Showing papers on "Lambda phage published in 2000"

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

The R‐type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F‐type is related to lambda phage

Abstract: Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.

Towards Single-Copy Gene Expression Systems Making Gene Cloning Physiologically Relevant: Lambda InCh, a Simple Escherichia coli Plasmid-Chromosome Shuttle System

Abstract: We describe a simple system for reversible, stable integration of plasmid-borne genes into the Escherichia coli chromosome. Most ordinary E. coli strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used. A single genetic element, a lambda phage, is the only specialized vector required. The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted.

Use of lambda phage S and R gene products in an inducible lysis system for Vibrio cholerae- and Salmonella enterica serovar typhimurium-based DNA vaccine delivery systems.

Abstract: Novel methods for adapting DNA vaccine technology to the prevention of mucosal diseases are greatly needed. Here we show that regulated expression of phage lambda lysis genes S and R causes dramatic lysis of both Vibrio cholerae and Salmonella enterica serovar Typhimurium cells with concomitant release of plasmid DNA into the surrounding media. We also used single and double DNase mutant strains to show that secreted V. cholerae DNases can adversely affect the integrity of DNA molecules released upon lysis. These results suggest that incorporation of lambda SR-mediated lysis constructs and DNA stabilizing mutations into candidate live attenuated bacterial vaccines offers a promising approach for the development of effective mucosal DNA delivery vectors for humans.

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12.

Abstract: Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.

A Bacteriophage Lambda-Based Genetic Screen for Characterization of the Activity and Phenotype of the Human Immunodeficiency Virus Type 1 Protease

Abstract: Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

Abstract: The O protein is a replication initiator that binds to the oriλ region and promotes assembly of the bacteriophage λ replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage λ is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, λ plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wild-type cells growing in a rich medium. Contrary to λ plasmid replication, the efficiency of lytic growth of bacteriophage λ was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major λ promoters operating during the lytic development, p R and p L, were found to be slightly dependent on the host growth rate. However, when p R activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of λ plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage λ lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

Functional analysis of heterologous holin proteins in a λΔS genetic background

Abstract: Holins are small hydrophobic proteins causing non-specific membrane lesions at the end of bacteriophage multiplication, to promote access of the murein hydrolase to their substrate. We have established a λΔS genetic system, which enables functional expression of holins from various phages in an isogenic phage λ background, and allows qualitative evaluation of their ability to support lysis of Escherichia coli cells. Synthesis of Holins is under control of native λ transcription and translation initiation signals, and the temperature-sensitive CIts857 repressor. A number of different holins were tested in this study. The opposing action of phage λ S105 and S107 holin variants in lysis timing could be confirmed, whereas we found evidence for a functionally non-homologous dual translational start motif in the Listeria phage Hol500 holin, i.e., the Hol500-96 polypeptide starting at Met-1 revealed a more distinct lytic activity as compared to the shorter product Hol500-93. The largest holin known, HolTW from a Staphylococcus aureus phage, revealed an early lysis phenotype in the λΔSthf background, which conferred a plaque forming defect due to premature lysis. Mutant analysis revealed that an altered C-terminus and/or a V52L substitution were sufficient to delay lysis and enable plaque formation. These results suggest that the extensively charged HolTW C-terminus may be important in regulation of lysis timing. The gene 17.5 product of E. coli phage T7 was found to support sudden, saltatory cell lysis in the λΔSthf background, which clearly confirms its holin character. In conclusion, λΔSthf offers a useful genetic tool for studying the structure–function relationship of the extremely heterogeneous group of holin protein orthologs.

Functional expression and affinity selection of single-chain cro by phage display: isolation of novel DNA-binding proteins.

Abstract: A robust selection system affording phage display of the DNA-binding helix-turn-helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage lambda Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.

Optimization of bacteriophage λ Q − -containing recombinant Escherichia coli fermentation process

Abstract: Q− mutant phage λ is deficient in the synthesis of the proteins involved in cell lysis and λ DNA packaging. As a result, the replicated Q−λ DNA containing a cloned gene is not easily coated by a phage head and remains naked for the ample expression of the cloned gene, and also the host cells do not lyse easily and larger amounts of cloned gene products are produced. In a two-phase operation, the first phase is operated at a low temperature to keep the phage in the lysogenic state for cell growth and cloned gene stability, while the second phase is operated at a high temperature to induce the lytic state for the amplification of the cloned gene and overproduction of its product. This two-phase operation was optimized by determining both the optimal temperatures for the growth and production phases and the optimal switching time between the growth to the production phase. The optimal temperatures for growth and production phases were 33 and 40 °C, respectively. The optimal switching time was 3 h. The recombinant β-galactosidase production using this optimal process was about 20 times higher than in the single-copy lysogenic state.

