Application of Denaturing High-Performance Liquid Chromatography in Microbial Ecology: Fermentor Sludge, Compost, and Soil Community Profiling

Genetic fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), are used in microbial ecology for the analysis of mixed microbial communities but are associated with various problems. In the present study we used a new alternative method: denaturing high-performance liquid chromatography (dHPLC). This method was previously shown to work with samples from water and gut flora but had not yet been applied to complex environmental samples. In contrast to other publications dealing with dHPLC, we used a commonly available HPLC system. Samples from different origins (fermentor sludge, compost, and soil), all ecologically significant, were tested, and the 16S rRNA gene was amplified via PCR. After optimization of the HPLC elution conditions, amplicons of pure cultures and mixed microbial populations could be separated successfully. Systematic differentiation was carried out by a cloning approach, since fraction collection of the peaks did not result in satisfactory fragment separation. dHPLC was evaluated as a tool for microbial community analysis on a genetic level and demonstrated major improvements compared to gel-based fingerprinting methods, such as DGGE, that are commonly used in microbial ecology.     

Identification of Distinct Communities of Sulfate-Reducing Bacteria in Oil Fields by Reverse Sample Genome Probing

Thirty-five different standards of sulfate-reducing bacteria, identified by reverse sample genome probing and defined as bacteria with genomes showing little or no cross-hybridization, were in part characterized by Southern blotting, using 16S rRNA and hydrogenase gene probes. Samples from 56 sites in seven different western Canadian oil field locations were collected and enriched for sulfate-reducing bacteria by using different liquid media containing one of the following carbon sources: lactate, ethanol, benzoate, decanoate, propionate, or acetate. DNA was isolated from the enrichments and probed by reverse sample genome probing using master filters containing denatured chromosomal DNAs from the 35 sulfate-reducing bacterial standards. Statistical analysis of the microbial compositions at 44 of the 56 sites indicated the presence of two distinct communities of sulfate-reducing bacteria. The discriminating factor between the two communities was the salt concentration of the production waters, which were either fresh water or saline. Of 34 standards detected, 10 were unique to the fresh water and 18 were unique to the saline oil field environment, while only 6 organisms were cultured from both communities.     

Denaturing High-Performance Liquid Chromatography Detects Reliably BRCA1 and BRCA2 Mutations

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.     

Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC)

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based denaturing high-performance liquid chromatography (DHPLC). An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.     

Diagnosis of Hemoglobinopathies by High-Performance Liquid Chromatography

This report describes a unique cation exchange high-performance liquidchromatography capable of separating more than 40 frequently encountered human hemoglobins and variants within 12 min. Some of these variants are unresolvable by the conventional electrophoretic methods and would thus lead to an incorrect diagnosis of hemoglobinpathy. The method provides high sensitivity, superior resolution and accurate quantitation of hemoglobin concentrations. It can also be fully automated thus make it an ideal methodology for the diagnosis of hemoglobin disorders in a routine clinical laboratory.     

Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.Over the last 2 decades, the development of molecular biology tools has led to the emergence of a new discipline, molecular microbial ecology. The overall structural diversity of microbial communities can be examined easily using PCR-based strategies (6), usually targeting the 16S rRNA gene as a universal genetic marker of prokaryotes. Genotyping approaches avoid current limitations of cultivation methods, which only poorly reflect the phylogenetic diversity of microbial communities (12). The principles, technical aspects, and limitations of commonly employed methods were recently reviewed (10). Among these methods, terminal restriction fragment length polymorphism (T-RFLP) has proved to be invaluable for rapid characterization of the composition and dynamics of species-rich samples (13). Compared to other approaches, T-RFLP is semiquantitative and combines high levels of sensitivity, resolution, and reproducibility (see Table S1 in the supplemental material). Taxonomic diversity of microbial communities is evaluated by using the strain-dependent variability of restriction sites within a conserved PCR-amplified DNA fragment. The terminal restriction fragments (T-RFs) of digested PCR products appear as chromatographic peaks after size-dependent electrophoretic separation due to a fluorescent tag attached to one of the primers used for PCR. The relative abundance of peaks is evaluated, and fragment lengths are estimated using a fluorescent internal size standard comigrating with the sample (5). The estimated lengths corresponding to the T-RFLP peaks obtained are compared to databases of T-RF sizes generated by in silico digestion of known 16S rRNA gene sequences with commonly used restriction enzymes for phylogenetic assignment (13). However, estimation of T-RF lengths from experimental chromatograms is biased by the fact that differences in the electrophoretic properties of the two different fluorescent dyes used to distinguish sample fragments from the size marker significantly affect fragment migration (7, 11). Discrepancies greater than 6 nucleotides (nt), depending on the length of the fragment, have been reported between expected and experimentally estimated fragment lengths (7). This causes errors in phylogenetic assignments and may in turn lead to erroneous inferences regarding the functional aspects of the microbial communities under investigation. Another drawback of T-RFLP is the difficulty of retrieving sequence information directly from experimental T-RFs, since additional construction of representative 16S rRNA gene libraries is required to obtain such information.Here we propose an experimental strategy to circumvent current limitations of T-RFLP and facilitate characterization of microbial communities. First, we propose an optimized protocol for T-RFLP that yields reliable T-RF sizes. Second, we describe use of denaturing high-performance liquid chromatography (D-HPLC) as an alternative to cloning in order to gain direct access to DNA sequence information. D-HPLC, an emerging technique for microbial community profiling (1, 4), enables collection of DNA fragments separated on the basis of differences in sequence, sequence length, and G+C content at a partially denaturing temperature. The unambiguous phylogenetic characterization of a model microbial assembly of five reference strains is described as proof of principle of the usefulness of the proposed strategy.     

Comparison of Bacterial Lipopolysaccharides by High-Performance Liquid Chromatography

A comparison of lipid-free polysaccharides from gram-negative bacteria was rapidly accomplished by using high-performance liquid chromatography of underivatized hydrolysates. Examination of a number of such products revealed that, contrary to earlier reports, Xanthomonas campestris lipopolysaccharide contained heptose, together with rhamnose and galactose, but not mannose. The polymers from the methanotrophs “Methylomonas albus” and “Methylosinus trichosporium” contained heptose and glucose, and that from a “Klebsiella aerogenes” strain contained heptose, glucose, and galactose. The absence of heptose from the lipopolysaccharide of Myxococcus xanthus was confirmed.     

Sequence-Dependent DNA Separation by Anion-Exchange High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) system with a new nonporous anion-exchange resin, DNA–NPR, made it possible to rapidly separate DNA fragments up to 20 kbp with high resolution. In order to further characterize this chromatographic DNA separation system, we prepared a mixture of double-stranded DNAs of constant length carrying a fully degenerated 50-bp region and analyzed their chromatographic behavior on the DNA–NPR column. The results indicated that the separation of DNA fragments on the anion-exchange HPLC was governed not only by size, but also by nucleotide sequence: even DNA fragments with the same size and the same base content could be separated on this column. Taking advantage of this characteristic feature of the anion-exchange HPLC, we could readily fractionate human cDNAs with practically acceptable recovery and high resolution. Furthermore, the combination of HPLC and gel electrophoresis realized separation of a mixture of DNA fragments in a two-dimensional pattern.     

Assay of Brain Calmodulin Levels Using High-Performance Liquid Chromatography

A simple, sensitive, and efficient HPLC method for the determination of calmodulin levels in brain tissue extracts is described. The assay is linear with respect to both calmodulin and protein concentrations. The specificity and validity of this assay for calmodulin is demonstrated by parallel radioimmunoassay determinations which give equivalent results. Determination of calmodulin levels in various brain regions revealed a high concentration of this protein in the hypothalamus, by comparison to other areas examined.     

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

Versatile Medium for the Enumeration of Sulfate-Reducing Bacteria

A lactate-yeast extract-sulfate medium, making use of both thioglycolate and ascorbate to poise the E_n gave valid colony counts of sulfate-reducing bacteria with both pure cultures and natural samples.     

Reversed-Phase High-Performance Liquid Chromatography of Products of Microbiological Degradation of Toluene

Conditions have been selected for reversed-phase high-performance liquid chromatographic assay of intermediate products formed in the course of utilization of toluene by Pseudomonas putida. The composition of products indicates that degradation of toluene by strain BS590-P proceeds primarily through the formation of benzoate and catechol. This is followed by degradation of catechol via ortho-cleavage. In strain BS3701-P, toluene oxidation involves both the side chain and the aromatic ring.     

