The evolution of Pyrosequencing® for microbiology: From genes to genomes.

Pyrosequencing is a unique sequencing method that was developed as an alternative to classical DNA sequencing for short- to medium-read applications. Compared to other methods, it is highly quantitative, fast and inexpensive. Additional advantages include high accuracy, flexibility and ability to automate sample preparation. This article presents a historical overview of pyrosequencing, improvements that have been made through the years and its evolution into a platform that vastly expanded the scope of genetic analysis that could be performed outside of a big sequencing center. In addition, we describe numerous applications in microbiology that have benefited from the pyrosequencing method.

Global analysis of ATM polymorphism reveals significant functional constraint.

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.

An automated hydrodynamic process for controlled, unbiased DNA shearing.

An automated, inexpensive, easy-to-use, and reproducible technique for controlled, random DNA fragmentation has been developed. The technique is based on point-sink hydrodynamics that result when a DNA sample is forced through a small hole by a syringe pump. Commercially available components are used to reduce the cost and complexity of the instrument. The design is optimized to reduce the volume of sample required and to speed processing time. Shearing of the samples can be completely automated by computer control. Ninety percent of sheared DNA fragments fall within a twofold size distribution that is highly reproducible. Three parameters are critical: the flow geometry, the flow rate, and a minimum number of iterations. Shearing is reproducible over a wide range of temperatures, DNA concentrations, and initial DNA size. The cloning efficiency of the sheared DNA is very good even without end repair, the distribution of assembled sequences is random, and there is no sequence bias at the ends of sheared fragments that have been cloned. The instrument, called the Point-sink Shearer (PtS), has already been exported successfully to many other laboratories.

Leaderless polypeptides efficiently extracted from whole cells by osmotic shock.

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation. A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E. coli when its native signal sequence was deleted. These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins. A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) beta-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient alpha complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA- or ftsZ-dependent mechanism. The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)--absent their normal mode of (signal sequence-dependent) translocation--was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins. Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization. As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens.

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.

The essential virulence protein VirB8 localizes to the inner membrane of Agrobacterium tumefaciens.

Agrobacterium tumefaciens genetically transforms plant cells by transferring a specific DNA fragment from the bacterium through several biological membranes to the plant nucleus where the DNA is integrated. This complex DNA transport process likely involves membrane-localized proteins in both the plant and the bacterium. The 11 hydrophobic or membrane-localized proteins of the virB operon are excellent candidates to have a role in DNA export from agrobacteria. Here, we show by TnphoA mutagenesis and immunogold electron microscopy that one of the VirB proteins, VirB8, is located at the inner membrane. The observation that a virB8::TnphoA fusion restores export of alkaline phosphatase to the periplasm suggests that VirB8 spans the inner membrane. Immunogold labeling of VirB8 was detected on the inner membrane of vir-induced A. tumefaciens by transmission electron microscopy. Compared with that of the controls, VirB8 labeling was significantly greater on the inner membrane than on the other cell compartments. These results confirm the inner membrane localization of VirB8 and strengthen the hypothesis that VirB proteins help form a transfer DNA export channel or gate.

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure.

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.

Effect of iodine disinfection products on higher plants.

Iodine is used to disinfect potable water on United States spacecraft. Iodinated potable water will likely be used to grow plants in space. Little is known about the effects of iodine disinfection products on plants. Seeds of select higher plants were germinated in water iodinated using the Shuttle Microbial Check Valve, and water to which measured amounts of iodide was added. Percent germination was decreased in seeds of most species germinated in iodinated water. Beans were most affected. Germination rates, determined from germination half-times, were decreased for beans germinated in iodinated water, and water to which iodide was added. Development was retarded and rootlets were conspicuously absent in bean and several other plant species germinated in iodinated water. Iodide alone did not elicit these responses. Clearly iodine disinfection products can affect higher plants. These effects must be carefully considered for plant experimentation and cultivation in space, and in design and testing of closed environmental life support systems.

Stable Carbon Isotope Composition (deltaC), Water Use Efficiency, and Biomass Productivity of Lycopersicon esculentum, Lycopersicon pennellii, and the F(1) Hybrid.

Three tomatoes, Lycopersicon esculentum Mill. cv UC82B, a droughttolerant wild related species, Lycopersicon pennellii (Cor.) D'Arcy, and their F(1) hybrid, were grown in containers maintained at three levels of soil moisture. Season-long water use was obtained by summing over the season daily weight losses of each container corrected for soil evaporation. Plant biomass was determined by harvesting and weighing entire dried plants. Season-long water use efficiency (gram dry weight/kilogram H(2)O) was calculated by dividing the dry biomass by the season-long water use. The season-long water use efficiency was greatest in the wild parent, poorest in the domestic parent, and intermediate (but closer to the wild parent) in the F(1) hybrid. Instantaneous water-use efficiency (micromole CO(2)/millimole H(2)O) determined by gas exchange measurements on individual leaves was poorly correlated with season-long water use efficiency. However, the relative abundance of stable carbon isotopes of leaf tissue samples was strongly correlated with the season-long water use efficiency. Also, the isotopic composition and the season-long water use efficiency of each genotype alone were strongly negatively correlated with plant dry weight when the dry weight varied as a function of soil moisture.

Effects of Air Pollutants on the Composition of Stable Carbon Isotopes, deltaC, of Leaves and Wood, and on Leaf Injury.

Air pollutants are known to cause visible leaf injury as well as impairment of photosynthetic CO(2) fixation. Here we evaluate whether the effects on photosynthesis are large enough to cause changes in the relative composition of stable carbon isotopes, delta(13)C, of plant tissue samples, and, if so, how the changes relate to visual leaf injury. For that purpose, several woody and herbaceous plant species were exposed to SO(2) + O(3) and SO(2) + O(3) + NO(2) for one month (8 hours per day, 5 days per week). At the end of the fumigations, the plants were evaluated for visual leaf lesions, and delta(13)C of leaf tissue was determined. Woody plants generally showed less visual leaf injury and smaller effects on delta(13)C of pollutant exposure than did herbaceous plants. If delta(13)C was affected by pollutants, it became, with few exceptions, less negative. The data from the fumigation experiments were consistent with delta(13)C analyses of whole wood of annual growth rings from two conifer tree species, Pseudotsuga menziesii and Pinus strobus. These trees had been exposed until 1977 to exhaust gases from a gas plant at Lacq, France. Wood of both conifer species formed in the polluted air of 1972 to 1976 had less negative delta(13)C values than had wood formed in the much cleaner air in 1982 to 1986. No similar, time-dependent differences in delta(13)C of wood were observed in trees which had been continuously growing in clean air. Our delta(13)C data from both relatively short-term artificial exposures and long-term natural exposure are consistent with greater stomatal limitation of photosynthesis in polluted air than in clean air.