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PubMed2006

From genomes to function: haloarchaea as model organisms.

Haloarchaea are adapted to high-salt environments and accumulate equally high salt concentrations in the cytoplasm. The genomes of representatives of six haloarchaeal genera have been fully or partially sequenced, allowing the analysis of haloarchaeal properties in silico. Transcriptome and proteome analyses have been established for Halobacterium salinarum and Haloferax volcanii. Genetic systems are available including methods that allow the fast in-frame deletion or modification of chromosomal genes. The high-efficiency transformation system of Hf. volcanii allows the isolation of genes essential for a biological process by complementation of loss-of-function mutants. For the analysis of haloarchaeal biology many molecular genetic, biochemical, structural and cell biological methods have been adapted to application at high salt concentrations. Recently it has become clear that several different mechanisms allow the adaptation of proteins to the high salt concentration of the cytoplasm. Taken together, the wealth of techniques available make haloarchaea excellent archaeal model species.

Adaptation, Physiological↗
PubMed2024

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

Journal Article↗
PubMed2001

Molecular motor based entirely on the Coulomb interaction.

A molecular motor is introduced which is composed of charges only and which does not contain any potential or interaction besides the Coulomb one. The motor is shown to transform efficiently a driven random rotation into a directed translational motion. The direction of translational motion can be chosen dynamically, so that a "forward gear," a "reverse gear," and even a "neutral gear" exist. The high efficiency stems from the fact that this motor only steps in the direction determined by the chosen gear.

Journal Article↗
PubMed2001

Dynamical encoding by networks of competing neuron groups: winnerless competition.

Following studies of olfactory processing in insects and fish, we investigate neural networks whose dynamics in phase space is represented by orbits near the heteroclinic connections between saddle regions (fixed points or limit cycles). These networks encode input information as trajectories along the heteroclinic connections. If there are N neurons in the network, the capacity is approximately e(N-1)!, i.e., much larger than that of most traditional network structures. We show that a small winnerless competition network composed of FitzHugh-Nagumo spiking neurons efficiently transforms input information into a spatiotemporal output.

Action Potentials↗
PubMed2004

Comparative genomics in salt tolerance between Arabidopsis and aRabidopsis-related halophyte salt cress using Arabidopsis microarray.

Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%-95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, beta-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.

Abscisic Acid↗
PubMed2006

Strategies for functional validation of genes involved in reproductive stages of orchids.

Plants in the largest family of angiosperms, Orchidaceae, are diverse in both specialized pollination and ecological strategies and provide a rich source for investigating evolutionary relationships and developmental biology. However, studies in orchids have been hindered by several challenges that include low transformation efficiency and long regeneration time. To overcome such obstacles, we selected a symptomless cymbidium mosaic virus (CymMV) isolate for constructing virus-induced gene-silencing vectors. The feasibility of the virus vectors was first assessed with use of an orchid phytoene desaturase gene. The vector was able to induce gene silencing in orchids; however, because of the slow growth of orchids, the commonly used phytoene desaturase gene was not a good visual marker in orchids. We inserted a 150-nucleotide unique region of a B-class MADS-box family gene, PeMADS6, into pCymMV-pro60. The transcription level of PeMADS6 in inoculated Phalaenopsis plants was reduced by up to 73%, but no effect was observed for other MADS-box family genes. In contrast, in Phalaenopsis plants inoculated with CymMV transcripts containing 500 nucleotides of PeMADS6, a conserved region among MADS-box genes, the transcription level of PeMADS6 and the B- and C-class MADS-box genes was reduced by up to 97.8% as compared with plants inoculated with the vector alone. Flower morphology was affected in the MADS-box family gene-silenced plants as well. This in vivo experiment demonstrates an efficient way to study genes involved in the reproductive stage of plants with a long life cycle.

Base Sequence↗
PubMed2006

Constitutive expression exposes functional redundancy between the Arabidopsis histone H2A gene HTA1 and other H2A gene family members.

The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agrobacterium tumefaciens. Disruption of this gene in the rat5 mutant results in decreased transformation. In Arabidopsis, histone H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in rat5 mutant plants. Overexpression of all tested HTA cDNAs restored transformation competence to the rat5 mutant. However, only the HTA1 gene, but not other HTA genes, could phenotypically complement rat5 mutant plants when expressed from their native promoters. Expression analysis of HTA promoters indicated that they had distinct but somewhat overlapping patterns of expression in mature plants. However, only the HTA1 promoter was induced by wounding or by Agrobacterium infection of root segments. Our data suggest that, with respect to Agrobacterium-mediated transformation, all tested histone H2A proteins are functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression patterns of the HTA genes.

Agrobacterium tumefaciens↗
PubMed1997

Transfer and Integration of T-DNA without Cell Injury in the Host Plant.

Agrobacterium colonizes plant cells via a gene transfer mechanism that results in plant tumorigenesis. Virulence (vir) genes are transcriptionally activated in the bacteria by plant metabolites released from the wound site. Hence, it is believed that agrobacteria use injuries to facilitate their entrance into the host plant and that the wounded state is required for plant cell competence for Agrobacterium-mediated gene delivery. However, our experiments using vir gene-activated bacteria sprayed onto tobacco plantlets demonstrated that cells in unwounded plants could also be efficiently transformed. The condition of the plant cells was monitored using [beta]-glucuronidase under the control of a wound-inducible promoter. Infection of leaf tissue is light dependent, and it is drastically reduced when abscisic acid is exogenously applied to the plant. Under these experimental conditions, stomatal opening seems to be used by Agrobacterium to circumvent the physical barrier of the cuticle. These results thus show that the proposed cellular responses evoked by wounding in higher plants are not essential for Agrobacterium-mediated transformation.