反相高效液相色谱法测定烟叶中的游离氨基酸

用不同浓度的乙醇溶液提取烟样中的游离氨基酸 ,结果显示 ,存在最佳的乙醇溶液浓度 ,使烟样中被提取的游离氨基酸总量最大 ;对比了活性炭、乙醚、5 %磺基水杨酸、阳离子交换柱的纯化效果 ,发现阳离子交换柱的纯化效果较其它三种试剂要好。在提取和纯化之后 ,采用OPA、FMOC联合在线衍生反相高效液相色谱法测定了烟样中的游离氨基酸 ,该方法使烟样中的氨基酸和亚氨基酸能被同时测定 ,并且分析方法的重现性和回收率均令人满意。最后用该方法对云南B2 F98(上部、橘黄、二等烟叶 ,98年产 )烟叶中的游离氨基酸进行了测定 ,有 15种氨基酸被测出 ,其中Pro含量最高 ,约占总量的 2 5 % ,Thr含量最低 ,约占总量的 1%。     

柱前衍生氨基酸的高效液相色谱分析

综述了目前柱前衍生氨基酸高效液相色谱分析技术的最新进展和现状,分别详细介绍了应用最广、技术最先进的几种柱前衍生氨基酸分析技术和方法。     

Reversed-Phase High-Performance Liquid Chromatography of Prenylquinones in Green Leaves Using an Electrochemical Detector

Ubiquinone-9, -10, plastoquinone-A, -B, -C, phylloquinone and-tocopherolquinone in spinach leaf extract were separated anddetermined by reversed-phase high-performance liquid chromatographyusing an electrochemical detector. These prenylquinones wereeluted with a mixture of ethanol and methanol containing 50mM NaClO₄ and 2 mM HClO₄from an octadecyl silica column. Theelectrochemical detector could selectively detect the quinonesin the eluate, and enabled to determine even the minor quinonessuch as PQ-B and PQ-C which had not been evaluated by HPLC withan optical detector. The method is simple and sensitive to thedegree that amounts of prenylquinones could be determined aslow as 0.1 nmol. (Received June 18, 1984; Accepted September 3, 1984)     

Identification and Quantification of Ascorbic Acid in Kiwi Fruit by High-Performance Liquid Chromatography

The content of ascorbic acid in kiwi fruit (Actinidia chinensisPlanch) of various cultivars was determined by high-performance liquid chromatography (HPLC). A minimal content of ascorbic acid was found in fruits of Gaivard cultivar: in juice – 5.44, skin – 1.14, and pulp – 4.20 mg/g.     

Complex Dielectric Properties of Sulfate-Reducing Bacteria Suspensions

Sulfate-reducing bacteria (SRB) can potentially enhance the remediation of heavy metals in the subsurface. Previous geophysical research has demonstrated the sensitivity of electrical measurements to SRB-mediated mineral transformation in porous media. However, the inherent dielectric properties of SRB and their direct contribution to the electrical properties of porous media are poorly understood. We studied the complex dielectric properties of SRB (Desulfovibrio vulgaris) suspensions at different concentrations and at different growth stages using a two-electrode dielectric spectroscopy measurement over the frequency range of 20 Hz to 1 MHz. Our results show higher dielectric responses (relative dielectric permittivity, real and imaginary conductivity) occurred with higher bacteria concentration at frequencies <10 kHz. Additionally, permittivity and conductivity both decreased as cells aged from mid-log phase to late stationary phase. Our results suggest that dielectric spectroscopy measurements can be used to noninvasively monitor biomass and various growth stages of SRB. Our work advances the interpretation of electrical signals associated with SRB observed in the subsurface.     

Metabolism of Explosive Compounds by Sulfate-Reducing Bacteria

The metabolism of various explosive compounds—1,3,5-trinitrobenzene (TNB), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)—by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied. The results indicated that the Desulfovibrio spp. used all of the explosive compounds studied as their sole source of nitrogen for growth. The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation. We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media. This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L. The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil. Received: 23 January 1998 / Accepted: 5 March 1998     

Oil Field Souring Control by Nitrate-Reducing Sulfurospirillum spp. That Outcompete Sulfate-Reducing Bacteria for Organic Electron Donors

Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including Sulfurospirillum and Thauera spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile Sulfurospirillum spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of Desulfovibrio sp. strain Lac3, Lac6, or Lac15 and Sulfurospirillum sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.