 (DE3) and the recombinant E. coli cells were used together with conventional Saccharomyces cerevisiae to produce ethanol from xylose by simultaneous xylose isomerisation and fermentation. When recombinant E. coli cells were used as the source of xylose isomerase, a significant amount of ethanol was produced from xylose, whereas the control without recombinant E. coli cells did not produce any detectable amount of ethanol from xylose. Ethanol production was increased by 38% by feeding more recombinant E. coli at 48 h compared to adding recombinant E. coli only in the beginning, resulting in more ethanol production than P. stipitis CBS6054 under the same conditions. The xylitol accumulation by the in situ process was only 57% of that produced by the P. stipitis CBS6054.     

Industrial biotransformations for the production of d-amino acids

Optically pure d-amino acids are industrially manufactured by biotransformations of cheap starting materials produced by chemical synthesis or fermentation in combination with the development of enzyme catalysts suitable for the starting materials. dl-Alaninamide, an intermediate of the chemical synthesis of dl-alanine, was efficiently converted to d-alanine by stereoselective hydrolysis with a d-isomer specific amidohydrolase produced by Arthrobacter sp. NJ-26. The total utilization system of dl-alaninamide for the production of optically pure d- and l-alanine was constructed by stereospecific amidohydrolases. On the other hand, d-amino acids were also produced from corresponding l-isomers, which are efficiently manufactured by fermentation. d-Glutamic acid was produced from l-glutamic acid. l-Glutamate was converted to the dl-form by the recombinant glutamate racemase of Lactobacillus brevis ATCC8287. Then l-glutamate in a racemic mixture was selectively decarboxylated to γ-aminobutyrate by the l-glutamate decarboxylase of E. coli ATCC11246. As a result of successive enzymatic reactions, d-glutamate was efficiently produced from l-glutamate by a one-pot reaction. d-Proline was produced by the same strategy from l-proline using the recombinant proline racemase of Clostridium sticklandii ATCC12262. In this case, l-proline was degraded by Candida sp. PRD-234. The strategy from l-amino acids to d-amino acids could be applicable to the manufacture of many d-amino acids.     

Identification of Cryptosporidium spp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.     

Characterization of levan produced by Serratia sp.

Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus.     

Microseiramide from the freshwater cyanobacterium Microseira sp. UIC 10445

Microseiramide (1), a cyclic heptapeptide, was isolated from a sample of the freshwater cyanobacterium Microseira sp. UIC 10445 collected in a shallow lake in Northern Indiana. Taxonomic identification of UIC 10445 was performed by a combination of morphological and phylogenetic characterization. Phylogenetic analysis revealed that UIC 10445 was a member of the recently described genus Microseira, which is phylogenetically distinct from the morphologically similar genera, Moorea and Lyngbya. The planar structure of microseiramide (1) was determined by extensive 1D and 2D NMR experiments as well as HRESIMS analysis. The absolute configurations of amino acid residues were determined using acid hydrolysis followed by the advanced Marfey’s analysis. Microseiramide (1) is the first cyclic peptide reported from a Microseira sp., and the structure of microseiramide (1) is distinct from the previously known metabolites from cyanobacteria of the genera Moorea and Lyngbya.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

Identification and distribution of ononitol in nodules of pisum sativum and glycine max

Ononitol (4-O-methyl-myo-inositol) was identified as a major carbohydrate in Pisum sativum nodules, comprising 25–34% of the total mono- plus disaccharides in nodules formed by two Rhizobium leguminosarum strains. Ononitol was purified from Glycine max nodules and was found to be a minor carbohydrate in these nodules. The distribution of ononitol in bacteroids and cytosol from soybean nodules suggests that it is not synthesized by bacteroids.     

Adding new pieces to the Azadinium (Dinophyceae) diversity and biogeography puzzle: Non-toxigenic Azadinium zhuanum sp. nov. from China,toxigenic A. poporum from the Mediterranean,and a non-toxigenic A. dalianense from the French Atlantic

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44 fg cell⁻¹. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.     

Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp. TE1826

The gene encoding sarcosine oxidase from Arthrobacter sp. TE1826 (soxA) was cloned in Escherichia coli by a convenient plate assay. It was located within a 1.7-kbp PstI-EcoRI fragment of the recombinant plasmid pSAOEP3. The purified sarcosine oxidase from the recombinant strain was found to be the same as that from the parental strain. The DNA sequence of soxA was determined, and an open reading frame composed of 389 amino acid residues was found. By Edman degradation of the enzyme, it was revealed that the amino-terminal amino acid (methionine) was eliminated in the parental strain and E. coli. The molecular weight (43,249) of the enzyme was consistent with the result from SDS-polyacrylamide gel electrophoresis. The FAD-binding site was found in the amino-terminal region of sarcosine oxidase by a homology search. The soxA gene was subcloned on a shuttle vector, pHY300PLK, and was expressed in both E. coli and Bacillus subtillis in the absence of an inducer, although the enzyme was induced with sarcosine in the parental strain.     

Phylogenetic and Functional Diversity Within Toluene-Degrading,Sulphate-Reducing Consortia Enriched from a Contaminated Aquifer

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.     

Enzymatic production of d-glutamate from l-glutamate by a glutamate racemase

d-Glutamate was produced from l-glutamate by two successive cellular reactions with a glutamate racemase produced by Escherichia coli TM93 harboring a plasmid containing a glutamate racemase gene from Lactobacillus brevis ATCC 8287 and a glutamate decarboxylase produced by E. coli ATCC 11246. l-Glutamate was first racemized to dl-glutamate at pH 8.5 and l-glutamate was then decarboxylated at pH 4.2. Starting from 100 g/l of l-glutamate, 50 g/l of d-glutamate remained after 15 h reaction.     

Adaptability and Persistence of the Emerging Pathogen Bordetella petrii

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient’s antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient’s serum. Finally, we characterize one strain that was poorly recognized by the patient’s antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.     

Solubilization of a functionally active proline carrier from membranes of Escherichia coli with an organic solvent.

A proline transport carrier was extracted from the membranes of Escherichia coli with acidic n-butanol. Vesicles reconstituted from the butanol extract and E. coli phospholipids and preloaded with K⁺ showed rapid uphill uptake of proline when energy was supplied as a membrane potential introduced by K⁺-diffusion via valinomycin. Proline uptake by the reconstituted vesicles, like that of intact cells and isolated membrane vesicles, was inhibited by 3,4-dehydroproline, SH reagents, and a proton conducting uncoupler. Reconstituted vesicles of mutants defective in proline transport showed little or no proline uptake. The proline carrier was partially purified from the extract and separated from the bulk of phospholipids on Sephadex LH-20.     

An S-adenosyl-l-methionine; coniine methyltransferase from Conium maculatum

Evidence is presented for a cell free system from Conium maculatum which catalyses the transfer of a methyl group from S-adenoysl-l-methionine to coniine with the formation of N-methyl coniine. Maximum enzyme activity which occurred in the unripe fruits was enhanced by dithiothreitol, and evidence for the role of sulphydryl groups of the enzyme was obtained from inhibition with p-CMB, iodoacetamide and N-methyl maleimide. A divalent metal cation dependency was not detected.     

Volatile constituents in leaves of parsley

Volatile chemicals obtained from the leaves of parsley, Petroselinum sativum by steam distillation, isopentane extraction, and head-space analysis were identified by GLC-MS. The presence in leaf oil of α-pinene, β-pinene, myrcene, β-phellandrene, trans-β-ocimene, γ-terpinene, 1-methyl-4-isopropenyl benzene, and 1,3,8-p-menthatriene as shown by earlier investigators was confirmed. In the present studies, the number of volatile chemicals detected in the leaves was extended by an additional 42. Sniffing tests of effluent from a gas chromatograph of a concentrate from parsley leaves showed that 1,3,8-p-menthatriene was only one of several compounds that gave a parsley-like aroma.     

(+)-5,17-dehydromatrine N-oxide,a new alkaloid in Euchresta japonica

A new lupin alkaloid, (+)-5,17-dehydromatrine N-oxide, was isolated from the fresh aerial parts of Euchresta japonica. Its structure was confirmed by spectrometric data and by direct comparison with a synthetic sample, prepared from (+)-sophoranol ((+)-5-hydroxymatrine). It was also concluded that (+)-5,17-dehydromatrine N-oxide and (+)-matrine N-oxide possess the same configuration with respect to the asymmetric nitrogen by NMR spectra.