Structure and Expression of Mitochondrial Citrate Synthases from Higher Plants

Mitochondrial citrate synthase (EC 4.1.3.7 [EC] ) represents the firstenzyme of the tricarboxylic acid cycle, catalyzing the condensationof acetyl-CoA and oxaloacetate, finally yielding citrate andCoA. We report here the isolation of cDNA clones encoding citratesynthase from Nicotiana tabacum, Beta vulgaris and Populus.Nucleotide and deduced amino acid sequences were compared withpreviously published sequences of mitochondrial citrate synthasesfrom Arabidopsis thaliana and potato, as well as with the sequenceof glyoxysomal citrate synthase from pumpkin. Homologies betweenthe various plant mitochondrial enzymes were in the range from77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco)on the nucleotide level (coding regions only), and in the rangefrom 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs.tobacco, respectively) on the amino acid level. Identities ofthe mitochondrial isozymes to the pumpkin glyoxysomal enzymewere below 30% on the nucleotide and amino acid level. In Northernblot experiments citrate synthase mRNA was detected in all tissuesanalyzed. However, levels of expression showed tissue dependencydespite the fact that citrate synthase is usually considereda house-keeping enzyme. Whether these different levels of expressionreflect tissuespecifc variations with respect to basic metabolismawaits further analysis. (Received May 20, 1996; Accepted August 20, 1996)     

Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k_cat = 8.3 s⁻¹; K_m = 14.1 and 4.3 μM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K⁺ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

A Rapid Method for Purifying Bacterial Deoxyribonucleic acid

DNA was purified from bacterial cell lysates by treatment with RNA ase and Pronase followed by chromatography on a column of Sepharose 4B with standard saline citrate buffer as eluant. It has the same base composition as DNA prepared by-Marmur's (1961) method.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.     

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.     

A Thin-Layer Chromatographic Technique for Rapid Estimation of Bacterial Lipases

S ummary . A thin-layer chromatographic technique for the estimation of lipase in Pseudomonas aeruginosa and staphylococci is described. The technique needs a short incubation period (4 h), while the subsequent procedure is quite simple and rapid. The validity of the technique was established by testing 56 strains of Ps. aeruginosa and 35 strains of staphylococci. The results strongly support the usefulness of the technique which can be quite easily applied to routine use.     

New Techniques for the Rapid Characterization of Oligonucleotides by Mass Spectrometry

Abstract

Recent advances in combined HPLC/electrospray ionization-mass spectrometry provide effective new capabilities for the rapid characterization of oligonucleotides. Accurate mass measurements with errors <0.3 Da, and determination of base and sugar modification and of nearest neighbor identities, can be routinely carried out on 10-100 component mixtures of RNA or DNA. These procedures are widely applicable in structural and analytical studies involving mixtures of oligonucleotides.     

Rapid Procedure for the Detection of Acid and Gas Production by Bacterial Cultures

A rapid miniaturized procedure, with a Microtiter plate and the Amojell-overlay technique, is described for the detection of acid and gas production by bacterial cultures.     

Rapid Microbiological Testing: Monitoring the Development of Bacterial Stress

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H₂O₂. The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

以犬2型腺病毒为载体进行疫苗和基因治疗的研究,是近年的研究热点.目前的焦点问题是新型载体的构建,经典的方法是体外连接,费时、费力.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp fight end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to fight fragment, obtaining a “rescue” plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstⅠ digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.     

Rapid Screening Method for Analyzing the Conjugated Linoleic Acid Production Capabilities of Bacterial Cultures

In this paper we describe a rapid method for identifying bacteria which convert free linoleic acid to conjugated linoleic acid (CLA). This method is based on spectrophotometric detection of CLA and compares well with the standard gas-liquid chromatography method. This method should facilitate high-throughput screening of bacterial isolates for the ability to produce conjugated fatty acids.     

Molecular Cloning of Plant Spermidine Synthases

Four cDNAs for spermidine synthase (SPDS), which converts thediamine putrescine to the higher polyamine spermidine usingdecarboxylated S-adenosylmethionine as the co-factor, were isolatedfrom Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsisthaliana. When the N. sylvestris SPDS cDNA was expressed ina SPDS-deficient E. coli mutant, the recombinant protein showedhigh SPDS activity, but did not have any spermine synthase activity.The plant SPDSs have molecular masses of about 34 kDa, possessthe co-factor binding motifs which have been proposed for S-adenosylmethionine,and are more homologous in amino acid sequence to tobacco putrescineN-methyltransferase (PMT) than to SPDSs from mammals and E.coli. The SPDS gene is expressed in root, stem, and leaf inN. sylvestris, whereas the PMT gene is expressed only in root.The potential evolution of plant SPDS and PMT, and their evolutionaryrelationships with animal SPDS are discussed. (Received September 3, 1997; Accepted November 5, 1997)     

Examination of Thermophilic Methane-Producing Digesters by Analysis of Bacterial Lipids

Thermophilic methane-producing digesters were examined by the analysis of lipids to determine the microbial biomass, community structure, and nutritional status of the microbes within the digesters. The digesters received a daily feedstock of cattle feed and Bermuda grass, with some digesters receiving additional supplements of propionate, butyrate, or nitrate. Microbial biomass, measured as total extractable lipid phosphate, was decreased in slurries from digesters receiving continuous addition of the fermentation intermediates propionate or butyrate as compared with slurries from control digesters receiving the feedstock alone. In slurries from digesters that received continuous addition of nitrate, the microbial biomass was higher than in the slurries from control digesters. The control digesters had ca. 2.5 × 10¹¹ bacteria per g (dry weight) as determined from total extractable lipid phosphate. Shifts in microbial community structure were observed by analysis of ester-linked phospholipid fatty acids. Statistical analysis of the patterns of phospholipid fatty acids indicated that the digesters receiving different supplements could be distinguished from the control digester and from each other. Poly-β-hydroxybutyric acid, an indicator of metabolic stress, was detected in slurries from all the digesters. Slurries from the nitrate-amended digester had the highest concentration of poly-β-hydroxybutyric acid, whereas slurries from the propionate-amended digester had the lowest concentration. These chemical analyses offer a quantitative means to correlate shifts in microbial biomass, community structure, and nutritional status in complex fermentation systems to the production of a specific end product.     

Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.     

Rapid Immunoassays for Detection of UV-Induced Cyclobutane Pyrimidine Dimers in Whole Bacterial Cells

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled (¹²⁵I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.     

Assessment of Robotic Patient Simulators for Training in Manual Physical Therapy Examination Techniques

Robots that simulate patients suffering from joint resistance caused by biomechanical and neural impairments are used to aid the training of physical therapists in manual examination techniques. However, there are few methods for assessing such robots. This article proposes two types of assessment measures based on typical judgments of clinicians. One of the measures involves the evaluation of how well the simulator presents different severities of a specified disease. Experienced clinicians were requested to rate the simulated symptoms in terms of severity, and the consistency of their ratings was used as a performance measure. The other measure involves the evaluation of how well the simulator presents different types of symptoms. In this case, the clinicians were requested to classify the simulated resistances in terms of symptom type, and the average ratios of their answers were used as performance measures. For both types of assessment measures, a higher index implied higher agreement among the experienced clinicians that subjectively assessed the symptoms based on typical symptom features. We applied these two assessment methods to a patient knee robot and achieved positive appraisals. The assessment measures have potential for use in comparing several patient simulators for training physical therapists, rather than as absolute indices for developing a standard.