Analysis of two-stage continuous operation of Escherichia coli containing bacteriophage λ vector

Abstract: Bacteriophage λ containing a cloned-gene is stably maintained in Escherichia coli in the lysogenic state while it is replicated and it overproduces a recombinant protein product in the lytic state. The host cell is eventually lysed in the lytic state. The kinetics of cell lysis and production induction were studied and are reported in this article through model equations. In two-stage continuous operation, the first tank is maintained in the lysogenic state for cell growth and cloned-gene stability while the second tank is in the lytic state for the overproduction of cloned-gene product. Individual cells in the second tank have different extent of the induction for product formation, since each has a different residence time. The different residence time for individual cells was taken into account using a population model. The numerical results show good agreement with the experimental data for the prediction of dilution rate in the second tank which gives the maximum product concentration.

Formation and Stability of the Bacteriophage λ Replication Complexes in UV-Irradiated Escherichia coli

Abstract: Bacteriophage λ replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter λ DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage λ, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the λ replication complex. We found that UV irradiation of the host cells prevented formation of the stable λ replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable λ replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of λ replication complexes is possible. Both stable and unstable λ replication complexes are functional because replication of λ DNA under conditions preventing formation of the stable complex proceeds efficiently.

Stability Puzzles in Phage Lambda

Abstract: The lysogeny maintenance switch in phage lambda is one of the simplest examples on the molecular level of computation, command and control in a living system. If, following infection of the bacterium E. coli, the virus enters the lysogenic pathway, it represses its developmental functions, and integrates its DNA into the host chromosome. In this state the prophage may be passively replicated for many generations of E. coli. In fact, this repressed state is intrinsically more stable than the gene encoding the repressor. We develop a mathematical formalism to predict the stability of such epigenetic states from affinities of the molecular components. We apply the model to the behavior of recently published mutants at the right operator complex of lambda, and find that the reported stability indicates that the current view of the switch is incomplete. The approach described here should be generally applicable to the stability of expressed states

The use of competitive PCR mimic to evaluate a Limulus lambda phage genomic DNA library.

Abstract: 1 A λ phage genomic DNA library for Limulus (L) polyphemus brain was constructed using the λGEM-12 vector and the host strain KW251 2 The primary library contained approximately 1275 × 106 independent clones, increasing upon amplfication to 666 × 109 pfu/ml in a total volume of 58 ml 3 A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library All clones contained inserts and the average size was 149 kb, ranging from 117 to 280 kb The library provides a 10-fold equivalent of the L polyphemus genome 4 A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA The putative protein kinase C e (PKCe) was selected as the target gene because its partial sequence of cDNA was recently cloned from L polyphemus brain in our laboratory (Cao et al, 1998) A 419-bp fragment of nonhomologous sequence derived from putative PKCe and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively 5 Within the genomic library DNA, a 08 value was obtained for the copy number of the putative PKCe gene that was detected in 01 amol of one equivalent L polyphemus genome in terms of the average recombinant molecular weight In the genomic DNA, a single copy of putative PKCe was found in 01 amol of one coverage for the L polyphemus genome Thus, it was implied that nearly 80% genetic resource was incorporated into the library This percentage was termed the incorporation rate 6 Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction

Cloning and Functional Analysis of ALS Family Genes from Candida albicans.

Abstract: With a 0.5 kb probe of CX2, distribution of CX2 tandem repeats was studied in different C.albicans strains. Results suggest that all the C.albicans strains tested contained the tandem repeat. In order to verify if the expression of CX2 was hyphal specific, its expression was analyzed under various inductive and non-inductive conditions. With CX2 0.5 kb probe, Northern hybridization analysis confirmed that it was specifically in hyphae. The result of chromosomal localizationtion and genomic Southern blot analysis suggested that there were other genes containing the tandem repeat besides of ALS1. A C.albicans s genomic DNA library was screened with the CX2 0.5 kb probe and several positive recombinant lambda phages were obtained. After endonuclease identification, subcloning, and sequence analysis, several ALS family genes were cloned. No.1 lambda phage DNA contained ALS4, No.4 lambda phage DNA contained ALS1, No.6 lambda phage DNA contained ALS3. To study the role of ALS family genes in yeast-hyphal transition, als1/ALS1 mutant was constructed by homologous recombination. The ability to form hyphae was tested in different inductive culturing conditions. Defective hyphal growth were observed in some solid media.