Journal Article↗
PubMed1992

Characterization of the minimal origin required for replication of the streptococcal plasmid pIP501 in Bacillus subtilis.

By using deletional analysis the origin of replication, oriR, of the streptococcal plasmid pIP501 in Bacillus subtilis has been mapped at a position immediately downstream of the repR gene. Determination of both the right and left border of oriR allowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading-strand synthesis of the closely related plasmid pAM beta 1 has been mapped at a position which is located exactly in the middle of this sequence (Bruand et al., 1991). The function of oriR did not depend on its location downstream of the repR gene. Translocation of oriR-containing fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of an Escherichia coli plasmid in B. subtilis when the RepR protein was supplied in trans from a repR gene integrated into the host chromosome. The transformation efficiency of plasmids carrying translocated oriR fragments showed a certain dependence on the fragment length and orientation. The DNA sequence of oriR included an inverted repeat, both branches of which appeared to be essential for oriR function. The repeats of oriR shared sequence similarity with a repeat located upstream of promoter pII, which has been suggested to be involved in autoregulation of repR expression.

Amino Acid Sequence↗
PubMed1994

Neither absence nor excess of lambda O initiator-digesting ClpXP protease affects lambda plasmid or phage replication in Escherichia coli.

Owing to rapid proteolysis of the coliphage lambda-coded initiator protein, lambda O, this protein is considered to carry a rate-limiting step in lambda DNA replication. The discovery of ClpXP protease responsible for lambda O protein turnover allowed an opportunity to verify this hypothesis. However, neither absence nor excess of this protease significantly affected the transformation efficiency and copy number of lambda plasmid, or the kinetics of the lambda phage growth. These results are also incompatible with the hypothesis that the stabilization of lambda O plays a role in the switch from early (circle-to-circle) to late (rolling-circle) lambda phage DNA replication. Transcriptional activation of ori lambda, probably assisted by the Escherichia coli DnaA function, remains as the possible rate-limiting step in lambda DNA replication.

ATP-Dependent Proteases↗
PubMed1990

High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions.

High efficiency transformation of Pseudomonas aeruginosa was achieved using frozen cell suspensions and high voltage electroporation. We have obtained frequencies as high as 5.8 x 10(8) transformants/micrograms of plasmid DNA using PA01 strain OT684 and a buffer of 15% glycerol-1 mM MOPS. The method allows for easy and reproducible production of frozen cell suspensions for rapid transformation of P. aeruginosa.

DNA, Bacterial↗
PubMed1984

Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene.

Avian erythroblastosis virus (AEV) contains two distinct oncogenes, erbA and erbB . The erbB oncogene, which is homologous to a portion of the epidermal growth factor receptor, is related to the src family of oncogenes and efficiently transforms erythroblasts, whereas erbA potentiates the effects of erbB by blocking the differentiation of erythroblasts at an immature stage. This "potentiator" was sequenced; the amino acid sequence deduced from it was clearly different from the sequences of other known oncogene products and was related to carbonic anhydrases. These enzymes participate in the transport of carbon dioxide by erythrocytes, the precursors of which are main targets of avian erythroblastosis virus. A src-related oncogene such as erbB in synergy with an activated specific cell-derived gene such as erbA can profoundly affect early erythroid differentiation.

Alpharetrovirus↗
PubMed1995

Superresolution three-dimensional images of fluorescence in cells with minimal light exposure.

Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.

Algorithms↗
PubMed2025

An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

Bacteriophage T7↗
PubMed1994

Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus.

A 6.4-kb DNA fragment containing the DNA gyrase gyrA and gyrB genes was cloned and sequenced from the quinolone-susceptible Staphylococcus aureus type strain ATCC 12600. An expression plasmid was constructed by inserting the cloned genes into the Escherichia coli-S. aureus shuttle vector pAT19, and deletion plasmids carrying only functional gyrA and gyrB genes were derived from this plasmid. An efficient transformation system for S. aureus RN4220 was established by using these plasmids. Quinolone-resistant mutants of S. aureus RN4220 were isolated by three-step selection with quinolones. The first- and second-step mutants were considered to be transport mutants, and the third-step mutants were divided into five groups with respect to their resistance patterns and transformation results with gyrA and gyrB genes. Sequencing analysis of the resulting mutant gyrase genes showed that they had the following point mutations: group 1, Ser-84 (TCA) to Leu (TTA) in GyrA; group 2, Ser-84 (TCA) to Ala (GCA), Ser-85 (TCT) to Pro (CCT), or Glu-88 (GAA) to Lys (AAA) in GyrA; group 3, Asp-437 (GAC) to Asn (AAC) in GyrB; group 4, Arg-458 (CGA) to Gln (CAA) in GyrB; and group 5, Ser-85 (TCT) to Pro (CCT) in GyrA and Asp-437 (GAC) to Asn (AAC) in GyrB. When the gyrA and/or gyrB mutants were transformed with the wild-type gyrA and/or gyrB plasmids, they became quinolone susceptible, but transformants with the plasmids having the same mutations on the gyrA and/or gyrB genes did not confer susceptibility. These results indicate that mutations in both gyrA and gyrB can be responsible for quinolone resistance in S. aureus.

Amino Acid Sequence↗
PubMed2003

Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation.

The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 x 10(-7) (20% of isolates) and 2.5 x 10(-8). The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and "normomutable" strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.

Bacterial Proteins↗
PubMed1995

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.

Animals↗